![[home]](http://www.molgen.mpg.de/pics/2001/mhead2001_home_off.gif){kind=small} |
![[contact]](http://www.molgen.mpg.de/pics/2001/mhead2001_contact_off.gif){kind=small} |
![[search]](http://www.molgen.mpg.de/pics/2001/mhead2001_search_off.gif){kind=small} |
![[back to Vertebrate Genomics]](http://www.molgen.mpg.de/pics/2001/mhead2001_titlesmall_vertegen.gif){kind=small} |
 |
|
|
|
|
|||
|
|
||||
|
|||||
|
Article (Adler2003) Adler, M.; Wacker, R. & Niemeyer, C.M. A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins. Biochem Biophys Res Commun, 2003, 308, 240-250 Abstract: A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.
Pubmed: ABSTRACT Article (Aerts2002) Aerts, J.; Wetzels, Y.; Cohen, N. & Aerssens, J. Data mining of public SNP databases for the selection of intragenic SNPs Hum Mutat, 2002, 20, 162-73 Abstract: Different strategies to search public single nucleotide polymorphism (SNP) databases for intragenic SNPs were evaluated. First, we assembled a strategy to annotate SNPs onto candidate genes based on a BLAST search of public SNP databases (Intragenic SNP Annotation by BLAST, ISAB). Only BLAST hits that complied with stringent criteria according to 1) percentage identity (minimum 98%), 2) BLAST hit length (the hit covers at least 98% of the length of the SNP entry in the database, or the hit is longer than 250 base pairs), and 3) location in non-repetitive DNA, were considered as valid SNPs. We assessed the intragenic context and redundancy of these SNPs, and demonstrated that the SNP content of the dbSNP and HGBASE/HGVbase databases are highly complementary but also overlap significantly. Second, we assessed the validity of intragenic SNP annotation available on the dbSNP and HGVbase websites by comparison with the results of the ISAB strategy. Only a minority of all annotated SNPs was found in common between the respective public SNP database websites and the ISAB annotation strategy. A detailed analysis was performed aiming to explain this discrepancy. As a conclusion, we recommend the application of an independent strategy (such as ISAB) to annotate intragenic SNPs, complementary to the annotation provided at the dbSNP and HGVbase websites. Such an approach might be useful in the selection process of intragenic SNPs for genotyping in genetic studies. Hum Mutat 20:162-173, 2002.
Pubmed: ABSTRACT Article (Ahmadian2001) Ahmadian, A.; Gharizadeh, B.; D, O.'.; Odeberg, J. & Lundeberg, J. Genotyping by apyrase-mediated allele-specific extension Nucleic Acids Res, 2001, 29, E121 Abstract: This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.
Pubmed: ABSTRACT Article (Altshuler2000) Altshuler, D.; Daly, M. & Kruglyak, L. Guilt by association Nat Genet, 2000, 26, 135-7
Pubmed: ABSTRACT Article (Angenendt2004) Angenendt, P.; Nyarsik, L.; Szaflarski, W.; Glokler, J.; Nierhaus, K.H.; Lehrach, H.; Cahill, D.J. & Lueking, A. Cell-free protein expression and functional assay in nanowell chip format Anal Chem, 2004, 76, 1844-9 Abstract: The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.
Pubmed: ABSTRACT Article (Antson2000) Antson, D.O.; Isaksson, A.; Landegren, U. & Nilsson, M. PCR-generated padlock probes detect single nucleotide variation in genomic DNA Nucleic Acids Res, 2000, 28, E58 Abstract: Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5'- and 3'-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.
Pubmed: ABSTRACT Article (Arakawa2003) Arakawa, K.; Mori, K.; Ikeda, K.; Matsuzaki, T.; Kobayashi, Y. & Tomita, M. G-language Genome Analysis Environment: a workbench for nucleotide sequence data mining Bioinformatics, 2003, 19, 305-6 Abstract: Summary: G-language Genome Analysis Environment (G-language GAE) is an open source generic software package aimed for higher efficiency in bioinformatics analysis. G-language GAE has an interface as a set of Perl libraries for software development, and a graphical user interface for easy manipulation. Both Windows and Linux versions are available. Availability: From http://www.g-language.org/ under GNU General Public License. CD-ROMs are distributed freely in major conferences. Contact: info@g-language.org
Pubmed: ABSTRACT Article (Ari2005) Ari, G.B.; Zenvirth, D.; Sherman, A.; Simchen, G.; Lavi, U. & Hillel, J. Application of SNPs for assessing biodiversity and phylogeny among yeast strains. Heredity, 2005, 95, 493-501 Abstract: We examined the efficacy of single-nucleotide polymorphism (SNP) markers for the assessment of the phylogeny and biodiversity of Saccharomyces strains. Each of 32 Saccharomyces cerevisiae strains was genotyped at 30 SNP loci discovered by sequence alignment of the S. cerevisiae laboratory strain SK1 to the database sequence of strain S288c. In total, 10 SNPs were selected from each of the following three categories: promoter regions, nonsynonymous and synonymous sites (in open reading frames). The strains in this study included 11 haploid laboratory strains used for genetic studies and 21 diploids. Three non-cerevisiae species of Saccharomyces (sensu stricto) were used as an out-group. A Bayesian clustering-algorithm, Structure, effectively identified four different strain groups: laboratory, wine, other diploids and the non-cerevisiae species. Analysing haploid and diploid strains together caused problems for phylogeny reconstruction, but not for the clustering produced by Structure. The ascertainment bias introduced by the SNP discovery method caused difficulty in the phylogenetic analysis; alternative options are proposed. A smaller data set, comprising only the nine most polymorphic loci, was sufficient to obtain most features of the results.
Pubmed: ABSTRACT Article (Ashburner2000) Ashburner, M.; Ball, C.A.; Blake, J.A.; Botstein, D.; Butler, H.; Cherry, J.M.; Davis, A.P.; Dolinski, K.; Dwight, S.S.; Eppig, J.T.; Harris, M.A.; Hill, D.P.; Issel-Tarver, L.; Kasarskis, A.; Lewis, S.; Matese, J.C.; Richardson, J.E.; Ringwald, M.; Rubin, G.M. & Sherlock, G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium Nat Genet, 2000, 25, 25-9
Pubmed: ABSTRACT Article (Auroux2002) Auroux, P.; Iossifidis, D.; Reyes, D.R. & Manz, A. Micro total analysis systems. 2. Analytical standard operations and applications. Anal Chem, 2002, 74, 2637-2652
Pubmed: ABSTRACT Article (Avi-Itzhak2003) Avi-Itzhak, H.I.; Su, X. & De La Vega, F.M. Selection of minimum subsets of single nucleotide polymorphisms to capture haplotype block diversity Pac Symp Biocomput, 2003, 466-77 Abstract: We present a simple numerical algorithm to select the minimal subset of SNPs required to capture the diversity of haplotype blocks or other genetic loci. This algorithm can be used to quickly select the minimum SNP subset with no loss of haplotype information. In addition, the method can be used in a more aggressive mode to further reduce the original SNP set, with minimal loss of information. We demonstrate the algorithm performance with data from over 11,000 SNPs with average spacing of 6 to 11 Kb, across all the genes of chromosomes 6, 21, and 22, genotyped on DNA samples of 45 unrelated African-Americans and 45 Caucasians from the Coriell Human Diversity Collection. With no loss of information, we reduced the number of SNPs required to capture the haplotype block diversity by 25% for the African-American and 36% for the Caucasian populations. With a maximum loss of 10% of haplotype distribution information, the SNP reduction was 38% and 49% respectively for the two populations. All computations were performed in less than 1 minute for the entire dataset used.
Pubmed: ABSTRACT Article (Bai2004) Bai, X.; Kim, S.; Li, Z.; Turro, N.J. & Ju, J. Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry Nucleic Acids Res, 2004, 32, 535-41 Abstract: We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2deoxyribouridine 5triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can be efficiently isolated by a streptavidin-coated surface and recovered by near-UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP-PC-Biotin to generate DNA products for MALDI-TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS-based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA-protein complexes under non-denaturing conditions.
Pubmed: ABSTRACT Article (Bai2003) Bai, X.; Li, Z.; Jockusch, S.; Turro, N.J. & Ju, J. Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5end of DNA Proc Natl Acad Sci U S A, 2003, 100, 409-13 Abstract: Three single-stranded DNA molecules of different lengths were synthesized and characterized, each containing a fluorescent dye (6-carboxyfluorescein) connected to the 5end via a photocleavable 2-nitrobenzyl linker and a biotin moiety at the 3end. UV irradiation (lambda approximately 340 nm) of solutions containing these fluorescent DNA molecules caused the complete cleavage of the nitrobenzyl linker, separating the fluorophore from the DNA. The photocleavage products were characterized by HPLC and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Our experimental results indicated that the proximity of the chromophore 6-carboxyfluorescein to the 2-nitrobenzyl linker did not hinder the quantitative photocleavage of the linker in the DNA molecules. The biotin moiety allowed immobilization of the fluorescent DNA on streptavidin-coated glass chips. The photocleavage of the immobilized DNA was investigated directly by fluorescence spectroscopy. The results demonstrated that close to 80% of the fluorophore was removed from the immobilized DNA after UV irradiation at 340 nm. These results strongly support the application of the 2-nitrobenzyl moiety as an efficient photocleavable linker, connecting fluorescent probes to DNA molecules for a variety of biological analyses such as DNA sequencing by synthesis.
Pubmed: ABSTRACT Article (Bailey2002) Bailey, J.A.; Gu, Z.; Clark, R.A.; Reinert, K.; Samonte, R.V.; Schwartz, S.; Adams, M.D.; Myers, E.W.; Li, P.W. & Eichler, E.E. Recent segmental duplications in the human genome Science, 2002, 297, 1003-7 Abstract: Primate-specific segmental duplications are considered important in human disease and evolution. The inability to distinguish between allelic and duplication sequence overlap has hampered their characterization as well as assembly and annotation of our genome. We developed a method whereby each public sequence is analyzed at the clone level for overrepresentation within a whole-genome shotgun sequence. This test has the ability to detect duplications larger than 15 kilobases irrespective of copy number, location, or high sequence similarity. We mapped 169 large regions flanked by highly similar duplications. Twenty-four of these hot spots of genomic instability have been associated with genetic disease. Our analysis indicates a highly nonrandom chromosomal and genic distribution of recent segmental duplications, with a likely role in expanding protein diversity.
Pubmed: ABSTRACT Article (Ball2003) Ball, T.B.; Plummer, F.A. & HayGlass, K.T. Improved mRNA quantitation in LightCycler RT-PCR Int Arch Allergy Immunol, 2003, 130, 82-6 Abstract: BACKGROUND: Real-time polymerase chain reaction (PCR) utilizing the LightCycler and similar systems is an increasingly used technique for quantitative reverse transcription (RT)-PCR of mRNA levels from genes of immunologic interest. A commonly encountered limitation with these systems is that the fluorescence induced by SYBR Green (a fluorophore that binds double-stranded DNA) can result from primer dimers (PDs) as well as the PCR product of interest, thus interfering with the ability to reproducibly quantitate mRNA levels. METHODS: We use a modification of the LightCycler PCR strategy to overcome this problem by altering the PCR strategy to take advantage of the LightCycler ability to measure fluorescence at a temperature greater than the melting point of PDs. The resulting measurements determine fluorescence of only the desired PCR product. RESULTS: We demonstrate that by using this modified PCR strategy, one can eliminate the fluorescence induced by PDs and obtain accurate product quantitation. CONCLUSIONS: This simple modification allows more precise quantitation of sample mRNA levels by eliminating the contaminating fluorescence induced by the formation of PCR PDs. This modification obviates the need to redesign PCR primers in RT-PCR experiments where this is impractical or impossible.
Pubmed: ABSTRACT Article (Bammler2005) Bammler, T.; Beyer, R.P.; Bhattacharya, S.; Boorman, G.A.; Boyles, A.; Bradford, B.U.; Bumgarner, R.E.; Bushel, P.R.; Chaturvedi, K.; Choi, D.; Cunningham, M.L.; Deng, S.; Dressman, H.K.; Fannin, R.D.; Farin, F.M.; Freedman, J.H.; Fry, R.C.; Harper, A.; Humble, M.C.; Hurban, P.; Kavanagh, T.J.; Kaufmann, W.K.; Kerr, K.F.; Jing, L.; Lapidus, J.A.; Lasarev, M.R.; Li, J.; Li, Y.; Lobenhofer, E.K.; Lu, X.; Malek, R.L.; Milton, S.; Nagalla, S.R.; O'malley, J.P.; Palmer, V.S.; Pattee, P.; Paules, R.S.; Perou, C.M.; Phillips, K.; Qin, L.; Qiu, Y.; Quigley, S.D.; Rodland, M.; Rusyn, I.; Samson, L.D.; Schwartz, D.A.; Shi, Y.; Shin, J.; Sieber, S.O.; Slifer, S.; Speer, M.C.; Spencer, P.S.; Sproles, D.I.; Swenberg, J.A.; Suk, W.A.; Sullivan, R.C.; Tian, R.; Tennant, R.W.; Todd, S.A.; Tucker, C.J.; Houten, B.V.; Weis, B.K.; Xuan, S.; Zarbl, H. & of the Toxicogenomics Research Consortium, M. Standardizing global gene expression analysis between laboratories and across platforms. Nat Methods, 2005, 2, 351-356 Abstract: To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.
Pubmed: ABSTRACT Article (Baner2003) Baner, J.; Isaksson, A.; Waldenstrom, E.; Jarvius, J.; Landegren, U. & Nilsson, M. Parallel gene analysis with allele-specific padlock probes and tag microarrays Nucleic Acids Res, 2003, 31, e103 Abstract: Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.
Pubmed: ABSTRACT Article (Baner2001) Baner, J.; Nilsson, M.; Isaksson, A.; Mendel-Hartvig, M.; Antson, D.O. & Landegren, U. More keys to padlock probes: mechanisms for high-throughput nucleic acid analysis Curr Opin Biotechnol, 2001, 12, 11-5 Abstract: With the impending availability of total information about nucleic acid sequences in humans and other organisms, tools to investigate these sequences on a large scale assume increasing importance. Methods currently in use, however, cannot offer the required combination of high-throughput, sensitivity and specificity of detection. Padlock probes, circularizing oligonucleotides, may provide a means to detect, distinguish, quantitate and also locate very large numbers of DNA or RNA sequences. Recent developments in areas such as the biochemistry of ligation and characterization of ligases, methods to replicate circularized probes and the development of assays based on these principles augment the potential of padlock probes.
Pubmed: ABSTRACT Article (Baner1998) Baner, J.; Nilsson, M.; Mendel-Hartvig, M. & Landegren, U. Signal amplification of padlock probes by rolling circle replication Nucleic Acids Res, 1998, 26, 5073-8 Abstract: Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.
Pubmed: ABSTRACT Article (Barker2000) Barker; Tarlov; Canavan; Hickman & Locascio Plastic microfluidic devices modified with polyelectrolyte multilayers Anal Chem, 2000, 72, 4899-4903 Abstract: Control of the polymer surface chemistry is a crucial aspect of development of plastic microfluidic devices. When commercially available plastic substrates are used to fabricate microchannels, differences in the EOF mobility from plastic to plastic can be very high. Therefore, we have used polyelectrolyte multilayers (PEMs) to alter the surface of microchannels fabricated in plastics. Optimal modification of the microchannel surfaces was obtained by coating the channels with alternating layers of poly(allylamine hydrochloride) and poly(styrene sulfonate). Polystyrene (PS) and poly(ethylene terephthalate) glycol (PETG) were chosen as substrate materials because of the significant differences in the polymer chemistries and in the EOF of channels fabricated in these two plastic materials. The efficacy of the surface modification has been evaluated using XPS and by measuring the EOF mobility. When microchannels prepared in both PS and PETG are modified with PEMs, they demonstrate very similar electroosmotic mobilities. The PEMs are easily fabricated and provide a means for controlling the flow direction and the electroosmotic mobility in the channels. The PEM-coated microchannels have excellent wettability, allowing facile filling of the channels. In addition, the PEMs produce reproducible results and are robust enough to withstand long-term storage.
Pubmed: ABSTRACT Article (Barnes2002) Barnes, M.R. Psychiatric genetics in silico: databases and tools for psychiatric geneticists Psychiatr Genet, 2002, 12, 67-73 Abstract: Bioinformatics can significantly impact the laboratory genetics process from the study design phase to conclusive identification of a disease gene. The present review will highlight key databases to enhance psychiatric genetic study design, based on full use of genomics data and the golden path sequence. It will address methods to ensure comprehensive genetic data mining, using the best available genomic and genetic databases such as the University of California Santa Cruz human genome browser, Ensembl, Mapview, dbSNP and GDB, and locus-specific databases such as Online Mendelian Inheritance In Man. Using the golden path sequence as a template, with the necessary quality checks, it is possible to design detailed genetic studies from sequence information alone. Drawing together this diverse information, it is possible to characterize a locus or gene in silico to a very detailed level. This in turn can have real cost and efficiency benefits by assisting in the identification of markers that are most likely to be informative, or by highlighting the best candidate genes for study.
Pubmed: ABSTRACT Article (Bauer2004) Bauer, O.; Guerasimova, A.; Sauer, S.; Thamm, S.; Steinfath, M.; Herwig, R.; Janitz, M.; Lehrach, H. & Radelof, U. Multiplexed hybridizations of positively charge-tagged peptide nucleic acids detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Rapid Commun Mass Spectrom, 2004, 18, 1821-9 Abstract: Peptide nucleic acid (PNA) is a novel class of DNA analogues in which the entire sugar-phosphate backbone is replaced by a pseudopeptide counterpart. Owing to its neutral character and the consequent lack of electrostatic repulsion, PNA exhibits very stable heteroduplex formation with complementary nucleic acid that is essentially ionic strength independent and enables hybridization under minimum salt conditions. This feature as well as its superior ion stability and easy ionization compared to DNA renders PNA very attractive for hybridization-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) applications. We have developed an approach to DNA characterization that takes advantage of multiplexed PNA hybridizations analyzed by MALDI-TOFMS. Our motivation was the further development of oligonucleotide fingerprinting, an efficient technique for cDNA and genomic DNA library characterization. Through positive harge-taggingof PNA the efficiency of detection in MALDI-TOFMS was considerably enhanced permitting an unparalleled degree of multiplexing. Results from the simultaneous hybridization of 21 charge-tagged PNA hexamer oligonucleotides showed that genomic DNA and cDNA clones are successfully characterized on the basis of their hybridization profiles. The degree of multiplexing achieved may render a significant increase in throughput and hence efficiency of oligonucleotide fingerprinting possible.
Pubmed: ABSTRACT Article (Beaudet2001) Beaudet, L.; Bedard, J.; Breton, B.; Mercuri, R.J. & Budarf, M.L. Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen Genome Res, 2001, 11, 600-8 Abstract: AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 microL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.
Pubmed: ABSTRACT Article (Becker2000) Becker, H. & Gärtner, C. Polymer microfabrication methods for microfluidic analytical applications. Electrophoresis, 2000, 21, 12-26 Abstract: A growing number of microsystem technology (MST) applications, particularly in the field of microfluidics with its applications in the life sciences, have a need for novel fabrication methods which account for substrates other than silicon or glass. We present in this paper an overview of existing polymer microfabrication technologies for microfluidic applications, namely replication methods such as hot embossing, injection molding and casting, and the technologies necessary to fabricate the molding masters. In addition, techniques such as laser ablation and layering techniques are examined. Methods for bonding and dicing of polymer materials, which are necessary for complete systems, are evaluated.
Pubmed: ABSTRACT Article (Beckman2006) Beckman, K.B.; Abel, K.J.; Braun, A. & Halperin, E. Using DNA pools for genotyping trios. Nucleic Acids Res, 2006 Abstract: The genotyping of mother-father-child trios is a very useful tool in disease association studies, as trios eliminate population stratification effects and increase the accuracy of haplotype inference. Unfortunately, the use of trios for association studies may reduce power, since it requires the genotyping of three individuals where only four independent haplotypes are involved. We describe here a method for genotyping a trio using two DNA pools, thus reducing the cost of genotyping trios to that of genotyping two individuals. Furthermore, we present extensions to the method that exploit the linkage disequilibrium structure to compensate for missing data and genotyping errors. We evaluated our method on trios from CEPH pedigree 66 of the Coriell Institute. We demonstrate that the error rates in the genotype calls of the proposed protocol are comparable to those of standard genotyping techniques, although the cost is reduced considerably. The approach described is generic and it can be applied to any genotyping platform that achieves a reasonable precision of allele frequency estimates from pools of two individuals. Using this approach, future trio-based association studies may be able to increase the sample size by 50% for the same cost and thereby increase the power to detect associations.
Pubmed: ABSTRACT Article (Beebe2002) Beebe, D.J.; Mensing, G.A. & Walker, G.M. Physics and applications of microfluidics in biology Annu Rev Biomed Eng, 2002, 4, 261-86 Abstract: Fluid flow at the microscale exhibits unique phenomena that can be leveraged to fabricate devices and components capable of performing functions useful for biological studies. The physics of importance to microfluidics are reviewed. Common methods of fabricating microfluidic devices and systems are described. Components, including valves, mixers, and pumps, capable of controlling fluid flow by utilizing the physics of the microscale are presented. Techniques for sensing flow characteristics are described and examples of devices and systems that perform bioanalysis are presented. The focus of this review is microscale phenomena and the use of the physics of the scale to create devices and systems that provide functionality useful to the life sciences.
Pubmed: ABSTRACT Article (Belgrader2001) Belgrader, P.; Young, S.; Yuan, B.; Primeau, M.; Christel, L.A.; Pourahmadi, F. & Northrup, M.A. A battery-powered notebook thermal cycler for rapid multiplex real-time PCR analysis Anal Chem, 2001, 73, 286-9 Abstract: A compact, real-time PCR instrument was developed for rapid, multiplex analysis of nucleic acids in an inexpensive, portable format. The instrument consists of a notebook computer, two reaction modules with integrated optics for four-color fluorescence detection, batteries, and a battery-charging system. The instrument weighs 3.3 kg, measures 26 x 22 x 7.5 cm, and can run continuously on the internal batteries for 4 h. Independent control of the modules allows differing temperature profiles and detection schemes to be run simultaneously. Results are presented that demonstrate rapid (1) detection and identification of Bacillus subtilis and Bacillus thuringensis spores and (2) characterization of a single nucleotide polymorphism for the hereditary hemochromatosis gene.
Pubmed: ABSTRACT Article (Bell2002) Bell, P.A.; Chaturvedi, S.; Gelfand, C.A.; Huang, C.Y.; Kochersperger, M.; Kopla, R.; Modica, F.; Pohl, M.; Varde, S.; Zhao, R.; Zhao, X. & Boyce-Jacino, M.T. SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery Biotechniques, 2002, Suppl, 70-2, 74, 76-7 Abstract: Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy
Pubmed: ABSTRACT Article (Bellin2001) Bellin, T.; Pulz, M.; Matussek, A.; Hempen, H.G. & Gunzer, F. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J Clin Microbiol, 2001, 39, 370-374 Abstract: In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.
Pubmed: ABSTRACT Article (Benes2003) Benes, V. & Muckenthaler, M. Standardization of protocols in cDNA microarray analysis. Trends Biochem Sci, 2003, 28, 244-249 Abstract: Systematic variations can occur at various steps of a cDNA microarray experiment and affect the measurement of gene expression levels. Accepted standards integrated into every cDNA microarray analysis can assess these variabilities and aid the interpretation of cDNA microarray experiments from different sources. A universally applicable approach to evaluate parameters such as input and output ratios, signal linearity, hybridization specificity and consistency across an array, as well as normalization strategies, is the utilization of exogenous control genes as spike-in and negative controls. We suggest that the use of such control sets, together with a sufficient number of experimental repeats, in-depth statistical analysis and thorough data validation should be made mandatory for the publication of cDNA microarray data.
Pubmed: ABSTRACT Article (Benson2003) Benson, D.A.; Karsch-Mizrachi, I.; Lipman, D.J.; Ostell, J. & Wheeler, D.L. GenBank Nucleic Acids Res, 2003, 31, 23-7 Abstract: GenBank (R) is a comprehensive sequence database that contains publicly available DNA sequences for more than 119 000 different organisms, obtained primarily through the submission of sequence data from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the BankIt (web) or Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in the UK and the DNA Data Bank of Japan helps ensure worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, go to the NCBI home page at: http://www.ncbi.nlm.nih.gov.
Pubmed: ABSTRACT Article (Bergen2005) Bergen, A.W.; Haque, K.A.; Qi, Y.; Beerman, M.B.; Closas, M.G.; Rothman, N. & Chanock, S.J. Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources. Hum Mutat, 2005, 26, 262-270 Abstract: The promise of whole genome amplification (WGA) is that genomic DNA (gDNA) quantity will not limit molecular genetic analyses. Multiple displacement amplification (MDA) and the OmniPlex PCR-based WGA protocols were evaluated using 4 and 5 ng of input gDNA from 60 gDNA samples from three tissue sources (mouthwash, buffy coat, and lymphoblast). WGA DNA (wgaDNA) yield and genotyping performance were evaluated using genotypes determined from gDNA and wgaDNA using the AmpFlSTR Identifiler assay and N = 49 TaqMan SNP assays. Short tandem repeat (STR) and SNP genotyping completion and concordance rates were significantly reduced with wgaDNA from all WGA methods compared with gDNA. OmniPlex wgaDNA exhibited a greater reduction in genotyping performance than MDA wgaDNA. Reduced wgaDNA genotyping performance was due to allelic (all protocols) and locus (OmniPlex) amplification bias leading to heterozygote and locus dropout, respectively, and %GC sequence content (%GC) was significantly correlated with TaqMan assay performance. Lymphoblast wgaDNA exhibited higher yield (OmniPlex), buffy coat wgaDNA exhibited higher STR genotyping completion (MDA), whereas mouthwash wgaDNA exhibited higher SNP genotyping discordance (MDA). Genotyping of wgaDNA generated from < or = 5 ng gDNA, e.g., from archaeological, forensic, prenatal diagnostic, or pathology samples, may require additional genotyping validation with gDNA and/or more sophisticated analysis of genotypes incorporating observed reductions in genotyping performance.
Pubmed: ABSTRACT Article (Beyer1995) Beyer Hietpas, P. & Ewing, A. On-column and post-column derivatization for capillary electrophoresis with laser-induced fluorescence for the analysis of single cells Journal of Liquid Chromatography, 1995, 18, 3557-76
Article (Bilitewski2003) Bilitewski, U.; Genrich, M.; Kadow, S. & Mersal, G. Biochemical analysis with microfluidic systems Anal Bioanal Chem, 2003, 377, 556-69 Abstract: Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.
Pubmed: ABSTRACT Article (Birch1996) Birch, D.E. Simplified hot start PCR Nature, 1996, 381, 445-6
Pubmed: ABSTRACT Article (Birney2006) Birney, E.; Andrews, D.; Caccamo, M.; Chen, Y.; Clarke, L.; Coates, G.; Cox, T.; Cunningham, F.; Curwen, V.; Cutts, T.; Down, T.; Durbin, R.; Suarez, X.M.F.; Flicek, P.; Gräf, S.; Hammond, M.; Herrero, J.; Howe, K.; Iyer, V.; Jekosch, K.; Kähäri, A.; Kasprzyk, A.; Keefe, D.; Kokocinski, F.; Kulesha, E.; London, D.; Longden, I.; Melsopp, C.; Meidl, P.; Overduin, B.; Parker, A.; Proctor, G.; Prlic, A.; Rae, M.; Rios, D.; Redmond, S.; Schuster, M.; Sealy, I.; Searle, S.; Severin, J.; Slater, G.; Smedley, D.; Smith, J.; Stabenau, A.; Stalker, J.; Trevanion, S.; Vidal, A.U.; Vogel, J.; White, S.; Woodwark, C. & Hubbard, T.J.P. Ensembl 2006. Nucleic Acids Res, 2006, 34, D556-D561 Abstract: The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased from 4 to 19, with the addition of the mammalian genomes of Rhesus macaque and Opossum, the chordate genome of Ciona intestinalis and the import and integration of the yeast genome. The year has also seen extensive improvements to both data analysis and presentation, with the introduction of a redesigned website, the addition of RNA gene and regulatory annotation and substantial improvements to the integration of human genome variation data.
Pubmed: ABSTRACT Article (Blanco1996) Blanco, L. & Salas, M. Relating structure to function in phi29 DNA polymerase. J Biol Chem, 1996, 271, 8509-8512
Pubmed: ABSTRACT Article (BlattlerAugust2006) Blattler, T.; Huwiler, C.; Ochsner, M.; Stadler, B.; Solak, H.; Voros, J. & Grandin, H.M. Nanopatterns with Biological Functions Journal of Nanoscience and Nanotechnology, August 2006, 6, 2237-2264(28) Abstract: Both curiosity and a desire for efficiency have advanced our ability to manipulate materials with great precision on the micrometer and, more recently, on the nanometer scale. Certainly, the semi-conductor and integrated circuit industry has put the pressure on scientist and engineers to develop better and faster nanofabrication techniques. Furthermore, our curiosity as to how life works, and how it can be improved from a medical perspective, stands to gain a great deal from advances in nanotechnology. Novel nanofabrication techniques are opening up the possibilities for mimicking the inherently nano-world of the cell, i.e., the nanotopographies of the extracellular matrix (ECM) and the nanochemistry presented on both the cell membrane and the ECM. In addition, biosensing applications that rely on fabrication of high-density, precision arrays, e.g., DNA or gene chips and protein arrays, will gain significantly in efficiency and, thus, in usefulness once it becomes possible to fabricate heterogeneous nanoarrays. Clearly, continued advances in nanotechnology are desired and required for advances in biotechnology. In this review, we describe the leading techniques for generating nanopatterns with biological function including parallel techniques such as extreme ultra-violet interference lithography (EUV-IL), soft-lithographic techniques (e.g., replica molding (RM) and microcontact printing (µCP)), nanoimprint lithography (NIL), nanosphere lithography (NSL) (e.g., colloid lithography or colloidal block-copolymer micelle lithography) and the nanostencil technique, in addition to direct-writing techniques including e-beam lithography (EBL), focused ion-beam lithography (FIBL) and dip-pen nanolithography (DPN). Details on how the patterns are generated, how biological function is imparted to the nanopatterns, and examples of how these surfaces can and are being used for biological applications will be presented. This review further illustrates the rapid pace by which advances are being made in the field of nanobiotechnology, owing to an increasing number of research endeavors, for an ever increasing number of applications.
DOI: http://www.ingentaconnect.com/content/asp/jnn/2006/00000006/00000008/art00003 Article (Blazej2003) Blazej, R.G.; Paegel, B.M. & Mathies, R.A. Polymorphism ratio sequencing: a new approach for single nucleotide polymorphism discovery and genotyping Genome Res, 2003, 13, 287-93 Abstract: Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.
Pubmed: ABSTRACT Article (Bochet2002) Bochet, C.G. Photolabile protecting groups and linkers J. Chem. Soc., Perkin Trans., 2002, 1, 125 - 142
Article (Bonnet1999) Bonnet, G.; Tyagi, S.; Libchaber, A. & Kramer, F.R. Thermodynamic basis of the enhanced specificity of structured DNA probes Proc Natl Acad Sci U S A, 1999, 96, 6171-6 Abstract: Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.
Pubmed: ABSTRACT Article (Borodina2003) Borodina, T.A.; Lehrach, H. & Soldatov, A.V. Ligation-based synthesis of oligonucleotides with block structure Anal Biochem, 2003, 318, 309-13 Abstract: We describe here a method for the synthesis of oligonucleotides with block structure (padlock probes, primers for multiplex polymerase chain reaction (PCR), and ligation-independent cloning), based on the ligation of presynthesized parts by T4 DNA ligase. The advantages of this approach are: (i) suitability of the technology for any producer-from synthesis company to laboratory, (ii) high quality and adjustable scale of synthesis, and (iii) possibility of including any modified bases inexpensively in the common part of the oligonucleotide. Clear difference of sizes of products and substrates makes the synthesis amenable to automation. For large series of padlock probes, the price per one primer approaches the price of the locus-specific parts. We demonstrate the application of this method to two different tasks: preparative-scale production of padlock probes and small-scale synthesis of PCR primers.
Pubmed: ABSTRACT Article (Bottema1993) Bottema, C.D. & Sommer, S.S. PCR amplification of specific alleles: rapid detection of known mutations and polymorphisms Mutat Res, 1993, 288, 93-102 Abstract: We review a method termed PCR Amplification of Specific Alleles (PASA), a generally applicable technique for the detection of known point mutations, small deletions and insertions, polymorphisms and other sequence variations. PASA is a modification of PCR that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. When the mismatch occurs near the 3end of the PCR primer, amplification is inefficient. Therefore, preferential amplification of the perfectly matched allele is obtained. The method should be generally applicable as our results indicate that with proper optimization all possible alleles can be reliably distinguished. The ease and technical simplicity of PASA make genetic analyses more accessible. PASA can be also adapted to accommodate specific requirements and can be extended by incorporating other techniques. Moreover, PASA shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amendable to automation.
Pubmed: ABSTRACT Article (Braem2004) Braem, A.D.; Terán, J.C. & Lindman, B. Influence of DNA adsorption and DNA/cationic surfactant coadsorption on the interaction forces between hydrophobic surfaces. Langmuir, 2004, 20, 6407-6413 Abstract: The forces between hydrophobic surfaces with physisorbed DNA are markedly and irreversibly altered by exposure to DNA/cetyltrimethylammonium bromide (CTAB) mixtures. In this colloidal probe atomic force microscopy study of the interactions between a hydrophobic polystyrene particle and an octadecyltrimethylethoxysilane-modified mica surface in sodium bromide solutions, we measure distinct changes in colloidal forces depending on the existence and state of an adsorbed layer of DNA or CTAB-DNA complexes. For bare hydrophobic surfaces, a monotonically attractive approach curve and very large adhesion are observed. When DNA is adsorbed at low bulk concentrations, a long-range repulsive force dominates the approach, but on retraction some adhesion persists and DNA bridging is clearly observed. When the DNA solution is replaced with a CTAB-DNA mixture at relative low CTAB concentration, the length scale of the repulsive force decreases, the adhesion due to hydrophobic interactions greatly decreases, and bridging events disappear. Finally, when the surface is rinsed with NaBr solution, the length scale of the repulsive interaction increases modestly, and only a very tiny adhesion remains. These pronounced changes in the force behavior are consistent with CTAB-induced DNA compaction accompanied by increased DNA adsorption, both of which are partially irreversible.
Pubmed: ABSTRACT Article (Brautigam1998) Brautigam, C.A. & Steitz, T.A. Structural principles for the inhibition of the 35exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates J Mol Biol, 1998, 277, 363-77 Abstract: A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 35exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
Pubmed: ABSTRACT Article (Breen2002) Breen, G. Novel and alternate SNP and genetic technologies Psychiatr Genet, 2002, 12, 83-8 Abstract: There are many different genotyping technologies and chemistries. Other articles in this special issue focus on nanotechnology, bioinformatics, DNA chips and genotyping methods. This article focuses on four method categories not featured elsewhere in this, or the following special issue: (1) melting curve-based technologies such as dynamic allele-specific hybridization (DASH), melting curve single nucleotide polymorphism (McSNP), fluorescent resonance energy transfer (FRET), hybridization-based melting curves, and homogeneous assay formats based on melting curves; (2) non-PCR-dependent assays such as the oligonucleotide ligation assay and Invader, and isothermal amplification techniques such as rolling circle amplification; (3) rapid whole genome sequencing with methods such as the use of single molecule arrays and molecular resonance sequencing; (4) and other promising novel technologies.
Pubmed: ABSTRACT Article (Brookes2000) Brookes, A.J.; Lehvaslaiho, H.; Siegfried, M.; Boehm, J.G.; Yuan, Y.P.; Sarkar, C.M.; Bork, P. & Ortigao, F. HGBASE: a database of SNPs and other variations in and around human genes Nucleic Acids Res, 2000, 28, 356-60 Abstract: Human genome polymorphism is expected to play a key role in defining the etiologic basis of phenotypic differences between individuals in aspects such as drug responses and common disease predisposition. Relevant functional DNA changes will probably be located in or near to transcribed sequences, and include many single nucleotide polymorphisms. To aid the future analysis of such genome variation, HGBASE (Human Genic Bi-Allelic SEquences) was constructed as a means to gather human gene-linked polymorphisms from all possible public sources, and show these as a non-redundant set of records in a standardized and user-friendly database endowed with text and sequence based search facilities. After 1 year of presence on the WWW, the HGBASE project has compiled data for over 22 000 records, and this number continues to triple every 6-12 months with data harvested or submitted from all major public genome databases and published literature from the previous decade. Extensive annotation enhancement, internal consistency checking and manual review of every record is undertaken to address potential errors and deficiencies sometimes present in the original source data. The fully polished and comprehensive database is made freely available to all at http://hgbase.cgr.ki.se
Pubmed: ABSTRACT Article (Brooksbank2003) Brooksbank, C.; Camon, E.; Harris, M.A.; Magrane, M.; Martin, M.J.; Mulder, N.; O'Donovan, C.; Parkinson, H.; Tuli, M.A.; Apweiler, R.; Birney, E.; Brazma, A.; Henrick, K.; Lopez, R.; Stoesser, G.; Stoehr, P. & Cameron, G. The European Bioinformatics Institute's data resources Nucleic Acids Res, 2003, 31, 43-50 Abstract: As the amount of biological data grows, so does the need for biologists to store and access this information in central repositories in a free and unambiguous manner. The European Bioinformatics Institute (EBI) hosts six core databases, which store information on DNA sequences (EMBL-Bank), protein sequences (SWISS-PROT and TrEMBL), protein structure (MSD), whole genomes (Ensembl) and gene expression (ArrayExpress). But just as a cell would be useless if it couldn't transcribe DNA or translate RNA, our resources would be compromised if each existed in isolation. We have therefore developed a range of tools that not only facilitate the deposition and retrieval of biological information, but also allow users to carry out searches that reflect the interconnectedness of biological information. The EBI's databases and tools are all available on our website at www.ebi.ac.uk.
Pubmed: ABSTRACT Article (Brownie1997) Brownie, J.; Shawcross, S.; Theaker, J.; Whitcombe, D.; Ferrie, R.; Newton, C. & Little, S. The elimination of primer-dimer accumulation in PCR Nucleic Acids Res, 1997, 25, 3235-41 Abstract: We attempted to produce primer-dimers (PDs) from a variety of primers with differing types and extents of complementarity. Where PDs were produced they were cloned and sequenced. We were unable to produce detectable PDs either with individual primers alone or with similar sequence primers even if they had 3'complementarity. These observations led to the hypothesis that a system could be developed whereby the accumulation of PDs in a PCR may be eliminated. We demonstrate a method for the general suppression of PD formation that uses a sequence of additional nucleotides (a Tail) at the 5' ends of amplimers. Tailed amplimers are present at low concentration and only participate during early cycles of PCR. In subsequent PCR cycles, amplification is achieved using a single primer that has the same sequence as that of the Tail portion of the early cycle primers, here we refer to this as a Tag. When products are small, as with PDs, there is a high local concentration of complementary sequences derived from the Tail. This favours the annealing of the complementary ends of a single strand produced by tailed primer interactions and gives rise to 'pan-handle' structures. The formation of these outcompetes the annealing of further Tag primers thereby preventing the accumulation of non-specific PD products. This aids the design of large multiplex reactions and provides a means of detecting specific amplicons directly in the reaction vessel by using an intercalating dye.
Pubmed: ABSTRACT Article (Bruchez1998) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S. & Alivisatos, A.P. Semiconductor nanocrystals as fluorescent biological labels Science, 1998, 281, 2013-6 Abstract: Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, symmetric emission spectrum and are photochemically stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
Pubmed: ABSTRACT Article (Buckley2005) Buckley, P.G.; Mantripragada, K.K.; Piotrowski, A.; de Ståhl, T.D. & Dumanski, J.P. Copy-number polymorphisms: mining the tip of an iceberg. Trends Genet, 2005, 21, 315-317 Abstract: Copy-number polymorphisms (CNPs) represent a greatly underestimated aspect of human genetic variation. Recently, two landmark studies reported genome-wide analyses of CNPs in normal individuals and represent the beginning of an understanding of this type of large-scale variation. Future array-CGH-based CNP analyses should include standard criteria on a common microarray platform. It is only when parallel analyses of CNPs and SNPs are performed in an integrated format that we will obtain a global picture of our genetic diversity.
Pubmed: ABSTRACT Article (Buetow2001) Buetow, K.H.; Edmonson, M.; MacDonald, R.; Clifford, R.; Yip, P.; Kelley, J.; Little, D.P.; Strausberg, R.; Koester, H.; Cantor, C.R. & Braun, A. High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Proc Natl Acad Sci U S A, 2001, 98, 581-4 Abstract: We describe here a system for the rapid identification, assay development, and characterization of gene-based single nucleotide polymorphisms (SNPs). This system couples informatics tools that mine candidate SNPs from public expressed sequence tag resources and automatically designs assay reagents with detection by a chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. As a proof of concept of this system, a genomewide collection of reagents for 9,115 gene-based SNP genetic markers was rapidly developed and validated. These data provide preliminary insights into patterns of polymorphism in a genomewide collection of gene-based polymorphisms.
Pubmed: ABSTRACT Article (Buetow1999) Buetow, K.H.; Edmonson, M.N. & Cassidy, A.B. Reliable identification of large numbers of candidate SNPs from public EST data Nat Genet, 1999, 21, 323-5 Abstract: High-resolution genetic analysis of the human genome promises to provide insight into common disease susceptibility. To perform such analysis will require a collection of high-throughput, high-density analysis reagents. We have developed a polymorphism detection system that uses public-domain sequence data. This detection system is called the single nucleotide polymorphism pipeline (SNPpipeline). The analytic core of the SNPpipeline is composed of three components: PHRED, PHRAP and DEMIGLACE. PHRED and PHRAP are components of a sequence analysis suite developed to perform the semi-automated analysis required for large-scale genomes (provided courtesy of P. Green). Using these informatics tools, which examine redundant raw expressed sequence tag (EST) data, we have identified more than 3,000 candidate single-nucleotide polymorphisms (SNPs). Empiric validation studies of a set of 192 candidates indicate that 82% identify variation in a sample of ten Centre d'Etudes Polymorphism Humain (CEPH) individuals. Our results suggest that existing sequence resources may serve as a valuable source for identifying genetic variation.
Pubmed: ABSTRACT Article (Bundock2006) Bundock, P.C.; Cross, M.J.; Shapter, F.M. & Henry, R.J. Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences. Theor Appl Genet, 2006, 112, 358-365 Abstract: Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.
Pubmed: ABSTRACT Article (Burgess1999) Burgess, L.C. & Hall, J.O. UV light irradiation of plastic reaction tubes inhibits PCR Biotechniques, 1999, 27, 252, 254-4, 256
Pubmed: ABSTRACT Article (Burgtorf2003) Burgtorf, C.; Kepper, P.; Hoehe, M.; Schmitt, C.; Reinhardt, R.; Lehrach, H. & Sauer, S. Clone-based systematic haplotyping (CSH): a procedure for physical haplotyping of whole genomes Genome Res, 2003, 13, 2717-24 Abstract: We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing approximately 10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual.
Pubmed: ABSTRACT Article (Burns1998) Burns, M.A.; Johnson, B.N.; Brahmasandra, S.N.; Handique, K.; Webster, J.R.; Krishnan, M.; Sammarco, T.S.; Man, P.M.; Jones, D.; Heldsinger, D.; Mastrangelo, C.H. & Burke, D.T. An integrated nanoliter DNA analysis device Science, 1998, 282, 484-7 Abstract: A device was developed that uses microfabricated fluidic channels, heaters, temperature sensors, and fluorescence detectors to analyze nanoliter-size DNA samples. The device is capable of measuring aqueous reagent and DNA-containing solutions, mixing the solutions together, amplifying or digesting the DNA to form discrete products, and separating and detecting those products. No external lenses, heaters, or mechanical pumps are necessary for complete sample processing and analysis. Because all of the components are made using conventional photolithographic production techniques, they operate as a single closed system. The components have the potential for assembly into complex, low-power, integrated analysis systems at low unit cost. The availability of portable, reliable instruments may facilitate the use of DNA analysis in applications such as rapid medical diagnostics and point-of-use agricultural testing.
Pubmed: ABSTRACT Article (Burns1996) Burns, M.A.; Mastrangelo, C.H.; Sammarco, T.S.; Man, F.P.; Webster, J.R.; Johnsons, B.N.; Foerster, B.; Jones, D.; Fields, Y.; Kaiser, A.R. & Burke, D.T. Microfabricated structures for integrated DNA analysis. Proc Natl Acad Sci U S A, 1996, 93, 5556-5561 Abstract: Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration.
Pubmed: ABSTRACT Article (Bustin2002) Bustin, S.A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems J Mol Endocrinol, 2002, 29, 23-39 Abstract: The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.
Pubmed: ABSTRACT Article (Bustin2000) Bustin, S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays J Mol Endocrinol, 2000, 25, 169-93 Abstract: The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Pubmed: ABSTRACT Article (Byun2007) Byun, C.; Wang, X.; Pu, Q. & Liu, S. Electroosmosis-Based Nanopipettor Analytical Chemistry, 2007 Abstract: Abstract: Decreasing the volume of reagent solutions consumed in each assay is an effective means to reduce the overall cost in high-throughput analysis laboratories. Recently, increasing attention has been paid to investigate the behavior of individual cells. If one wishes to transfer solution to or from a single cell, a picoliter pipettor is needed since the entire cell volume is commonly less than 1 nL. While pressure ejection and iontophoresis have been used to deliver picoliter volumes of solutions, these techniques cannot yield routine pipettors which perform both solution "picking up" and "dispensing" functions. The state-of-the-art pipettors can handle liquids down to ~100 nL, although the pipetting accuracy and precision deteriorate considerably from microliters to nanoliters. If one wishes to pipet reagents of less than 100 nL, new pipettors need to be developed. Electroosmosis has been utilized to pump solutions at flow rates of nanoliters to approximately picoliters per second, which is ideal for nanopipettors. The issue is how to arrange fluidic/electrical connections so that pipetting functions can be performed conveniently. In this paper, we present the results of our initial attempt to develop an electroosmosis-based nanopipettor. The first version of this pipettor consists of a microfabricated electroosmotic (EO) flow pump, a polyacrylamide grounding interface, and a nanoliter-to-picoliter pipet tip. The detailed configuration and fabrication process of the pipettor are discussed. An excellent feature of an EO-driven pipettor is that it has no moving parts. Good reproducibilities (RSD = 6% at 140 pL, 2% at 950 pL, and 2% at 13 nL) and accuracies (9% at 0.13 nL, 4% at 1.0 nL, and 3% at 10 nL) of this pipettor have been demonstrated to aliquot/transport nanoliter-to-picoliter solutions.
PDF: Byun2007.pdf Article (Cai2006) Cai, L.; Friedman, N. & Xie, X.S. Stochastic protein expression in individual cells at the single molecule level. Nature, 2006, 440, 358-362 Abstract: In a living cell, gene expression--the transcription of DNA to messenger RNA followed by translation to protein--occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. These stochastic events of protein production are difficult to observe directly with measurements on large ensembles of cells owing to lack of synchronization among cells. Measurements so far on single cells lack the sensitivity to resolve individual events of protein production. Here we demonstrate a microfluidic-based assay that allows real-time observation of the expression of beta-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with the number of molecules per burst following an exponential distribution. We show that the two key parameters of protein expression--the burst size and frequency--can be either determined directly from real-time monitoring of protein production or extracted from a measurement of the steady-state copy number distribution in a population of cells. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrates its generality. Many important proteins are expressed at low levels, and are thus inaccessible by current genomic and proteomic techniques. This microfluidic single cell assay opens up possibilities for system-wide characterization of the expression of these low copy number proteins.
Pubmed: ABSTRACT Article (Callegaro2006) Callegaro, A.; Spinelli, R.; Beltrame, L.; Bicciato, S.; Caristina, L.; Censuales, S.; Bellis, G.D. & Battaglia, C. Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data. Nucleic Acids Res, 2006, 34, e56 Abstract: Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.
Pubmed: ABSTRACT Article (Callow2004) Callow, M.J.; Drmanac, S. & Drmanac, R. Selective DNA amplification from complex genomes using universal double-sided adapters Nucleic Acids Res, 2004, 32, e21 Abstract: There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest.
Pubmed: ABSTRACT Article (Canales2006) Canales, R.D.; Luo, Y.; Willey, J.C.; Austermiller, B.; Barbacioru, C.C.; Boysen, C.; Hunkapiller, K.; Jensen, R.V.; Knight, C.R.; Lee, K.Y.; Ma, Y.; Maqsodi, B.; Papallo, A.; Peters, E.H.; Poulter, K.; Ruppel, P.L.; Samaha, R.R.; Shi, L.; Yang, W.; Zhang, L. & Goodsaid, F.M. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol, 2006, 24, 1115-1122 Abstract: We have evaluated the performance characteristics of three quantitative gene expression technologies and correlated their expression measurements to those of five commercial microarray platforms, based on the MicroArray Quality Control (MAQC) data set. The limit of detection, assay range, precision, accuracy and fold-change correlations were assessed for 997 TaqMan Gene Expression Assays, 205 Standardized RT (Sta)RT-PCR assays and 244 QuantiGene assays. TaqMan is a registered trademark of Roche Molecular Systems, Inc. We observed high correlation between quantitative gene expression values and microarray platform results and found few discordant measurements among all platforms. The main cause of variability was differences in probe sequence and thus target location. A second source of variability was the limited and variable sensitivity of the different microarray platforms for detecting weakly expressed genes, which affected interplatform and intersite reproducibility of differentially expressed genes. From this analysis, we conclude that the MAQC microarray data set has been validated by alternative quantitative gene expression platforms thus supporting the use of microarray platforms for the quantitative characterization of gene expression.
Pubmed: ABSTRACT Article (Cargill1999) Cargill, M.; Altshuler, D.; Ireland, J.; Sklar, P.; Ardlie, K.; Patil, N.; Shaw, N.; Lane, C.R.; Lim, E.P.; Kalyanaraman, N.; Nemesh, J.; Ziaugra, L.; Friedland, L.; Rolfe, A.; Warrington, J.; Lipshutz, R.; Daley, G.Q. & Lander, E.S. Characterization of single-nucleotide polymorphisms in coding regions of human genes Nat Genet, 1999, 22, 231-8 Abstract: A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.
Pubmed: ABSTRACT Article (Carlson2004) Carlson, C.S.; Eberle, M.A.; Kruglyak, L. & Nickerson, D.A. Mapping complex disease loci in whole-genome association studies. Nature, 2004, 429, 446-452 Abstract: Identification of the genetic polymorphisms that contribute to susceptibility for common diseases such as type 2 diabetes and schizophrenia will aid in the development of diagnostics and therapeutics. Previous studies have focused on the technique of genetic linkage, but new technologies and experimental resources make whole-genome association studies more feasible. Association studies of this type have good prospects for dissecting the genetics of common disease, but they currently face a number of challenges, including problems with multiple testing and study design, definition of intermediate phenotypes and interaction between polymorphisms.
Pubmed: ABSTRACT Article (Carlson2003) Carlson, C.S.; Eberle, M.A.; Rieder, M.J.; Smith, J.D.; Kruglyak, L. & Nickerson, D.A. Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans. Nat Genet, 2003, 33, 518-521 Abstract: More than 5 million single-nucleotide polymorphisms (SNPs) with minor-allele frequency greater than 10% are expected to exist in the human genome. Some of these SNPs may be associated with risk of developing common diseases. To assess the power of currently available SNPs to detect such associations, we resequenced 50 genes in two ethnic samples and measured patterns of linkage disequilibrium between the subset of SNPs reported in dbSNP and the complete set of common SNPs. Our results suggest that using all 2.7 million SNPs currently in the database would detect nearly 80% of all common SNPs in European populations but only 50% of those common in the African American population and that efficient selection of a minimal subset of SNPs for use in association studies requires measurement of allele frequency and linkage disequilibrium relationships for all SNPs in dbSNP.
Pubmed: ABSTRACT Article (Chabert2006) Chabert, M.; Dorfman, K.; Cremoux, P.; Roeraade, J. & Viovy, J. Automated Microdroplet Platform for Sample Manipulation and Polymerase Chain Reaction. Anal Chem, 2006, 78, 7722-7728 Abstract: We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.
Pubmed: ABSTRACT Article (Chambers2002) Chambers, R.C. Gene expression profiling: good housekeeping and a clean message Thorax, 2002, 57, 754-6
Pubmed: ABSTRACT Article (Chen2005) Chen, C.; Ridzon, D.A.; Broomer, A.J.; Zhou, Z.; Lee, D.H.; Nguyen, J.T.; Barbisin, M.; Xu, N.L.; Mahuvakar, V.R.; Andersen, M.R.; Lao, K.Q.; Livak, K.J. & Guegler, K.J. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 2005, 33, e179 Abstract: A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
Pubmed: ABSTRACT Article (Chen2003) Chen, S.H.; Lin, C.Y.; Cho, C.S.; Lo, C.Z. & Hsiung, C.A. Primer Design Assistant (PDA): A web-based primer design tool Nucleic Acids Res, 2003, 31, 3751-4 Abstract: Primer Design Assistant (PDA) is a web interface primer design service combined with thermodynamic theory to evaluate the fitness of primers. It runs in a Linux-Apache-MySQL-PHP structure on a PC equipped with dual CPU (Intel Pentium III 1.4 GHz) and 512 Mb of RAM. A succinct user interface of PDA is accomplished by built-in parameters setting. Advanced options on 5GC content, 3GC content, dimer check and hairpin check are available. The option of covered region constrains the PCR product to cover a user-defined segment. PDA accepts single sequence query or multiple ones in FASTA format. It produces optimal and homogenous primer pairs that meet the need in experimental design with large-scaled PCR amplifications. Considering the system loading, the size of a submitted sequence is limited to 10 kb and the total sequence number in a query is limited to 20. The authors may be contacted regarding other requirements for primer design. The web application can be found at http://dbb.nhri.org.tw/primer/.
Pubmed: ABSTRACT Article (Cheng1996) Cheng, J.; Shoffner, M.A.; Hvichia, G.E.; Kricka, L.J. & Wilding, P. Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips Nucleic Acids Res, 1996, 24, 380-5 Abstract: We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip.
Pubmed: ABSTRACT Article (Cheung2003) Cheung, J.; Estivill, X.; Khaja, R.; MacDonald, J.R.; Lau, K.; Tsui, L.C. & Scherer, S.W. Genome-wide detection of segmental duplications and potential assembly errors in the human genome sequence Genome Biol, 2003, 4, R25 Abstract: BACKGROUND: Previous studies have suggested that recent segmental duplications, which are often involved in chromosome rearrangements underlying genomic disease, account for some 5% of the human genome. We have developed rapid computational heuristics based on BLAST analysis to detect segmental duplications, as well as regions containing potential sequence misassignments in the human genome assemblies. RESULTS: Our analysis of the June 2002 public human genome assembly revealed that 107.4 of 3,043.1 megabases (Mb) (3.53%) of sequence contained segmental duplications, each with size equal or more than 5 kb and 90% identity. We have also detected that 38.9 Mb (1.28%) of sequence within this assembly is likely to be involved in sequence misassignment errors. Furthermore, we have identified a significant subset (199,965 of 2,327,473 or 8.6%) of single-nucleotide polymorphisms (SNPs) in the public databases that are not true SNPs but are potential paralogous sequence variants. CONCLUSION: Using two distinct computational approaches, we have identified most of the sequences in the human genome that have undergone recent segmental duplications. Near-identical segmental duplications present a major challenge to the completion of the human genome sequence. Potential sequence misassignments detected in this study would require additional efforts to resolve.
Pubmed: ABSTRACT Article (Chou2002) Chou, C.F.; Changrani, R.; Roberts, P.; Sadler, D.; Burdon, J.; Zenhausern, F.; Lin, S.; Mulholland, A.; Swami, N. & Terbrueggen, R. A miniaturized cyclic PCR device-modeling and experiments Microelectron. Eng, 2002, 61-62, 921-925
PDF: Chou2002.pdf Article (Chou1992) Chou, Q.; Russell, M.; Birch, D.E.; Raymond, J. & Bloch, W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res, 1992, 20, 1717-1723 Abstract: A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).
Pubmed: ABSTRACT Article (Chun2007) Chun, J.; Kim, K.; Hwang, I.; Kim, Y.; Lee, D.; Lee, I. & Kim, J. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res, 2007, 35, e40 Abstract: Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.
Pubmed: ABSTRACT Article (Clifford2004) Clifford, R.J.; Edmonson, M.N.; Nguyen, C. & Buetow, K.H. Large-scale analysis of non-synonymous coding region single nucleotide polymorphisms Bioinformatics, 2004, 20, 1006-14 Abstract: MOTIVATION: Single nucleotide polymorphisms (SNPs) are the most common form of genetic variant in humans. SNPs causing amino acid substitutions are of particular interest as candidates for loci affecting susceptibility to complex diseases, such as diabetes and hypertension. To efficiently screen SNPs for disease association, it is important to distinguish neutral variants from deleterious ones. RESULTS: We describe the use of Pfam protein motif models and the HMMER program to predict whether amino acid changes in conserved domains are likely to affect protein function. We find that the magnitude of the change in the HMMER E-value caused by an amino acid substitution is a good predictor of whether it is deleterious. We provide internet-accessible display tools for a genomewide collection of SNPs, including 7391 distinct non-synonymous coding region SNPs in 2683 genes. AVAILABILITY: http://lpgws.nci.nih.gov/cgi-bin/GeneViewer.cgi
Pubmed: ABSTRACT Article (Colella2003) Colella, S.; Shen, L.; Baggerly, K.A.; Issa, J.P. & Krahe, R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites Biotechniques, 2003, 35, 146-50
Pubmed: ABSTRACT Article (Compton1991) Compton, J. Nucleic acid sequence-based amplification Nature, 1991, 350, 91-2 Abstract: Nucleic acid sequence-based amplification (NASBA) is a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature.
Pubmed: ABSTRACT Article (Conner1983) Conner, B.J.; Reyes, A.A.; Morin, C.; Itakura, K.; Teplitz, R.L. & Wallace, R.B. Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci U S A, 1983, 80, 278-282 Abstract: Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.
Pubmed: ABSTRACT Article (Connolly1995) Connolly, A.R.; Cleland, L.G. & Kirkham, B.W. Mathematical considerations of competitive polymerase chain reaction J Immunol Methods, 1995, 187, 201-11 Abstract: Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1-5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.
Pubmed: ABSTRACT Article (Consortium2004) Consortium, I.H.G.S. Finishing the euchromatic sequence of the human genome. Nature, 2004, 431, 931-945 Abstract: The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.
Pubmed: ABSTRACT Article (Cooper1985) Cooper, D.N.; Smith, B.A.; Cooke, H.J.; Niemann, S. & Schmidtke, J. An estimate of unique DNA sequence heterozygosity in the human genome. Hum Genet, 1985, 69, 201-205 Abstract: Fifteen different restriction fragment length polymorphisms (RFLPs) were detected in the human genome using 19 cloned DNA segments, derived from flow-sorted metaphase chromosomes or total genomic DNA, as hybridization probes. Since these clones were selected at random with respect to their coding potential, their analysis permitted an unbiased estimate of single-copy DNA sequence heterozygosity in the human genome. Since our estimate (h = 0.0037) is an order of magnitude higher than previous estimates derived from protein data, most of the polymorphic variation present in the genome must occur in non-coding sequences. In addition, it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequence detect more polymorphic variation than those that do not contain CpG.
Pubmed: ABSTRACT Article (Crawford2004) Crawford, D.C.; Carlson, C.S.; Rieder, M.J.; Carrington, D.P.; Yi, Q.; Smith, J.D.; Eberle, M.A.; Kruglyak, L. & Nickerson, D.A. Haplotype Diversity across 100 Candidate Genes for Inflammation, Lipid Metabolism, and Blood Pressure Regulation in Two Populations Am J Hum Genet, 2004, 74, 610-22 Abstract: Recent studies have suggested that a significant fraction of the human genome is contained in blocks of strong linkage disequilibrium, ranging from ~5 to >100 kb in length, and that within these blocks a few common haplotypes may account for >90% of the observed haplotypes. Furthermore, previous studies have suggested that common haplotypes in candidate genes are generally shared across populations and represent the majority of chromosomes in each population. The conclusions drawn from these preliminary studies, however, are based on an incomplete knowledge of the variation in the regions examined. To bridge this gap in knowledge, we have completely resequenced 100 candidate genes in a population of African descent and one of European descent. Although these genes have been well studied because of their medical importance, we demonstrate that a large amount of sequence variation has not yet been described. We also report that the average number of inferred haplotypes per gene, when complete data is used, is higher than in previous reports and that the number and proportion of all haplotypes represented by common haplotypes per gene is variable. Furthermore, we demonstrate that haplotypes shared between the two populations constitute only a fraction of the total number of haplotypes observed and that these shared haplotypes represent fewer of the African-descent chromosomes than was expected from previous studies. Finally, we show that restricting variation discovery to coding regions does not adequately describe all common haplotypes or the true haplotype block structure observed when all common variation is used to infer haplotypes. These data, derived from complete knowledge of genetic variation in these genes, suggest that the haplotype architecture of candidate genes across the human genome is more complex than previously suggested, with important implications for candidate gene and genomewide association studies.
Pubmed: ABSTRACT Article (Croucher2003) Croucher, P.J.; Mascheretti, S.; Hampe, J.; Huse, K.; Frenzel, H.; Stoll, M.; Lu, T.; Nikolaus, S.; Yang, S.K.; Krawczak, M.; Kim, W.H. & Schreiber, S. Haplotype structure and association to Crohn disease of CARD15 mutations in two ethnically divergent populations Eur J Hum Genet, 2003, 11, 6-16 Abstract: Current debate focuses on the relevance of linkage disequilibrium (LD), ethnicity and underlying haplotype structure to the search for genes involved in complex disorders. The recently described association between single nucleotide polymorphisms (SNPs) of the CARD15 (NOD2) gene and Crohn disease (CD) in populations of north-European descent provides a test case that we have subjected to detailed SNP haplotype based analyses. We examined 23 SNPs spanning 290 kb, including CARD15, in large North-European and Korean samples of patients with Crohn disease and normal controls. In Europeans we confirmed that the three disease-associated SNPs occur independently but share a common background haplotype. This suggests a common origin and the possibility of an undiscovered more strongly predisposing mutation. Korean CD patients present a phenotype identical to the European patients and have not previously been screened for CARD15. The three disease-associated SNPs were absent and there was no evidence of association between CARD15 and CD. Consequently, the disease-associated mutations in the Europeans, which are rare, have arisen recently (after the Asian-European split). Our results highlight important issues relevant to mapping the genes that predispose to complex disorders. First, although ethnically divergent populations may present identical phenotypes they do not necessarily share the same set of predisposing genes. Second, although single-locus tests of association showed consistent association with markers throughout the gene, pair-wise LD between markers (r(2) and D yielded very little information about actual disease-association. Third, a population comparative approach allowed refining of the marker set through the examination of shared polymorphisms and common LD-groups. This approach, in conjunction with the examination of the mutational steps in a haplotype network, allows unambiguous identification of the potentially causative mutations.
Pubmed: ABSTRACT Article (Crowe2003) Crowe, M.L.; Serizet, C.; Thareau, V.; Aubourg, S.; Rouze, P.; Hilson, P.; Beynon, J.; Weisbeek, P.; van Hummelen, P.; Reymond, P.; Paz-Ares, J.; Nietfeld, W. & Trick, M. CATMA: a complete Arabidopsis GST database Nucleic Acids Res, 2003, 31, 156-8 Abstract: The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.org/.
Pubmed: ABSTRACT Article (Curcio2003) Curcio, M. & Roeraade, J. Continuous segmented-flow polymerase chain reaction for high-throughput miniaturized DNA amplification Anal Chem, 2003, 75, 1-7 Abstract: A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300-nL volumes after 30 passes through the heated zones. The total reaction time was approximately 45 min, and the detection interval between individual samples was 1 min. Automation and the possibility to further reduce sample volumes, as well as to employ many reaction columns simultaneously, should provide a platform for an extremely high throughput.
Pubmed: ABSTRACT Article (Cardenas2005) Cárdenas, M.; Dreiss, C.A.; Nylander, T.; Chan, C.P.; Cosgrove, T. & Lindman, B. SANS study of the interactions among DNA, a cationic surfactant, and polystyrene latex particles. Langmuir, 2005, 21, 3578-3583 Abstract: The compaction of DNA by a cationic surfactant both in the bulk and adsorbed on the surface of latex particles was followed for the first time by SANS. In the bulk, a decrease in the overall size of the DNA coil in the presence of the cationic surfactant was observed at a negative-to-positive charge ratio far below the phase separation region, at a negative-to-positive charge ratio of 18. Additionally, large surfactant aggregates seem to form within the DNA-surfactant complex. On the other hand, DNA adsorbs onto the surface of latex particles, forming a thick layer, as evidenced by the fitting of the SANS data to a core-shell form factor. Addition of a cationic surfactant to the DNA-coated latex particles at a negative-to-positive charge ratio of 38 induces a slight decrease in the size of the particle layer, where the cationic surfactant is evenly distributed within the adsorbed layer. A further decrease of the negative-to-positive charge ratio to 18 induces a dramatic change in the SANS data that suggests significant compaction of the adsorbed layer and the formation of large surfactant aggregates, similar to those detected in the bulk.
Pubmed: ABSTRACT Article (Cardenas2005a) Cárdenas, M.; Schillén, K.; Pebalk, D.; Nylander, T. & Lindman, B. Interaction between DNA and charged colloids could be hydrophobically driven. Biomacromolecules, 2005, 6, 832-837 Abstract: The interaction of DNA with amino-functionalized polystyrene particles has been studied by using a dynamic light scattering (DLS) technique. In 10 mM NaBr solution the particles have a hydrodynamic radius of 76 nm and the DNA macromolecule investigated (double stranded) has a hydrodynamic radius of 107 nm. At very low DNA concentrations, DNA adopts a flat conformation on the particle surface. If the DNA concentration is increased above 0.1 microg/mL, the thickness of the DNA layer increases, suggesting the presence of large loops and tails. Although the particles contain primary amino groups, they have a negative net charge under the conditions used in this work. Thus, the driving force for DNA adsorption is not of electrostatic origin but rather due to a hydrophobic effect. Addition of cationic surfactant to the DNA-precoated amino-functionalized particles induces changes in the adsorbed layer conformation, in agreement with the coadsorption of cationic surfactant.
Pubmed: ABSTRACT Article (Cardenas2004) Cárdenas, M.; Terán, J.C.; Nylander, T. & Lindman, B. DNA and cationic surfactant complexes at hydrophilic surfaces. An ellipsometry and surface force study. Langmuir, 2004, 20, 8597-8603 Abstract: The adsorption and formation of DNA and cationic surfactant complexes at the silica-aqueous interface have been studied by ellipsometry. The interaction between the DNA-surfactant complexes at the mica-aqueous interface has been determined by the interferometric surface force apparatus. Adsorption was as expected not observed on negatively charged hydrophilic surfaces for DNA and when DNA-cationic surfactant complexes were negatively charged. However, adsorption was observed when there is an excess of cationic surfactant, just below the point of phase separation. The adsorption process requires hours to reach steady state. The adsorbed layer thickness is large at low surface coverage but becomes more compact and thinner at high coverage. A long-range repulsive force was observed between adsorbed layers of DNA-cationic surfactant complexes, which was suggested to be of both electrostatic and steric origin. The forces were found to be dependent on the equilibration time and the experimental pathway.
Pubmed: ABSTRACT Article (Dahl2007) Dahl, A.; Sultan, M.; Jung, A.; Schwartz, R.; Lange, M.; Steinwand, M.; Livak, K.; Lehrach, H. & Nyarsik, L. Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips. Biomed Microdevices, 2007 Abstract: The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (muPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the muPCR chip down to 5 starting target molecules using TaqMan ((R)) chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 mul assays. The high sensitivity and quantification capability of the muPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.
Pubmed: ABSTRACT Article (Daly2001) Daly, M.J.; Rioux, J.D.; Schaffner, S.F.; Hudson, T.J. & Lander, E.S. High-resolution haplotype structure in the human genome Nat Genet, 2001, 29, 229-32 Abstract: Linkage disequilibrium (LD) analysis is traditionally based on individual genetic markers and often yields an erratic, non-monotonic picture, because the power to detect allelic associations depends on specific properties of each marker, such as frequency and population history. Ideally, LD analysis should be based directly on the underlying haplotype structure of the human genome, but this structure has remained poorly understood. Here we report a high-resolution analysis of the haplotype structure across 500 kilobases on chromosome 5q31 using 103 single-nucleotide polymorphisms (SNPs) in a European-derived population. The results show a picture of discrete haplotype blocks (of tens to hundreds of kilobases), each with limited diversity punctuated by apparent sites of recombination. In addition, we develop an analytical model for LD mapping based on such haplotype blocks. If our observed structure is general (and published data suggest that it may be), it offers a coherent framework for creating a haplotype map of the human genome.
Pubmed: ABSTRACT Article (Dang2005) Dang, F.; Tabata, O.; Kurokawa, M.; Ewis, A.A.; Zhang, L.; Yamaoka, Y.; Shinohara, S.; Shinohara, Y.; Ishikawa, M. & Baba, Y. High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding. Anal Chem, 2005, 77, 2140-2146 Abstract: We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.
Pubmed: ABSTRACT Article (Daniel1998) Daniel, J.; Iqbal, S.; Millington, R.; Moore, D.; Lowe, C.; Leslie, D.; Lee, M. & Pearce, M. Silicon microchambers for DNA amplification Sensors and Actuators A: Physical, 1998, 71, 81-88 Abstract: Microchambers have been fabricated in silicon by bulk micromachining using anisotropic wet etching. Rapid temperature cycling and control at low power is achieved using a special thermal design of the chambers, combined with thin film platinum resistors as temperature sensors and heaters. The chambers have been used for the polymerase chain reaction (PCR) in order to amplify DNA. Rapid temperature cycling and small size are promising attributes for portable high efficiency analytical systems.
PDF: Daniel1998.pdf Article (Darhuber2003) Darhuber, A.A.; Valentino, J.P.; Davis, J.M.; Troian, S.M. & Wagner, S. Microfluidic actuation by modulation of surface stresses Applied Physics Letters, AIP, 2003, 82, 657-659
PDF: Darhuber2003.pdf Article (Day1999) Day, J.P.; Bergstrom, D.; Hammer, R.P. & Barany, F. Nucleotide analogs facilitate base conversion with 3mismatch primers Nucleic Acids Res, 1999, 27, 1810-8 Abstract: We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3base. To determine the distribution of bases inserted opposite eight different analogs, 3analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced.Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed onvertides The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.
Pubmed: ABSTRACT Article (Day2006) Day, P.J.R. Miniaturization applied to analysis of nucleic acids in heterogeneous tissues. Expert Rev Mol Diagn, 2006, 6, 23-28 Abstract: Despite huge efforts in sample analysis, the measurement of marker nucleic acids within tissues remains largely nonquantitative. Gene analyses have benefited from sensitivity gains through in vitro gene amplification, including PCR. However, whilst these processes are intrinsically suited to highly reproducible, accurate and precise gene measurement, the term semiquantitative analysis is still commonly used, suggesting that other fundamental limitations preclude a generic quantitative basis to gene analysis. The most poorly defined aspect of gene analysis relates to the sample itself. The amount of cells and, particularly, cell subtype composition are rarely annotated before analysis; indeed, they are often extrapolated after analysis. To advance our understanding of pathogenesis, assay formats will benefit from resembling the dimensions of the cell, to assist in the analysis of cellular components of tissue complexes. This review is partly a perspective on how current miniaturization technologies, in association with molecular biology, microfluidics and surface chemistries, may evolve from the parts of a paradigm to enable the unambiguous quantitative analysis of complex biologic matter.
Pubmed: ABSTRACT Article (Dean2001) Dean, F.B.; Nelson, J.R.; Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res, 2001, 11, 1095-1099 Abstract: We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.
Pubmed: ABSTRACT Article (Deindl2002) Deindl, E.; Boengler, K.; van Royen, N. & Schaper, W. Differential expression of GAPDH and beta3-actin in growing collateral arteries Mol Cell Biochem, 2002, 236, 139-46 Abstract: Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin are often used as internal standards for quantitative RNA analysis. In our study we analyzed the relative expression level of GAPDH and beta-actin as well as of the 18S rRNA and the Poly (A)+ RNA in growing collateral arteries in a rabbit model of arteriogenesis which is not associated with ischemia. Relative quantitation of the housekeeping genes displayed a significant upregulation of the beta-actin- and GAPDH mRNA during the first 24 h of vessel growth. For day 3 our results revealed an even stronger upregulation of the beta-actin mRNA (140%) but a significant downregulation of the GAPDH mRNA (50% of control). The 18S rRNA, however, showed for the same periods only minor alterations compared to the Poly (A)+ RNA. From these results we conclude that the 18S rRNA, but not the GAPDH- or beta-actin mRNA is an appropriate internal control for relative quantitation of gene expression under conditions of cell proliferation in growing vessels.
Pubmed: ABSTRACT Article (DeMello2006) DeMello, A.J. Control and detection of chemical reactions in microfluidic systems. Nature, 2006, 442, 394-402 Abstract: Recent years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Much development has been driven by a need to perform rapid measurements on small sample volumes. However, at a more primary level, interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micrometre scale. Such systems define new operational paradigms and provide predictions about how molecular synthesis might be revolutionized in the fields of high-throughput synthesis and chemical production.
Pubmed: ABSTRACT Article (DeMello2003) DeMello, A.J. Microfluidics: DNA amplification moves on Nature, 2003, 422, 28-9
Pubmed: ABSTRACT Article (DeMello2001) DeMello, A.J. DNA amplification: does small really mean efficient? Lab Chip, 2001, 1, 24N
PDF: DeMello2001.pdf Article (DeMello2004) DeMello, J. & DeMello, A.J. Microscale reactors: nanoscale products. Lab Chip, 2004, 4, 11N-15N
Pubmed: ABSTRACT Article (Desai2003) Desai, N.A. & Shankar, V. Single-strand-specific nucleases FEMS Microbiol Rev, 2003, 26, 457-91 Abstract: Single-strand-specific nucleases are multifunctional enzymes and widespread in distribution. Their ability to act selectively on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids has led to their extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition. Although more than 30 single-strand-specific nucleases from various sources have been isolated till now, only a few enzymes (S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum and nucleases from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean) have been characterized to a significant extent. Recently, some of these enzymes have been cloned, their crystal structures solved and their interactions with different substrates have been established. The detection, purification, characteristics, structure-function correlations, biological role and applications of single-strand-specific nucleases are reviewed.
Pubmed: ABSTRACT Article (Detter2002) Detter, J.C.; Jett, J.M.; Lucas, S.M.; Dalin, E.; Arellano, A.R.; Wang, M.; Nelson, J.R.; Chapman, J.; Lou, Y.; Rokhsar, D.; Hawkins, T.L. & Richardson, P.M. Isothermal strand-displacement amplification applications for high-throughput genomics Genomics, 2002, 80, 691-8 Abstract: Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.
Pubmed: ABSTRACT Article (Deutsch2001) Deutsch, S.; Iseli, C.; Bucher, P.; Antonarakis, S.E. & Scott, H.S. A cSNP map and database for human chromosome 21 Genome Res, 2001, 11, 300-7 Abstract: Single nucleotide polymorphisms (SNPs) are likely to contribute to the study of complex genetic diseases. The genomic sequence of human chromosome 21q was recently completed with 225 annotated genes, thus permitting efficient identification and precise mapping of potential cSNPs by bioinformatics approaches. Here we present a human chromosome 21 (HC21) cSNP database and the first chromosome-specific cSNP map. Potential cSNPs were generated using three approaches: (1) Alignment of the complete HC21 genomic sequence to cognate ESTs and mRNAs. Candidate cSNPs were automatically extracted using a novel program for context-dependent SNP identification that efficiently discriminates between true variation, poor quality sequencing, and paralogous gene alignments. (2) Multiple alignment of all known HC21 genes to all other human database entries. (3) Gene-targeted cSNP discovery. To date we have identified 377 cSNPs averaging ~1 SNP per 1.5 kb of transcribed sequence, covering 65% of known genes in the chromosome. Validation of our bioinformatics approach was demonstrated by a confirmation rate of 78% for the predicted cSNPs, and in total 32% of the cSNPs in our database have been confirmed. The database is publicly available at http://csnp.unige.ch or http://csnp.isb-sib.ch. These SNPs provide a tool to study the contribution of HC21 loci to complex diseases such as bipolar affective disorder and allele-specific contributions to Down syndrome phenotypes.
Pubmed: ABSTRACT Article (Di2003) Di Giusto, D. & King, G.C. Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays Nucleic Acids Res, 2003, 31, e7 Abstract: Model single base extension (SBE) genotyping reactions with individual deoxy-, dideoxy- and acyclonucleoside triphosphates are monitored by MALDI-TOF mass spectrometry. Three non-proofreading DNA polymerases display remarkably high misincorporation (up to 64% of correct incorporation) when extending primers with single substrates at saturating concentrations. Introduction of one phosphorothioate (PS) linkage into the primer 3terminus reduces misincorporation by these enzymes an average 1.4-fold (range 0- to 3.5-fold) versus correct incorporation. Combined use of 3PS primers with strongly proofreading DNA polymerases yields order of magnitude improvements in SBE fidelity over those produced by the equivalent non-proofreading enzymes. Errors are reduced to below MALDI-TOF detectable levels in almost all cases. The Sp diastereomer of the 3PS primer, which can be prepared in situ by incubation with proofreading polymerase, is stable to 3exonuclease activity over periods longer than 16 h. Products of correct extension by T7 DNAP are retained over 30-60 min during idling turnover at a dNTP concentration of 2.5 micro M, indicating that the assay can be applied over a broad range of substrate concentrations. These results suggest that the use of PS primers and proofreading polymerases will offer a simple and cost-effective means to improve fidelity in a range of single-substrate SBE assay formats.
Pubmed: ABSTRACT Article (Ding2003) Ding, C. & Cantor, C.R. Direct molecular haplotyping of long-range genomic DNA with M1-PCR Proc Natl Acad Sci U S A, 2003, 100, 7449-53 Abstract: Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for ultiplexand 1 for ingle-copy DNA molecules that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.
Pubmed: ABSTRACT Article (Dirks2004) Dirks, R.M.; Lin, M.; Winfree, E. & Pierce, N.A. Paradigms for computational nucleic acid design Nucleic Acids Res, 2004, 32, 1392-1403 Abstract: The design of DNA and RNA sequences is critical for many endeavors, from DNA nanotechnology, to PCR-based applications, to DNA hybridization arrays. Results in the literature rely on a wide variety of design criteria adapted to the particular requirements of each application. Using an extensively studied thermodynamic model, we perform a detailed study of several criteria for designing sequences intended to adopt a target secondary structure. We conclude that superior design methods should explicitly implement both a positive design paradigm (optimize affinity for the target structure) and a negative design paradigm (optimize specificity for the target structure). The commonly used approaches of sequence symmetry minimization and minimum free-energy satisfaction primarily implement negative design and can be strengthened by introducing a positive design component. Surprisingly, our findings hold for a wide range of secondary structures and are robust to modest perturbation of the thermodynamic parameters used for evaluating sequence quality, suggesting the feasibility and ongoing utility of a unified approach to nucleic acid design as parameter sets are refined further. Finally, we observe that designing for thermodynamic stability does not determine folding kinetics, emphasizing the opportunity for extending design criteria to target kinetic features of the energy landscape.
Pubmed: ABSTRACT Article (Dittrich2006) Dittrich, P.S. & Manz, A. Lab-on-a-chip: microfluidics in drug discovery. Nat Rev Drug Discov, 2006, 5, 210-218 Abstract: Miniaturization can expand the capability of existing bioassays, separation technologies and chemical synthesis techniques. Although a reduction in size to the micrometre scale will usually not change the nature of molecular reactions, laws of scale for surface per volume, molecular diffusion and heat transport enable dramatic increases in throughput. Besides the many microwell-plate- or bead-based methods, microfluidic chips have been widely used to provide small volumes and fluid connections and could eventually outperform conventionally used robotic fluid handling. Moreover, completely novel applications without a macroscopic equivalent have recently been developed. This article reviews current and future applications of microfluidics and highlights the potential of 'lab-on-a-chip' technology for drug discovery.
Pubmed: ABSTRACT Article (Douabin-Gicquel2001) Douabin-Gicquel, V.; Soriano, N.; Ferran, H.; Wojcik, F.; Palierne, E.; Tamim, S.; Jovelin, T.; McKie, A.T.; Le Gall, J.Y.; David, V. & Mosser, J. Identification of 96 single nucleotide polymorphisms in eight genes involved in iron metabolism: efficiency of bioinformatic extraction compared with a systematic sequencing approach Hum Genet, 2001, 109, 393-401 Abstract: Single nucleotide polymorphisms (SNPs) can significantly contribute to the characterization of the genes predisposing to iron overloads or deficiencies. We report an SNP survey of coding and non-coding regions of eight genes involved in iron metabolism, by two successive methods. First, we made use of the public domain sequence data, by using assembled expressed sequence tags, non-redundant sequences, and SNP database screening. We extracted 77 potential SNPs of which only 31 could be further validated by sequencing DNA from 44 unrelated multi-ethnic individuals. Our results indicate that a bioinformatic approach may be effective only in those cases where candidate SNPs are extracted from two different data sources or in cases of experimentally confirmed SNPs. Second, additional systematic sequencing of DNA from 24 unrelated Breton subjects increased the number of SNPs over a total length of 86 kb to 96. The average distance between the SNPs and minor allele frequencies were higher than reported by others authors; this discrepancy may reflect the nature of the genes studied and the ethnic homogeneity of our test population.
Pubmed: ABSTRACT Article (Doublie1998) Doublie, S. & Ellenberger, T. The mechanism of action of T7 DNA polymerase Curr Opin Struct Biol, 1998, 8, 704-12 Abstract: The recent crystal structure determination of T7 DNA polymerase complexed to a deoxynucleoside triphosphate and primer-template DNA has provided the first glimpse of a replicative DNA polymerase in a catalytic complex. The structure complements many functional and structural studies of this and other DNA polymerases, allowing a detailed evaluation of proposals for the mechanism of nucleotidyl transfer and the exploration of the basis for the high fidelity of template-directed DNA synthesis.
Pubmed: ABSTRACT Article (Douglas2001) Douglas, J.A.; Boehnke, M.; Gillanders, E.; Trent, J.M. & Gruber, S.B. Experimentally-derived haplotypes substantially increase the efficiency of linkage disequilibrium studies Nat Genet, 2001, 28, 361-4 Abstract: The study of complex genetic traits in humans is limited by the expense and difficulty of ascertaining populations of sufficient sample size to detect subtle genetic contributions to disease. Here we introduce an application of a somatic cell hybrid construction strategy called conversion that maximizes the genotypic information from each sampled individual. The approach permits direct observation of individual haplotypes, thereby eliminating the need for collecting and genotyping DNA from family members for haplotype-based analyses. We describe experimental data that validate the use of conversion as a whole-genome haplotyping tool and evaluate the theoretical efficiency of using conversion-derived haplotypes instead of conventional genotypes in the context of haplotype-frequency estimation. We show that, particularly when phenotyping is expensive, conversion-based haplotyping can be more efficient and cost-effective than standard genotyping.
Pubmed: ABSTRACT Article (Dowell2001) Dowell, R.D.; Jokerst, R.M.; Day, A.; Eddy, S.R. & Stein, L. The Distributed Annotation System BMC Bioinformatics, 2001, 2, 7 Abstract: BACKGROUND: Currently, most genome annotation is curated by centralized groups with limited resources. Efforts to share annotations transparently among multiple groups have not yet been satisfactory. RESULTS: Here we introduce a concept called the Distributed Annotation System (DAS). DAS allows sequence annotations to be decentralized among multiple third-party annotators and integrated on an as-needed basis by client-side software. The communication between client and servers in DAS is defined by the DAS XML specification. Annotations are displayed in layers, one per server. Any client or server adhering to the DAS XML specification can participate in the system; we describe a simple prototype client and server example. CONCLUSIONS: The DAS specification is being used experimentally by Ensembl, WormBase, and the Berkeley Drosophila Genome Project. Continued success will depend on the readiness of the research community to adopt DAS and provide annotations. All components are freely available from the project website http://www.biodas.org/.
Pubmed: ABSTRACT Article (Drabek2001) Drabek, J. A commented dictionary of techniques for genotyping Electrophoresis, 2001, 22, 1024-45 Abstract: Several tools, differing in their technical and practical parameters, are available for the detection of point mutations as well as small deletions and insertions. In this article, a dictionary featuring over fifty methods for detection of mutation is presented. The distinguishing principle for each method is briefly explained. Sorting of and discussion on the methods give the reader a brief introduction to the field of genotyping.
Pubmed: ABSTRACT Article (Dressman2003) Dressman, D.; Yan, H.; Traverso, G.; Kinzler, K.W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations Proc Natl Acad Sci U S A, 2003, 100, 8817-22 Abstract: Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.
Pubmed: ABSTRACT Article (Duggan1999) Duggan, D.J.; Bittner, M.; Chen, Y.; Meltzer, P. & Trent, J.M. Expression profiling using cDNA microarrays. Nat Genet, 1999, 21, 10-14 Abstract: cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. DNA targets, in the form of 3' expressed sequence tags (ESTs), are arrayed onto glass slides (or membranes) and probed with fluorescent- or radioactively-labelled cDNAs. Here, we review technical aspects of cDNA microarrays, including the general principles, fabrication of the arrays, target labelling, image analysis and data extraction, management and mining.
Pubmed: ABSTRACT Article (Dunn2000) Dunn, D.A. & Feygin, I. Challenges and solutions to ultra-high-throughput screening assay miniaturization: Submicroliter fluid handling Drug Discovery Today, 2000, 5, S84-S91 Abstract: The miniaturization of HTS assays is an important objective in the pharmaceutical industry. The ability to perform primary screening assays in high-density micro-well plates at volumes of 1-2 [mu]l will accelerate the early stages of drug discovery and reduce costs. Ultra-HTS (uHTS) assays require an accurate and reliable means of fluid handling in the submicroliter volume range. This relates to the design of instrumentation for dispensing fluids, as well as assay plates. Fluid handling has been a major obstacle to the full implementation of miniaturized assays. This report focusses on current approaches to submicroliter fluid handling in high-density multi-well plates.
PDF: Dunn2000.pdf Article (Eisenstein2006) Eisenstein, M. Tiny droplets make a big splash. Nat Methods, 2006, 3, 71
Pubmed: ABSTRACT Article (Ekstrom2000) Ekstrom, S.; Onnerfjord, P.; Nilsson, J.; Bengtsson, M.; Laurell, T. & Marko-Varga, G. Integrated microanalytical technology enabling rapid and automated protein identification Anal Chem, 2000, 72, 286-93 Abstract: Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The mu-chip IMER provides on-line enzymatic digestion of protein samples (1 microL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).
Pubmed: ABSTRACT Article (Elbashir2001) Elbashir, S.M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K. & Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature, 2001, 411, 494-8 Abstract: RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
Pubmed: ABSTRACT Article (Erickson2005) Erickson, D.; Liu, X.; Venditti, R.; Li, D. & Krull, U.J. Electrokinetically based approach for single-nucleotide polymorphism discrimination using a microfluidic device. Anal Chem, 2005, 77, 4000-4007 Abstract: In this work, we describe and implement an electrokinetic approach for single-nucleotide polymorphism (SNP) discrimination using a PDMS/glass-based microfluidic chip. The technique takes advantage of precise control of the coupled thermal (Joule heating), shear (electroosmosis), and electrical (electrophoresis) energies present at an array of probes afforded by the application of external electrical potentials. Temperature controllers and embedded thermal devices are not required. The chips can be easily and inexpensively fabricated using standard microarray printing methods combined with soft-lithography patterned PDMS fluidics, making these systems easily adaptable to applications using higher density arrays. Extensive numerical simulations of the coupled flow and thermal properties and microscale thermometry experiments are described and used to characterize the in-channel conditions. It was found that optimal conditions for SNP detection occur at a lower temperature on-chip than for typical microarray experiments, thereby revealing the importance of the electrical and shear forces to the overall process. To demonstrate the clinical utility of the technique, the detection of single-base pair mutations in the survival motor neuron gene, associated with the childhood disease spinal muscular atrophy, is conducted.
Pubmed: ABSTRACT Article (Erill2003) Erill, I.; Campoy, S.; Erill, N.; Barbe, J. & J., A. Biochemical analysis and optimization of inhibition and adsorption phenomena in glass-silicon PCR-chips Sensors and Actuators B: Chemical, 2003, 96, 685-692
Article (Erlich1999) Erlich, H.A. Principles and applications of the polymerase chain reaction Rev Immunogenet, 1999, 1, 127-34 Abstract: The polymerase chain reaction (PCR), a primer-directed in vitro enzymatic reaction for the production of a specific DNA fragment, has dramatically facilitated molecular biology approaches to fundamental research questions, as well introduced DNA testing into a variety of clinical diagnostic areas. Some of the principles of PCR, several recent advances in PCR technology and selected applications are reviewed. A variety of methods for analyzing allelic sequence diversity in amplified DNA have been developed. Genetic testing of the HLA loci, and other genes encoding cytokines and cytokine receptors, has had a significant impact on disease susceptibility studies and in clinical transplantation.
Pubmed: ABSTRACT Article (Estivill2002) Estivill, X.; Cheung, J.; Pujana, M.A.; Nakabayashi, K.; Scherer, S.W. & Tsui, L.C. Chromosomal regions containing high-density and ambiguously mapped putative single nucleotide polymorphisms (SNPs) correlate with segmental duplications in the human genome Hum Mol Genet, 2002, 11, 1987-95 Abstract: We have explored the National Center for Biotechnology Information (NCBI) single nucleotide polymorphisms (SNPs) database for a correlation between the density of putative SNPs, as well as SNPs that map to different chromosomal locations (ambiguously mapped SNPs), and segmental duplications of DNA in chromosome regions involved in genomic disorders. A high density of SNPs (14.4 and 12.4 SNPs per kb) was detected in the low copy repeats (LCRs) responsible for the chromosome 17p12 duplication and deletion that cause peripheral neuropathies. None of the SNPs at the PMP22 gene were ambiguously mapped, but 93% of the SNPs at LCRs mapped on both LCR copies, indicating that they are in fact variants in paralogous sequences. Similarly, a high SNP density was found in the LCR regions flanking the neurofibromatosis type 1 (NF1) gene, with 80% of SNPs mapping on both LCR copies. A high density of SNPs was found within LCR sequences involved in the deletions that mediate contiguous gene syndromes on chromosomes 7q11, 15q11-q13 and 22q11. We have analyzed the whole sequence of chromosome 22, which contains 14% of ambiguously mapped SNPs, and have found a good correlation between these SNPs and segmental duplications detected by BLAST analysis. We have identified several segments of ambiguously mapped SNPs, four corresponding to LCRs involved in the chromosome 22q11 microdeletion syndromes. Our data indicate that most SNPs in LCR segments are in fact paralogous sequence variants (PSVs), and suggest that a significant proportion of the SNPs in the NCBI database correspond to PSVs within segmental duplications of the human genome sequence.
Pubmed: ABSTRACT Article (Evans1999) Evans, W.E. & Relling, M.V. Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 1999, 286, 487-491 Abstract: Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual differences in the efficacy and toxicity of many medications. Pharmacogenomic studies are rapidly elucidating the inherited nature of these differences in drug disposition and effects, thereby enhancing drug discovery and providing a stronger scientific basis for optimizing drug therapy on the basis of each patient's genetic constitution.
Pubmed: ABSTRACT Article (Falkowski2004) Falkowski, P.G. & de Vargas, C. Genomics and evolution. Shotgun sequencing in the sea: a blast from the past? Science, 2004, 304, 58-60
Pubmed: ABSTRACT Article (Fan2000) Fan, J.B.; Chen, X.; Halushka, M.K.; Berno, A.; Huang, X.; Ryder, T.; Lipshutz, R.J.; Lockhart, D.J. & Chakravarti, A. Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res, 2000, 10, 853-860 Abstract: Large scale human genetic studies require technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy. We describe a highly parallel method for genotyping single nucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags. First, marker-specific primers are used in PCR amplifications of genomic regions containing SNPs. Second, the amplification products are used as templates in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specific probes, or tags, synthesized on the array. The SBE primers, terminating one base before the polymorphic site, are extended in the presence of labeled dideoxy NTPs, using a different label for each of the two SNP alleles, and hybridized to the tag array. Third, genotypes are deduced from the fluorescence intensity ratio of the two colors. This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage. We illustrate and test this method by genotyping 44 individuals for 142 human SNPs identified previously in 62 candidate hypertension genes. Because the hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples.
Pubmed: ABSTRACT Article (Fan2002) Fan, R. & Xiong, M. High resolution mapping of quantitative trait loci by linkage disequilibrium analysis Eur J Hum Genet, 2002, 10, 607-15 Abstract: Two methods, linkage analysis and linkage disequilibrium (LD) mapping or association study, are usually utilised for mapping quantitative trait loci (QTL). Linkage mapping is appropriate for low resolution mapping to localise trait loci to broad chromosome regions within a few cM (<10 cM), and is based on family data. Linkage disequilibrium mapping, on the other hand, is useful in high resolution or fine mapping, and is based on both population and family data. Using only one marker, one may carry out single-point linkage analysis and linkage disequilibrium mapping. Using two or more markers, it is possible to flank the QTL by multipoint analysis. The development and thus availability of dense marker maps, such as single nucleotide polymorphisms (SNP) in human genome, presents a tremendous opportunity for multipoint fine mapping. In this article, we propose a regression approach of mapping QTL by linkage disequilibrium mapping based on population data. Assuming that two marker loci flank one quantitative trait locus, a two-point linear regression is proposed to analyse population data. We derive analytical formulas of parameter estimations, and non-centrality parameters of appropriate tests of genetic effects and linkage disequilibrium coefficients. The merit of the method is shown by the power calculation and comparison. The two-point regression model can capture much more linkage and linkage disequilibrium information than that derived when only one marker is used. For a complex disease with heritability h(2)> or =0.15, a study with sample size of 250 can provide high power for QTL detection under moderate linkage disequilibria.
Pubmed: ABSTRACT Article (Fara2003) Fara, P. Perceptions of science. Face values: how portraits win friends and influence people Science, 2003, 299, 831-2
Pubmed: ABSTRACT Article (Farinas2005) Fariñas, M.S.; Pellegrino, M.; Wittkowski, K.M. & Magnasco, M.O. Harshlight: a "corrective make-up" program for microarray chips. BMC Bioinformatics, 2005, 6, 294 Abstract: BACKGROUND: Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans do show similar artifacts, which might affect subsequent analysis. Although all but the starkest blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs), few tools are available to help with the detection of those defects. RESULTS: We develop a novel tool, Harshlight, for the automatic detection and masking of blemishes in HDONA microarray chips. Harshlight uses a combination of statistic and image processing methods to identify three different types of defects: localized blemishes affecting a few probes, diffuse defects affecting larger areas, and extended defects which may invalidate an entire chip. CONCLUSION: We demonstrate the use of Harshlight can materially improve analysis of HDONA chips, especially for experiments with subtle changes between samples. For the widely used MAS5 algorithm, we show that compact blemishes cause an average of 8 gene expression values per chip to change by more than 50%, two of them by more than twofold; our masking algorithm restores about two thirds of this damage. Large-scale artifacts are successfully detected and eliminated.
Pubmed: ABSTRACT Article (Findlay1993) Findlay, J.B.; Atwood, S.M.; Bergmeyer, L.; Chemelli, J.; Christy, K.; Cummins, T.; Donish, W.; Ekeze, T.; Falvo, J.; Patterson, D. & e. al. Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction Clin Chem, 1993, 39, 1927-33 Abstract: An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.
Pubmed: ABSTRACT Article (Fisher2003) Fisher, S.A.; Moody, A.; Mirza, M.M.; Cuthbert, A.P.; Hampe, J.; Macpherson, A.; Sanderson, J.; Forbes, A.; Mansfield, J.; Schreiber, S.; Lewis, C.M. & Mathew, C.G. Genetic variation at the chromosome 16 chemokine gene cluster: development of a strategy for association studies in complex disease Ann Hum Genet, 2003, 67, 377-90 Abstract: The chemokine gene cluster [CCL22, CX3CL1, CCL17] (previously known as [SCYA22, SCYD1, SCYA17]) is a candidate locus for one of the susceptibility genes for inflammatory bowel disease that are located in the peri-centromeric region of chromosome 16. Screening for sequence variation at this locus led to the detection of 14 single nucleotide polymorphisms (SNPs). An efficient experimental and computational approach was developed to estimate allele frequencies and pairwise linkage disequilibrium relationships between SNPs at this locus, and to test them for association with inflammatory bowel disease. The 12 common SNPs were assigned to 5 distinct linkage disequilibrium groups. Genotyping of one SNP from each linkage disequilibrium group in a large cohort of families with inflammatory bowel disease did not provide convincing evidence of association with either Crohn disease or ulcerative colitis. We describe an efficient experimental design from SNP screening to association testing. This strategy can be used to test candidate genes for involvement in susceptibility to complex disease.
Pubmed: ABSTRACT Article (Fixe2004) Fixe, F.; Dufva, M.; Telleman, P. & Christensen, C.B.V. Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays. Nucleic Acids Res, 2004, 32, e9 Abstract: A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray trade mark slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.
Pubmed: ABSTRACT Article (Fredman2002) Fredman, D.; Siegfried, M.; Yuan, Y.P.; Bork, P.; Lehvaslaiho, H. & Brookes, A.J. HGVbase: a human sequence variation database emphasizing data quality and a broad spectrum of data sources Nucleic Acids Res, 2002, 30, 387-91 Abstract: HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.
Pubmed: ABSTRACT Article (Fredman2004) Fredman, D.; White, S.J.; Potter, S.; Eichler, E.E.; Den Dunnen, J.T. & Brookes, A.J. Complex SNP-related sequence variation in segmental genome duplications Nat Genet, 2004, 36, 861-6 Abstract: There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.
Pubmed: ABSTRACT Article (Fredrickson2004) Fredrickson, C.K. & Fan, Z.H. Macro-to-micro interfaces for microfluidic devices Lab Chip, 2004, 4, 526-33 Abstract: Since the concept of miniaturized total analysis systems (microTAS) was invented, a great number of microfluidic devices have been demonstrated for a variety of applications. However, an important hurdle that still needs to be cleared is the connection of a microfluidic device with the rest of the world, which is often referred to as the macro-to-micro interface, interconnect, or world-to-chip interface. In this review, we will examine the methods used by pioneers in the field and other investigators, review the approaches for capillary electrophoresis-based devices and those using pneumatic pumping, and present additional discussion on interface standardization and choosing and designing interconnects for your applications.
Pubmed: ABSTRACT Article (Fredriksson2007) Fredriksson, S.; Banér, J.; Dahl, F.; Chu, A.; Ji, H.; Welch, K. & Davis, R.W. Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res, 2007 Abstract: Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.
Pubmed: ABSTRACT Article (Freeman2006) Freeman, J.L.; Perry, G.H.; Feuk, L.; Redon, R.; McCarroll, S.A.; Altshuler, D.M.; Aburatani, H.; Jones, K.W.; Smith, C.T.; Hurles, M.E.; Carter, N.P.; Scherer, S.W. & Lee, C. Copy number variation: new insights in genome diversity. Genome Res, 2006, 16, 949-961 Abstract: DNA copy number variation has long been associated with specific chromosomal rearrangements and genomic disorders, but its ubiquity in mammalian genomes was not fully realized until recently. Although our understanding of the extent of this variation is still developing, it seems likely that, at least in humans, copy number variants (CNVs) account for a substantial amount of genetic variation. Since many CNVs include genes that result in differential levels of gene expression, CNVs may account for a significant proportion of normal phenotypic variation. Current efforts are directed toward a more comprehensive cataloging and characterization of CNVs that will provide the basis for determining how genomic diversity impacts biological function, evolution, and common human diseases.
Pubmed: ABSTRACT Article (Freeman1999) Freeman, W.M.; Walker, S.J. & Vrana, K.E. Quantitative RT-PCR: pitfalls and potential Biotechniques, 1999, 26, 112-22, 124-5 Abstract: Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.
Pubmed: ABSTRACT Article (Fronhoffs2002) Fronhoffs, S.; Totzke, G.; Stier, S.; Wernert, N.; Rothe, M.; Bruning, T.; Koch, B.; Sachinidis, A.; Vetter, H. & Ko, Y. A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction Mol Cell Probes, 2002, 16, 99-110 Abstract: Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression - offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCycler - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR.
Pubmed: ABSTRACT Article (Fullerton2000) Fullerton, S.M.; Clark, A.G.; Weiss, K.M.; Nickerson, D.A.; Taylor, S.L.; Stengard, J.H.; Salomaa, V.; Vartiainen, E.; Perola, M.; Boerwinkle, E. & Sing, C.F. Apolipoprotein E variation at the sequence haplotype level: implications for the origin and maintenance of a major human polymorphism Am J Hum Genet, 2000, 67, 881-900 Abstract: Three common protein isoforms of apolipoprotein E (apoE), encoded by the epsilon2, epsilon3, and epsilon4 alleles of the APOE gene, differ in their association with cardiovascular and Alzheimer's disease risk. To gain a better understanding of the genetic variation underlying this important polymorphism, we identified sequence haplotype variation in 5.5 kb of genomic DNA encompassing the whole of the APOE locus and adjoining flanking regions in 96 individuals from four populations: blacks from Jackson, MS (n=48 chromosomes), Mayans from Campeche, Mexico (n=48), Finns from North Karelia, Finland (n=48), and non-Hispanic whites from Rochester, MN (n=48). In the region sequenced, 23 sites varied (21 single nucleotide polymorphisms, or SNPs, 1 diallelic indel, and 1 multiallelic indel). The 22 diallelic sites defined 31 distinct haplotypes in the sample. The estimate of nucleotide diversity (site-specific heterozygosity) for the locus was 0.0005+/-0.0003. Sequence analysis of the chimpanzee APOE gene showed that it was most closely related to human epsilon4-type haplotypes, differing from the human consensus sequence at 67 synonymous (54 substitutions and 13 indels) and 9 nonsynonymous fixed positions. The evolutionary history of allelic divergence within humans was inferred from the pattern of haplotype relationships. This analysis suggests that haplotypes defining the epsilon3 and epsilon2 alleles are derived from the ancestral epsilon4s and that the epsilon3 group of haplotypes have increased in frequency, relative to epsilon4s, in the past 200,000 years. Substantial heterogeneity exists within all three classes of sequence haplotypes, and there are important interpopulation differences in the sequence variation underlying the protein isoforms that may be relevant to interpreting conflicting reports of phenotypic associations with variation in the common protein isoforms.
Pubmed: ABSTRACT Article (Gabriel2002) Gabriel, S.B.; Schaffner, S.F.; Nguyen, H.; Moore, J.M.; Roy, J.; Blumenstiel, B.; Higgins, J.; DeFelice, M.; Lochner, A.; Faggart, M.; Liu-Cordero, S.N.; Rotimi, C.; Adeyemo, A.; Cooper, R.; Ward, R.; Lander, E.S.; Daly, M.J. & Altshuler, D. The structure of haplotype blocks in the human genome Science, 2002, 296, 2225-9 Abstract: Haplotype-based methods offer a powerful approach to disease gene mapping, based on the association between causal mutations and the ancestral haplotypes on which they arose. As part of The SNP Consortium Allele Frequency Projects, we characterized haplotype patterns across 51 autosomal regions (spanning 13 megabases of the human genome) in samples from Africa, Europe, and Asia. We show that the human genome can be parsed objectively into haplotype blocks: sizable regions over which there is little evidence for historical recombination and within which only a few common haplotypes are observed. The boundaries of blocks and specific haplotypes they contain are highly correlated across populations. We demonstrate that such haplotype frameworks provide substantial statistical power in association studies of common genetic variation across each region. Our results provide a foundation for the construction of a haplotype map of the human genome, facilitating comprehensive genetic association studies of human disease.
Pubmed: ABSTRACT Article (Gallup2006) Gallup, J.M. & Ackermann, M.R. Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions. Biol Proced Online, 2006, 8, 87-152 Abstract: The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time - calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional.
Pubmed: ABSTRACT Article (Galvin2002) Galvin, P. A nanobiotechnology roadmap for high-throughput single nucleotide polymorphism analysis Psychiatr Genet, 2002, 12, 75-82 Abstract: Genetic analysis based on single nucleotide polymorphisms (SNPs) has the potential to enable identification of genes associated with disease susceptibility, to facilitate improved understanding and diagnosis of those diseases, and should ultimately contribute to the provision of new therapies. To achieve this end, new technology platforms are required that can increase genotyping throughput, while simultaneously reducing costs by as much as two orders of magnitude. Development of a variety of genotyping platforms with the potential to resolve this dilemma is already well advanced through research in the field of nanobiotechnology. Novel approaches to DNA extraction and amplification have reduced the times required for these processes to seconds. Microfluidic devices enable polymorphism detection through very rapid fragment separation using capillary electrophoresis and high-performance liquid chromatography, together with mixing and transport of reagents and biomolecules in integrated systems. The potential for application of established microelectronic fabrication processes to genetic analyses systems has been demonstrated (e.g. photolithography-based in situ synthesis of oligonucleotides on microarrays). Innovative application of state-of-the-art photonics and integrated circuitry are leading to improved detection capabilities. The diversity of genotyping applications envisaged in the future, ranging from the very high-throughput requirements for drug discovery through to rapid and cheap near-patient genotype analysis, suggests that several SNP genotyping platforms will be necessary to optimally address the different niches.
Pubmed: ABSTRACT Article (Gao2006) Gao, D.; Agayan, R.; Xu, H.; Philbert, M. & Kopelman, R. Nanoparticles for Two-Photon Photodynamic Therapy in Living Cells Nano Letters, 2006, 6, 2383-2386
PDF: Gao2006.pdf Article (Gascoyne2004) Gascoyne, P.; Satayavivad, J. & Ruchirawat, M. Microfluidic approaches to malaria detection. Acta Trop, 2004, 89, 357-369 Abstract: Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.
Pubmed: ABSTRACT Article (Gaunt2003) Gaunt, T.R.; Hinks, L.J.; Rassoulian, H. & Day, I.N. Manual 768 or 384 well microplate gel ryelectrophoresis for PCR checking and SNP genotyping Nucleic Acids Res, 2003, 31, E48 Abstract: Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid-protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient rygel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5-10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.
Pubmed: ABSTRACT Article (Gentle2001) Gentle, A.; Anastasopoulos, F. & McBrien, N.A. High-resolution semi-quantitative real-time PCR without the use of a standard curve Biotechniques, 2001, 31, 502, 504-6, 508 Abstract: The repeatability and sensitivity of a simple, adaptable, semi-quantitative, real-time RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the LightCycler real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.
Pubmed: ABSTRACT Article (Germer2000) Germer, S.; Holland, M.J. & Higuchi, R. High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR Genome Res, 2000, 10, 258-66 Abstract: We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r(2) = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
Pubmed: ABSTRACT Article (Gibbs1990) Gibbs, R.A. DNA amplification by the polymerase chain reaction Anal Chem, 1990, 62, 1202-14 Abstract: The polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences via repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension. PCR has revolutionized the practice of DNA technology as it allows virtually any nucleic acid sequence to be readily generated in vitro in relatively great abundance, so that subsequent analyses are not confounded by the presence of other DNA fragments or a lack of material with which to work. PCR also enables the sequence of individual DNA fragments to be altered. The method has advantages over conventional procedures for DNA cloning and analysis in many circumstances because it is faster, simpler, and more flexible. The total range and number of applications that have evolved in the short time since the first report of PCR are enormous. This review describes some of the history of PCR, the principle of the method, practical considerations for performing PCR, and a variety of applications.
Pubmed: ABSTRACT Article (Gibbs2003) Gibbs, R.A.; Belmont, J.W.; Hardenbol, P.; Willis, T.D.; Yu, F.; Yang, H.; Cháng, L.Y.; Huang, W.; Liu, B.; Shen, Y.; Tam, P.K.; Tsui, L.C.; Waye, M.M.; Wong, J.T.; Zeng, C.; Zhang, Q.; Chee, M.S.; Galver, L.M.; Kruglyak, S.; Murray, S.S.; Oliphant, A.R.; Montpetit, A.; Hudson, T.J.; Chagnon, F.; Ferretti, V.; Leboeuf, M.; Phillips, M.S.; Verner, A.; Kwok, P.Y.; Duan, S.; Lind, D.L.; Miller, R.D.; Rice, J.P.; Saccone, N.L.; Taillon-Miller, P.; Xiao, M.; Nakamura, Y.; Sekine, A.; Sorimachi, K.; Tanaka, T.; Tanaka, Y.; Tsunoda, T.; Yoshino, E.; Bentley, D.R.; Deloukas, P.; Hunt, S.; Powell, D.; Altshuler, D.; Gabriel, S.B.; Zhang, H.; Matsuda, I.; Fukushima, Y.; Macer, D.R.; Suda, E.; Rotimi, C.N.; Adebamowo, C.A.; Aniagwu, T.; Marshall, P.A.; Matthew, O.; Nkwodimmah, C.; Royal, C.D.; Leppert, M.F.; Dixon, M.; Stein, L.D.; Cunningham, F.; Kanani, A.; Thorisson, G.A.; Chakravarti, A.; Chen, P.E.; Cutler, D.J.; Kashuk, C.S.; Donnelly, P.; Marchini, J.; McVean, G.A.; Myers, S.R.; Cardon, L.R.; Abecasis, G.R.; Morris, A.; Weir, B.S.; Mullikin, J.C.; Sherry, S.T.; Feolo, M.; Altshuler, D.; Daly, M.J.; Schaffner, S.F.; Qiu, R.; Kent, A.; Dunston, G.M.; Kato, K.; Niikawa, N.; Knoppers, B.M.; Foster, M.W.; Clayton, E.W.; Wang, V.O.; Watkin, J.; Gibbs, R.A.; Belmont, J.W.; Sodergren, E.; Weinstock, G.M.; Wilson, R.K. & others The International HapMap Project Nature, 2003, 426, 789-96 Abstract: The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention.
Pubmed: ABSTRACT Article (Gilbert2002) Gilbert, D. Pise: software for building bioinformatics webs Brief Bioinform, 2002, 3, 405-9 Abstract: Pise is interface construction software for bioinformatics applications that run by command-line operations. It creates common, easy-to-use interfaces to these applications for the Web, or other uses. It is adaptable to new bioinformatics tools, and offers program chaining, Unix system batch and other controls, making it an attractive method for building and using your own bioinformatics web services.
Pubmed: ABSTRACT Article (Gilgen1995) Gilgen, M.; Hofelein, C.; Luthy, J. & Hubner, P. Hydroxyquinoline overcomes PCR inhibition by UV-damaged mineral oil Nucleic Acids Res, 1995, 23, 4001-2
Pubmed: ABSTRACT Article (Giordano2001) Giordano, B.C.; Ferrance, J.; Swedberg, S.; Huhmer, A.F. & Landers, J.P. Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds Anal Biochem, 2001, 291, 124-32 Abstract: There is much interest in developing methods amenable to amplifying nucleic acids by the polymerase chain reaction (PCR) in small volumes in microfabricated devices. The use of infrared-mediated temperature control to accurately thermocycle microliter volumes in microchips fabricated from polyimide is demonstrated. Amplification of a 500-base-pair fragment of lambda-phage DNA was achieved in a 1.7-microl chamber containing a thermocouple that allowed for accurate control of temperature. While previous work showed that Taq polymerase was inactivated when in direct contact with the thermocouple, this was circumvented with the polyimide chip by the addition of polyethylene glycol as a buffer additive. This, consequently, allowed for adequate amounts of PCR product to be observed after only 15 cycles, with a total time for amplification of 240 s.
Pubmed: ABSTRACT Article (Gitton2002) Gitton, Y.; Dahmane, N.; Baik, S.; i Altaba, A.R.; Neidhardt, L.; Scholze, M.; Herrmann, B.G.; Kahlem, P.; Benkahla, A.; Schrinner, S.; Yildirimman, R.; Herwig, R.; Lehrach, H.; Yaspo, M. & expression map initiative, H.S.A. A gene expression map of human chromosome 21 orthologues in the mouse. Nature, 2002, 420, 586-590 Abstract: The DNA sequence of human chromosome 21 (HSA21) has opened the route for a systematic molecular characterization of all of its genes. Trisomy 21 is associated with Down's syndrome, the most common genetic cause of mental retardation in humans. The phenotype includes various organ dysmorphies, stereotypic craniofacial anomalies and brain malformations. Molecular analysis of congenital aneuploidies poses a particular challenge because the aneuploid region contains many protein-coding genes whose function is unknown. One essential step towards understanding their function is to analyse mRNA expression patterns at key stages of organism development. Seminal works in flies, frogs and mice showed that genes whose expression is restricted spatially and/or temporally are often linked with specific ontogenic processes. Here we describe expression profiles of mouse orthologues to HSA21 genes by a combination of large-scale mRNA in situ hybridization at critical stages of embryonic and brain development and in silico (computed) mining of expressed sequence tags. This chromosome-scale expression annotation associates many of the genes tested with a potential biological role and suggests candidates for the pathogenesis of Down's syndrome.
Pubmed: ABSTRACT Article (Giulietti2001) Giulietti, A.; Overbergh, L.; Valckx, D.; Decallonne, B.; Bouillon, R. & Mathieu, C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression Methods, 2001, 25, 386-401 Abstract: The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.
Pubmed: ABSTRACT Article (Goldberg2006) Goldberg, S.M.D.; Johnson, J.; Busam, D.; Feldblyum, T.; Ferriera, S.; Friedman, R.; Halpern, A.; Khouri, H.; Kravitz, S.A.; Lauro, F.M.; Li, K.; Rogers, Y.; Strausberg, R.; Sutton, G.; Tallon, L.; Thomas, T.; Venter, E.; Frazier, M. & Venter, J.C. A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. Proc Natl Acad Sci U S A, 2006, 103, 11240-11245 Abstract: Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.
Pubmed: ABSTRACT Article (Golenberg1996) Golenberg, E.M.; Bickel, A. & Weihs, P. Effect of highly fragmented DNA on PCR. Nucleic Acids Res, 1996, 24, 5026-5033 Abstract: We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.
Pubmed: ABSTRACT Article (Gorelenkov2001) Gorelenkov, V.; Antipov, A.; Lejnine, S.; Daraselia, N. & Yuryev, A. Set of novel tools for PCR primer design Biotechniques, 2001, 31, 1326-30 Abstract: We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.
Pubmed: ABSTRACT Article (Gresham2006) Gresham, D.; Ruderfer, D.M.; Pratt, S.C.; Schacherer, J.; Dunham, M.J.; Botstein, D. & Kruglyak, L. Genome-wide detection of polymorphisms at nucleotide resolution with a single DNA microarray. Science, 2006, 311, 1932-1936 Abstract: A central challenge of genomics is to detect, simply and inexpensively, all differences in sequence among the genomes of individual members of a species. We devised a system to detect all single-nucleotide differences between genomes with the use of data from a single hybridization to a whole-genome DNA microarray. This allowed us to detect a variety of spontaneous single-base pair substitutions, insertions, and deletions, and most (>90%) of the approximately 30,000 known single-nucleotide polymorphisms between two Saccharomyces cerevisiae strains. We applied this approach to elucidate the genetic basis of phenotypic variants and to identify the small number of single-base pair changes accumulated during experimental evolution of yeast.
Pubmed: ABSTRACT Article (Guetens2000) Guetens, G.; Van Cauwenberghe, K.; De Boeck, G.; Maes, R.; Tjaden, U.R.; van der Greef, J.; Highley, M.; van Oosterom, A.T. & de Bruijn, E.A. Nanotechnology in bio/clinical analysis J Chromatogr B Biomed Sci Appl, 2000, 739, 139-50 Abstract: Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries. Chip technology has come of age in the field of genomics, allowing faster analyses, and will fulfil an important role in RNA and peptide/protein analysis. The introduction of nanotechnology in LC-MS and CE-MS has resulted in new findings in the study of DNA adduct formation caused by carcinogenic substances, including anticancer drugs. Sample handling and introduction also can benefit from nanotechnology: The downscaling of sample volumes to the picoliter level has resulted in zeptomole (10(-21)) detection limits in the single-shot mass spectrum of proteins.
Pubmed: ABSTRACT Article (Guijt2003) Guijt, R.M.; Dodge, A.; van Dedem, G.W.K.; de Rooij, N.F. & Verpoorte, E. Chemical and physical processes for integrated temperature control in microfluidic devices. Lab Chip, 2003, 3, 1-4 Abstract: Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature. In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool a microchannel. Additionally, heating of a microchannel was achieved by dissolution of concentrated sulfuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allows control of the position and the magnitude of the thermal effect.
Pubmed: ABSTRACT Article (Guilfoyle1997) Guilfoyle, R.A.; Leeck, C.L.; Kroening, K.D.; Smith, L.M. & Guo, Z. Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest Nucleic Acids Res, 1997, 25, 1854-8 Abstract: A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide índexing sequence.The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.
Pubmed: ABSTRACT Article (Gulliksen2005) Gulliksen, A.; Anders Solli, L.; Stefan Drese, K.; Sorensen, O.; Karlsen, F.; Rogne, H.; Hovig, E. & Sirevag, R. Parallel nanoliter detection of cancer markers using polymer microchips Lab Chip, 2005, 5, 416-20 Abstract: A general multipurpose microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10(-6)[micro sign]M for artificial HPV 16 sequences, and 20 cells [micro sign]l(-1) for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer microchips using NASBA is therefore possible.
Pubmed: ABSTRACT Article (Gulliksen2004) Gulliksen, A.; Solli, L.; Karlsen, F.; Rogne, H.; Hovig, E.; Nordstrom, T. & Sirevag, R. Real-time nucleic acid sequence-based amplification in nanoliter volumes Anal Chem, 2004, 76, 9-14 Abstract: Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.
Pubmed: ABSTRACT Article (Gunderson2005) Gunderson, K.L.; Steemers, F.J.; Lee, G.; Mendoza, L.G. & Chee, M.S. A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet, 2005, 37, 549-554 Abstract: Oligonucleotide probe arrays have enabled massively parallel analysis of gene expression levels from a single cDNA sample. Application of microarray technology to analyzing genomic DNA has been stymied by the sequence complexity of the entire human genome. A robust, single base-resolution direct genomic assay would extend the reach of microarray technology. We developed an array-based whole-genome genotyping assay that does not require PCR and enables effectively unlimited multiplexing. The assay achieves a high signal-to-noise ratio by combining specific hybridization of picomolar concentrations of whole genome-amplified DNA to arrayed probes with allele-specific primer extension and signal amplification. As proof of principle, we genotyped several hundred previously characterized SNPs. The conversion rate, call rate and accuracy were comparable to those of high-performance PCR-based genotyping assays.
Pubmed: ABSTRACT Article (Gut2004) Gut, I.G. & Lathrop, G.M. Duplicating SNPs Nat Genet, 2004, 36, 789-90
Pubmed: ABSTRACT Article (Haas1998) Haas, S.; Vingron, M.; Poustka, A. & Wiemann, S. Primer design for large scale sequencing Nucleic Acids Res, 1998, 26, 3006-12 Abstract: We have developed PRIDE, a primer design program that automatically designs primers in single contigs or whole sequencing projects to extend the already known sequence and to double strand single-stranded regions. The program is fully integrated into the Staden package (GAP4) and accessible with a graphical user interface. PRIDE uses a fuzzy logic-based system to calculate primer qualities. The computational performance of PRIDE is enhanced by using suffix trees to store the huge amount of data being produced. A test set of 110 sequencing primers and 11 PCR primer pairs has been designed on genomic templates, cDNAs and sequences containing repetitive elements to analyze PRIDE's success rate. The high performance of PRIDE, combined with its minimal requirement of user interaction and its fast algorithm, make this program useful for the large scale design of primers, especially in large sequencing projects.
Pubmed: ABSTRACT Article (Haber2006) Haber, C. Microfluidics in commercial applications; an industry perspective. Lab Chip, 2006, 6, 1118-1121
Pubmed: ABSTRACT Article (Hacia1999) Hacia, J.G.; Fan, J.B.; Ryder, O.; Jin, L.; Edgemon, K.; Ghandour, G.; Mayer, R.A.; Sun, B.; Hsie, L.; Robbins, C.M.; Brody, L.C.; Wang, D.; Lander, E.S.; Lipshutz, R.; Fodor, S.P. & Collins, F.S. Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays. Nat Genet, 1999, 22, 164-167 Abstract: Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.
Pubmed: ABSTRACT Article (Haga2002) Haga, H.; Yamada, R.; Ohnishi, Y.; Nakamura, Y. & Tanaka, T. Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome. Single-nucleotide polymorphism J Hum Genet, 2002, 47, 605-10 Abstract: To construct an infrastructure for genome-wide association studies of common diseases or drug sensitivities, we have been systematically exploring common variants by resequencing genomic regions containing genes in DNA from 24 Japanese individuals. We have analyzed a total of 154 Mb, corresponding to approximately 5% of the human genome, and so far have identified 174,269 single-nucleotide polymorphisms and 16,293 insertion/deletion polymorphisms within gene regions, i.e., one polymorphism in 807 bp on average. Our data are freely available via our web site (http://snp.ims.u-tokyo.ac.jp) and will facilitate studies to identify genes associated with susceptibility to common diseases and genes involved in sensitivity to therapeutic drugs.
Pubmed: ABSTRACT Article (Halford2004) Halford, S.E. & Marko, J.F. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res, 2004, 32, 3040-52 Abstract: Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore earchingthrough megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the acilitated diffusionmodel of Berg, Winter and von Hippel, showing how non-specific DNA-protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional lidingmotion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive oppingmotions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of approximately 100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.
Pubmed: ABSTRACT Article (Hampe2001) Hampe, J.; Cuthbert, A.; Croucher, P.J.; Mirza, M.M.; Mascheretti, S.; Fisher, S.; Frenzel, H.; King, K.; Hasselmeyer, A.; MacPherson, A.J.; Bridger, S.; van Deventer, S.; Forbes, A.; Nikolaus, S.; Lennard-Jones, J.E.; Foelsch, U.R.; Krawczak, M.; Lewis, C.; Schreiber, S. & Mathew, C.G. Association between insertion mutation in NOD2 gene and Crohn disease in German and British populations Lancet, 2001, 357, 1925-8 Abstract: Background Genetic predisposition to inflammatory bowel disease (IBD) has been shown by epidemiological and linkage studies. Genetic linkage of IBD to chromosome 16 has been previously observed and replicated in independent populations. The recently identified NOD2 gene is a good positional and functional candidate gene since it is located in the region of linkage on chromosome 16q12, and activates nuclear factor (NF) kappaB in response to bacterial lipopolysaccharides.Methods We sequenced the coding region of the NOD2 gene and genotyped an insertion polymorphism affecting the leucine-rich region of the protein product in 512 individuals with IBD from 309 German or British families, 369 German trios (ie, German patients with sporadic IBD and their unaffected parents), and 272 normal controls. We then tested for association with Crohn disease and ulcerative colitis.Findings Family-based association analyses were consistently positive in 95 British and 99 German affected sibling pairs with Crohn disease (combined p<0.0001); the association was confirmed in the 304 German trios with Crohn disease. No association was seen in the 115 sibling pairs and 65 trios with ulcerative colitis. The genotype-specific disease risks conferred by heterozygous and homozygous mutant genotypes were 2.6 (95% CI 1.5-4.5) and 42.1 (4.3-infinity), respectively.Interpretation The insertion mutation in the NOD2 gene confers a substantially increased susceptibility to Crohn disease but not to ulcerative colitis.
Pubmed: ABSTRACT Article (Hampe2002) Hampe, J.; Grebe, J.; Nikolaus, S.; Solberg, C.; Croucher, P.J.; Mascheretti, S.; Jahnsen, J.; Moum, B.; Klump, B.; Krawczak, M.; Mirza, M.M.; Foelsch, U.R.; Vatn, M. & Schreiber, S. Association of NOD2 (CARD 15) genotype with clinical course of Crohn disease: a cohort study Lancet, 2002, 359, 1661-5 Abstract: BACKGROUND: Crohn disease is a heterogeneous disorder for which NOD2 (CARD 15) has been identified as a susceptibility gene. We investigate the relation between NOD2 genotype and phenotypic characteristics of patients with Crohn disease. METHODS: Hypotheses about the relation between NOD2 genotype and Crohn disease phenotype were generated retrospectively from a group of 446 German patients with this disorder. Positive findings (p<0.10) were verified in prospectively established cohorts of 106 German and 55 Norwegian patients with Crohn disease. All patients were genotyped for the main coding mutations in NOD2, denoted SNP8, SNP12, and SNP13, with Taqman technology. FINDINGS: In the retrospective cohort, six clinical characteristics showed noteworthy haplotype association: fistulising, ileal, left colonic and right colonic disease, stenosis, and resection. In the German prospective cohort, these haplotype associations could be replicated for ileal (p=0.006) and right colonic disease (p < or =0.001). A similar trend was noted in the Norwegian patients. INTERPRETATION: We recorded a distinct relation between NOD2 genotype and phenotype of Crohn disease. Test strategies with NOD2 variations to predict the clinical course of Crohn disease could lead to the development of new therapeutic paradigms.
Pubmed: ABSTRACT Article (Hampe2001a) Hampe, J.; Wollstein, A.; Lu, T.; Frevel, H.J.; Will, M.; Manaster, C. & Schreiber, S. An integrated system for high throughput TaqMan based SNP genotyping Bioinformatics, 2001, 17, 654-5 Abstract: We have developed an integrated laboratory information system that allows the flexible handling of pedigree, phenotype and genotype information. Specifically, it includes client applications for an integrated data import from TaqMan typing files, Mendel checking, data export, handling of pedigree and phenotype information and analysis features. AVAILABILITY: The SQL source code, sources and binaries of the client applications (NT and Windows95/98 platforms) and additional documentation are available at http://www.mucosa.de/.
Pubmed: ABSTRACT Article (Hardenbol2003) Hardenbol, P.; Baner, J.; Jain, M.; Nilsson, M.; Namsaraev, E.A.; Karlin-Neumann, G.A.; Fakhrai-Rad, H.; Ronaghi, M.; Willis, T.D.; Landegren, U. & Davis, R.W. Multiplexed genotyping with sequence-tagged molecular inversion probes Nat Biotechnol, 2003 Abstract: We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.
Pubmed: ABSTRACT Article (Harris1997) Harris, S. & Jones, D.B. Optimisation of the polymerase chain reaction Br J Biomed Sci, 1997, 54, 166-73 Abstract: The polymerase chain reaction (PCR) is a method by which specific sequences of DNA can be copied many times, allowing detailed molecular studies to be performed on as little as a single cell. Numerous and diverse applications of PCR are being developed across all disciplines of diagnostic pathology and research, and no single protocol is appropriate for all situations. Optimising PCR requires a delicate balance between the amplification of specific products and avoiding the production of non-specific products. Each step, from DNA template extraction to cycling times and temperatures, needs to be considered carefully. The aim of this study is to assess which parameters influence DNA amplification efficiency and specificity. The parameters evaluated are the denaturation, annealing and extension temperatures, the number of cycles performed, and the primer, magnesium chloride, dNTP, Taq DNA polymerase and DNA template concentrations. The important parameters for efficient, specific amplification were denaturation time and temperature, stringent annealing temperatures and magnesium chloride concentration. The importance of DNA concentration was found to depend upon the source from which the DNA was extracted.
Pubmed: ABSTRACT Book (Hartung2005) Hartung, J.; Elpelt, B. & Kloesener, K. Statistik. Lehr- und Handbuch der angewandten Statistik Oldenbourg, 2005
Article (Hashimoto2004) Hashimoto, M.; Chen, P.; Mitchell, M.W.; Nikitopoulos, D.E.; Soper, S.A. & Murphy, M.C. Rapid PCR in a continuous flow device. Lab Chip, 2004, 4, 638-645 Abstract: Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 degrees C) and renaturation (55 degrees C-68 degrees C) zones; above 6 mm s(-1) the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from lambda-DNA. Amplifications at velocities ranging from 1 mm s(-1) to 20 mm s(-1) were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle(-1)) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle(-1)). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).
Pubmed: ABSTRACT Article (Hashimoto2005) Hashimoto, M.; Hupert, M.L.; Murphy, M.C.; Soper, S.A.; Cheng, Y. & Barany, F. Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations. Anal Chem, 2005, 77, 3243-3255 Abstract: We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.
Pubmed: ABSTRACT Article (Hauser2006) Hauser, N.C.; Martinez, R.; Jacob, A.; Rupp, S.; Hoheisel, J.D. & Matysiak, S. Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform. Nucleic Acids Res, 2006 Abstract: L-DNA is the perfect mirror-image form of the naturally occurring d-conformation of DNA. Therefore, L-DNA duplexes have the same physical characteristics in terms of solubility, duplex stability and selectivity as D-DNA but form a left-helical double-helix. Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however. We analysed some of the properties that are typical for L-DNA. For all the differences, L-DNA is chemically compatible with the D-form of DNA, so that chimeric molecules can be synthesized. We take advantage of the characteristics of L-DNA toward the establishment of a universal microarray that permits the analysis of different kinds of molecular diagnostic information in a single experiment on a single platform, in various combinations. Typical results for the measurement of transcript level variations, genotypic differences and DNA-protein interactions are presented. However, on the basis of the characteristic features of L-DNA, also other applications of this molecule type are discussed.
Pubmed: ABSTRACT Article (Hawkins2002) Hawkins, J.R.; Khripin, Y.; Valdes, A.M. & Weaver, T.A. Miniaturized sealed-tube allele-specific PCR Hum Mutat, 2002, 19, 543-53 Abstract: DNA diagnostics has been progressively moving from expensive, low-throughput, multi-step methods towards inexpensive, robust, and high-throughput methods. Here we describe the further validation and refinement of a recently described novel genotyping method that has the latter characteristics. An evolved form of allele-specific PCR, the method generates a fluorescent signal through the use of universal labeled primers, which can be quantified directly from microplates using standard plate readers. We have applied the method successfully to a test set of 12 novel single nucleotide polymorphisms (SNPs) on a panel of 47 individuals using low reaction volumes. We demonstrate that the method is extremely accurate, robust, and can be optimized in a simple and predictable manner. By conducting the assay in closed-tube format, the potential for contamination is reduced to a minimum. By virtue of its simplicity, the method is versatile and cost-effective with potential for use in industrial-scale genetic studies or in the clinical diagnostic setting.
Pubmed: ABSTRACT Article (Heid1996) Heid, C.A.; Stevens, J.; Livak, K.J. & Williams, P.M. Real time quantitative PCR Genome Res, 1996, 6, 986-94 Abstract: We have developed a novel eal timequantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
Pubmed: ABSTRACT Article (Heil2002) Heil, J.; Glanowski, S.; Scott, J.; Deen, E.W.; McMullen, I.; Wu, L.; Gire, C. & Sprague, A. An automated computer system to support ultra high throughput SNP genotyping. Pac Symp Biocomput, 2002, 30-40 Abstract: Celera Genomics has constructed an automated computer system to support ultra high-throughput SNP genotyping that satisfies the increasing demand that disease association studies are placing on current genotyping facilities. This system consists of the seamless integration of target SNP selection, automated oligo design, in silico assay quality validation, laboratory management of samples, reagents and plates, automated allele calling, optional manual review of autocalls, regular status reports, and linkage disequilibrium analysis. Celera has proven the system by generating over 2.5 million genotypes from more than 10,000 SNPs, and is approaching the target capacity of over 10,000 genotypes per machine per hour using limited human intervention with state of the art laboratory hardware.
Pubmed: ABSTRACT Article (Hein2001) Hein, I.; Lehner, A.; Rieck, P.; Klein, K.; Brandl, E. & Wagner, M. Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. Appl Environ Microbiol, 2001, 67, 3122-3126 Abstract: Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.
Pubmed: ABSTRACT Article (Heller2000) Heller, M.J.; Forster, A.H. & Tu, E. Active microeletronic chip devices which utilize controlled electrophoretic fields for multiplex DNA hybridization and other genomic applications Electrophoresis, 2000, 21, 157-64 Abstract: Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.
Pubmed: ABSTRACT Article (Heng2006) Heng, X.; Erickson, D.; Baugh, L.R.; Yaqoob, Z.; Sternberg, P.W.; Psaltis, D. & Yang, C. Optofluidic microscopy-a method for implementing a high resolution optical microscope on a chip. Lab Chip, 2006, 6, 1274-1276 Abstract: We report a novel microfluidics-based lensless imaging technique, termed optofluidic microscopy (OFM), and demonstrate Caenorhabditis elegans imaging with an OFM prototype that gives comparable resolution to a conventional microscope and a measured resolution limit of 490 +/- 40 nm.
Pubmed: ABSTRACT Article (Hengen1997) Hengen, P.N. Optimizing multiplex and LA-PCR with betaine Trends Biochem Sci, 1997, 22, 225-6 Abstract: Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the internet. This month column discusses the use of additives for optimizing the amount and quality of product obtained through multiplex and ong and accuratepolymerase chain reaction (LA-PCR). For details on how to partake in the newsgroup, see the accompanying box.
Pubmed: ABSTRACT Article (Higuchi1993) Higuchi, R.; Fockler, C.; Dollinger, G. & Watson, R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions Biotechnology (N Y), 1993, 11, 1026-30 Abstract: We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.
Pubmed: ABSTRACT Article (Hinds2005) Hinds, D.A.; Stuve, L.L.; Nilsen, G.B.; Halperin, E.; Eskin, E.; Ballinger, D.G.; Frazer, K.A. & Cox, D.R. Whole-genome patterns of common DNA variation in three human populations. Science, 2005, 307, 1072-1079 Abstract: Individual differences in DNA sequence are the genetic basis of human variability. We have characterized whole-genome patterns of common human DNA variation by genotyping 1,586,383 single-nucleotide polymorphisms (SNPs) in 71 Americans of European, African, and Asian ancestry. Our results indicate that these SNPs capture most common genetic variation as a result of linkage disequilibrium, the correlation among common SNP alleles. We observe a strong correlation between extended regions of linkage disequilibrium and functional genomic elements. Our data provide a tool for exploring many questions that remain regarding the causal role of common human DNA variation in complex human traits and for investigating the nature of genetic variation within and between human populations.
Pubmed: ABSTRACT Article (Hirakawa2002) Hirakawa, M.; Tanaka, T.; Hashimoto, Y.; Kuroda, M.; Takagi, T. & Nakamura, Y. JSNP: a database of common gene variations in the Japanese population Nucleic Acids Res, 2002, 30, 158-62 Abstract: JSNP is a repository of Japanese Single Nucleotide Polymorphism (SNP) data, begun in 2000 and developed through the Prime Minister Millennium Project. The aim of this undertaking is to identify and collate up to 150 000 SNPs from the Japanese population, located in genes or in adjacent regions that might influence the coding sequence of the genes. The project has been carried out by a collaboration between the Human Genome Center (HGC) in the Institute of Medical Science (IMS) at the University of Tokyo and the Japan Science and Technology Corporation (JST). JSNP serves as both a storage site for the Japanese SNPs obtained from the ongoing project and as a facility for public dissemination to allow researchers access to high quality SNP data. A primary motivation of the project is the construction of a basic data set to identify relationships between polymorphisms and common diseases or the reaction to drugs. As such, emphasis has been placed on the identification of SNPs that lie in candidate regions which may affect phenotype but which would not necessarily directly cause disease. Unrestricted access to JSNP and any associated files is available at http://snp.ims.u-tokyo.ac.jp/.
Pubmed: ABSTRACT Article (Hoehe2003) Hoehe, M.R. Haplotypes and the systematic analysis of genetic variation in genes and genomes Pharmacogenomics, 2003, 4, 547-70 Abstract: Haplotypes have been used in various fields of genetics for a long time, in a variety of contexts, and for different purposes. Now, haplotype-based approaches to the analysis of candidate genes and genome-wide linkage disequilibrium (LD) mapping have gained center stage. It is time to explicitly distinguish the different concepts implied in the present haplotype approaches: haplotypes are not haplotypes, after all. The distinction of three different categories, ancestral, common haplotypes or haplotype blocks, gene-based haplotypes as complex genetic markers and gene-based functional haplotypes, is proposed. These categories serve as framework to review and analyze in particular the recent work suggesting evidence for a haplotype block structure of the human genome and the body of comparative sequencing studies addressing haplotype and LD structures at the gene level. Haplotype approaches will be evaluated along the dimensions preselection of variants versus complete DNA sequence information, role of LD and stages in the process of disease gene identification. Overall, the content of haplotypes is conceived as a function of available technologies to evaluate genetic variation and general advances in human genome research.
Pubmed: ABSTRACT Article (Hoh2003) Hoh, J.; Matsuda, F.; Peng, X.; Markovic, D.; Lathrop, M.G. & Ott, J. SNP haplotype tagging from DNA pools of two individuals BMC Bioinformatics, 2003, 4, 14 Abstract: BACKGROUND: DNA pooling is a technique to reduce genotyping effort while incurring only minor losses in accuracy of allele frequency estimates for single nucleotide polymorphism (SNP) markers. RESULTS: We present an algorithm for reconstructing haplotypes (alleles for multiple SNPs on same chromosome) from pools of two individual DNAs, in which Hardy-Weinberg equilibrium conditions or other assumptions are not required. The program outputs, in addition to inferred haplotypes, a minimal number of haplotype-tagging SNPs that are identified after an exhaustive search procedure. CONCLUSION: Our method and algorithms lead to a significant reduction in genotyping effort, for example, in case-control disease association studies while maintaining the possibility of reconstructing haplotypes under very general conditions.
Pubmed: ABSTRACT Article (Holbrook2005) Holbrook, J.F.; Stabley, D. & Church, K.S. Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies. J Biomol Tech, 2005, 16, 125-133 Abstract: The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.
Pubmed: ABSTRACT Article (Holden2002) Holden, A.L. The SNP consortium: summary of a private consortium effort to develop an applied map of the human genome Biotechniques, 2002, Suppl, 22-4, 26
Pubmed: ABSTRACT Article (Holland1991) Holland, P.M.; Abramson, R.D.; Watson, R. & Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5---3exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci U S A, 1991, 88, 7276-80 Abstract: The 5---3exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3end, labeled at the 5end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5---3exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
Pubmed: ABSTRACT Article (Hong2001) Hong, J.W.; Fujii, T.; Seki, M.; Yamamoto, T. & Endo, I. Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip. Electrophoresis, 2001, 22, 328-333 Abstract: We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.
Pubmed: ABSTRACT Article (Howald2006) Howald, C.; Merla, G.; Digilio, M.C.; Amenta, S.; Lyle, R.; Deutsch, S.; Choudhury, U.; Bottani, A.; Antonarakis, S.E.; Fryssira, H.; Dallapiccola, B. & Reymond, A. Two high throughput technologies to detect segmental aneuploidies identify new Williams-Beuren syndrome patients with atypical deletions. J Med Genet, 2006, 43, 266-273 Abstract: OBJECTIVE: To develop and compare two new technologies for diagnosing a contiguous gene syndrome, the Williams-Beuren syndrome (WBS). METHODS: The first proposed method, named paralogous sequence quantification (PSQ), is based on the use of paralogous sequences located on different chromosomes and quantification of specific mismatches present at these loci using pyrosequencing technology. The second exploits quantitative real time polymerase chain reaction (QPCR) to assess the relative quantity of an analysed locus. RESULTS: A correct and unambiguous diagnosis was obtained for 100% of the analysed samples with either technique (n = 165 and n = 155, respectively). These methods allowed the identification of two patients with atypical deletions in a cohort of 182 WBS patients. Both patients presented with mild facial anomalies, mild mental retardation with impaired visuospatial cognition, supravalvar aortic stenosis, and normal growth indices. These observations are consistent with the involvement of GTF2IRD1 or GTF2I in some of the WBS facial features. CONCLUSIONS: Both PSQ and QPCR are robust, easy to interpret, and simple to set up. They represent a competitive alternative for the diagnosis of segmental aneuploidies in clinical laboratories. They have advantages over fluorescence in situ hybridisation or microsatellites/SNP genotyping for detecting short segmental aneuploidies as the former is costly and labour intensive while the latter depends on the informativeness of the polymorphisms.
Pubmed: ABSTRACT Article (Hsu2001) Hsu, T.M.; Chen, X.; Duan, S.; Miller, R.D. & Kwok, P.Y. Universal SNP genotyping assay with fluorescence polarization detection Biotechniques, 2001, 31, 560, 562, 564-8, passim Abstract: The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.
Pubmed: ABSTRACT Article (Hu1998) Hu, Y.W.; Balaskas, E.; Kessler, G.; Issid, C.; Scully, L.J.; Murphy, D.G.; Rinfret, A.; Giulivi, A.; Scalia, V. & Gill, P. Primer specific and mispair extension analysis (PSMEA) as a simple approach to fast genotyping Nucleic Acids Res, 1998, 26, 5013-5 Abstract: A simple method, primer specific and mispair extension analysis (PSMEA) with pfu DNA polymerase was developed for genotyping. PSMEA is based on the unique properties of 3->5exonuclease proofreading activity. In the presence of an incomplete set of dNTPs, pfu was found to be extremely discriminative in nucleotide incorporation and proofreading at the initiation step of DNA synthesis, completely preventing primer extension when mispair(s) are found adjacent to the 3end of the primer. This has allowed us to accurately detect nucleotide variations, deletions and insertions for fast genotyping.
Pubmed: ABSTRACT Article (Hubbard2002) Hubbard, T. Biological information: making it accessible and integrated (and trying to make sense of it) Bioinformatics, 2002, 18 Suppl 2, S140 Abstract: The availability of the genome sequences of human and mouse, human sequence variation data and other large genetic data sets will lead to a revolution in understanding of the human machine and the treatment of its diseases. The success of the international genome sequencing consortiums shows what can be achieved by well coordinated large scale public domain projects and the benefits of data access to all. It is already clear that the availability of this sequence is having a huge impact on research worldwide. Complete genome sequences provide a framework to pull all biological data together such that each piece has the potential to say something about biology as a whole. Biology is too complex for any organisation to have a monopoly of ideas or data, so the collection, analysis and access to this data can be contributed to by research institutes around the world. However, although it is possible for all this data to be accessible to all through the internet, the more organisations provide data or analysis separately, the harder it becomes for anyone to collect and integrate the results. To address these problems of intergration of data, open standards for biological data exchange, such as the 'Distributed Annotation System' (DAS) are being developed and bioinformatics (Dowell et al., 2001) as a whole is now being strongly driven by the open source software (OSS) model for collaborative software development (Hubbard and Birney, 1999). The leading provider of human genome annotation, the Ensembl project (http://www.ensembl.org), is entirely an OSS project and has been widely adopted by academic and commerical organisations alike (Hubbard et al., 2002). Accurate automatic annotation of features such as genes in vertebrate genomes currently relies on supporting evidence in the form of homologies to mRNAs, ESTs or protein. However, it appears that sufficient high quality experimentally curated annotation now exists to be used as a substrate for machine learning algorithms to create effective models of biological signal sequences (Down and Hubbard, 2002). Is there hope for ab initio prediction methods after all?
Pubmed: ABSTRACT Article (Hubbard2002a) Hubbard, T.; Barker, D.; Birney, E.; Cameron, G.; Chen, Y.; Clark, L.; Cox, T.; Cuff, J.; Curwen, V.; Down, T.; Durbin, R.; Eyras, E.; Gilbert, J.; Hammond, M.; Huminiecki, L.; Kasprzyk, A.; Lehvaslaiho, H.; Lijnzaad, P.; Melsopp, C.; Mongin, E.; Pettett, R.; Pocock, M.; Potter, S.; Rust, A.; Schmidt, E.; Searle, S.; Slater, G.; Smith, J.; Spooner, W.; Stabenau, A.; Stalker, J.; Stupka, E.; Ureta-Vidal, A.; Vastrik, I. & Clamp, M. The Ensembl genome database project Nucleic Acids Res, 2002, 30, 38-41 Abstract: The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.
Pubmed: ABSTRACT Article (Huebner2007) Huebner, A.; Art, M.S.; Holt, D.; Abell, C.; Hollfelder, F.; deMello, A.J. & Edel, J.B. Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Commun (Camb), 2007, 1218-1220 Abstract: We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.
Pubmed: ABSTRACT Article (Hughes1996) Hughes, C.M.; Lewis, S.E.; Martin, V.J.M. & Thompson, W. A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. Mol Hum Reprod, 1996, 2, 613-619 Abstract: Baseline DNA damage in spermatozoa from fertile and infertile men was compared using a modified alkali single cell gel electrophoresis (comet) assay. Semen from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile (World Health Organization criteria, 1992) samples were studied. No significant difference was observed in levels of baseline damage between the three groups. A median value for baseline damage of approximately 20% (80% head DNA) was obtained in all samples. Irradiation with X-rays (5-30 Gy) produced no additional damage in fertile samples when median values were examined. However, irradiation with 30 Gy X-rays produced significant damage in both infertile groups. Hydrogen peroxide (40 microM) treatment induced significant damage in the asthenozoospermic group, whereas 100 microM H2O2 was required to cause significant damage in the normozoospermic fertile and infertile samples. Within the fertile population a subgroup in which percentage head DNA was greater than 80% was observed in both treated and untreated specimens. This subgroup significantly decreased with treatment in both infertile groups. We conclude that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.
Pubmed: ABSTRACT Article (Huh2005) Huh, D.; Gu, W.; Kamotani, Y.; Grotberg, J.B. & Takayama, S. Microfluidics for flow cytometric analysis of cells and particles. Physiol Meas, 2005, 26, R73-R98 Abstract: This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.
Pubmed: ABSTRACT Article (Huhmer2000) Huhmer, A.F. & Landers, J.P. Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes Anal Chem, 2000, 72, 5507-12 Abstract: We demonstrate that accurate thermocycling of nanoliter volumes is possible using infrared-mediated temperature control. Thermocycling in the presence of Taq polymerase and the appropriate primers for amplification of lambda-DNA in a total volume of 160 nL is shown to result in the successful amplification of a 500-base pair fragment of lambda-DNA. The efficiency of the amplification is sufficiently high so that as few as 10 cycles were required to amplify an adequate mass of DNA for analysis by capillary electrophoresis. This indicates that, as expected, PCR amplification of DNA in nanoliter volumes should not only require less Taq polymerase but require less cycling time to produce a detectable amount of product. This sets the stage for microchip integration of the PCR process in the nanoliter volumes routinely manipulated in electrophoretic microchips.
Pubmed: ABSTRACT Article (Ibrahim1997) Ibrahim, M.S.; Esposito, J.J.; Jahrling, P.B. & Lofts, R.S. The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus. Mol Cell Probes, 1997, 11, 143-147 Abstract: A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.
Pubmed: ABSTRACT Article (Innis1988) Innis, M.A.; Myambo, K.B.; Gelfand, D.H. & Brow, M.A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA Proc Natl Acad Sci U S A, 1988, 85, 9436-40 Abstract: The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.
Pubmed: ABSTRACT Article (Irawan2005) Irawan, R.; Tjin, S.C.; Yager, P. & Zhang, D. Cross-talk problem on a fluorescence multi-channel microfluidic chip system. Biomed Microdevices, 2005, 7, 205-211 Abstract: Development of a compact fluorescence-based detection system for use in a micro-analytical system, such as a point-of-care diagnostic system, often requires a multi-channel microfluidic chip system. Since the materials used for microfluidic chips usually are transparent in the visible region and have a refractive indices higher than that of air or the surrounding environment, the fluorescence emission and scattered excitation light can propagate through the chip. We observed that such propagation can cause cross-talk between adjacent channels, and may become the major source of noise in the system and/or photo bleach the fluorescent samples in the adjacent channels, particularly for the small distances between the channels found in microfluidic chips, usually in order of several micro m. We monitored this cross-talk using fluorescein as a fluorescent sample and Mylar sheeting as a microfluidic chip material. We then discuss how this cross-talk can be avoided using a simple, inexpensive and effective method.
Pubmed: ABSTRACT Article (Irizarry2000) Irizarry, K.; Kustanovich, V.; Li, C.; Brown, N.; Nelson, S.; Wong, W. & Lee, C.J. Genome-wide analysis of single-nucleotide polymorphisms in human expressed sequences Nat Genet, 2000, 26, 233-6 Abstract: Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations-comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing-verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).
Pubmed: ABSTRACT Article (Irizarry2005) Irizarry, R.A.; Warren, D.; Spencer, F.; Kim, I.F.; Biswal, S.; Frank, B.C.; Gabrielson, E.; Garcia, J.G.N.; Geoghegan, J.; Germino, G.; Griffin, C.; Hilmer, S.C.; Hoffman, E.; Jedlicka, A.E.; Kawasaki, E.; Murillo, F.M.; Morsberger, L.; Lee, H.; Petersen, D.; Quackenbush, J.; Scott, A.; Wilson, M.; Yang, Y.; Ye, S.Q. & Yu, W. Multiple-laboratory comparison of microarray platforms. Nat Methods, 2005, 2, 345-350 Abstract: Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC-Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.
Pubmed: ABSTRACT Article (Isaksson1999) Isaksson, A. & Landegren, U. Accessing genomic information: alternatives to PCR Curr Opin Biotechnol, 1999, 10, 11-5 Abstract: The growing abundance of genomic sequence data invites increasingly large-scale genetic analyses. Studies of genetic variation in large sets of genes can illuminate important disease mechanisms and serve to identify novel drug targets or predict therapeutic responses. At present mostly a concern in extensive research projects, large-scale genetic analyses will gradually also find their way into clinical practice as an aid to the physician. It is timely, therefore, to take stock of methods that are becoming available for analyses of large sets of gene sequences. Clearly PCR remains the workhorse for molecular genetic analysis, and several modifications such as homogenous amplification assays and parallel detection on DNA microarrays further increase throughput. Recent developments, however, also offer hope that other methods will become available for genomic investigations, providing substantially increased analytical capacity.
Pubmed: ABSTRACT Article (Jackman1998) Jackman, R.; Duffy, D.; Ostuni, E.; Willmore, N. & Whitesides, G. Fabricating Large Arrays of Microwells with Arbitrary Dimensions and Filling Them Using Discontinuous Dewetting Analytical Chemistry, 1998, 70, 2280-2287
PDF: Jackman1998.pdf Article (Jain2006) Jain, K.K. Challenges of drug discovery for personalized medicine. Curr Opin Mol Ther, 2006, 8, 487-492 Abstract: Personalized medicine--the treatment best suited for an individual patient--is based on genomic as well as other factors that influence the response to drugs. Besides matching existing drugs to appropriate patients, 'personalization' is being extended to the drug-discovery stage. Several 'omics' technologies are being increasingly used for this purpose and personalized drug-discovery efforts are in progress in major therapeutic areas. Biomarkers are an important link between drug discovery efforts and diagnostics. The concept of personalized medicine is also a driver for the integration of various biotechnologies, such as RNA interference and nanobiotechnology, which are also being applied to drug discovery. The limitations of various approaches to personalized medicine have been identified, as well as the financial implications of fragmenting the markets for drugs for the biopharmaceutical industry, which remains focused on the development of blockbuster drugs.
Pubmed: ABSTRACT Article (Janasek2006) Janasek, D.; Franzke, J. & Manz, A. Scaling and the design of miniaturized chemical-analysis systems. Nature, 2006, 442, 374-380 Abstract: Micrometre-scale analytical devices are more attractive than their macroscale counterparts for various reasons. For example, they use smaller volumes of reagents and are therefore cheaper, quicker and less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction and detection devices before they are fabricated. Some devices designed using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to arise.
Pubmed: ABSTRACT Article (Jebbink2003) Jebbink, J.; Bai, X.; Rogers, B.B.; Dawson, D.B.; Scheuermann, R.H. & Saad, R.D. Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons. J Mol Diagn, 2003, 5, 15-20 Abstract: Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.
Pubmed: ABSTRACT Article (Jeffreys2001) Jeffreys, A.J.; Kauppi, L. & Neumann, R. Intensely punctate meiotic recombination in the class II region of the major histocompatibility complex Nat Genet, 2001, 29, 217-22 Abstract: There is considerable interest in understanding patterns of linkage disequilibrium (LD) in the human genome, to aid investigations of human evolution and facilitate association studies in complex disease. The relative influences of meiotic crossover distribution and population history on LD remain unclear, however. In particular, it is uncertain to what extent crossovers are clustered into ot spots, that might influence LD patterns. As a first step to investigating the relationship between LD and recombination, we have analyzed a 216-kb segment of the class II region of the major histocompatibility complex (MHC) already characterized for familial crossovers. High-resolution LD analysis shows the existence of extended domains of strong association interrupted by patchwork areas of LD breakdown. Sperm typing shows that these areas correspond precisely to meiotic crossover hot spots. All six hot spots defined share a remarkably similar symmetrical morphology but vary considerably in intensity, and are not obviously associated with any primary DNA sequence determinants of hot-spot activity. These hot spots occur in clusters and together account for almost all crossovers in this region of the MHC. These data show that, within the MHC at least, crossovers are far from randomly distributed at the molecular level and that recombination hot spots can profoundly affect LD patterns.
Pubmed: ABSTRACT Article (Jia1996) Jia, Y.; Kumar, A. & Patel, S.S. Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes. J Biol Chem, 1996, 271, 30451-30458 Abstract: The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, [formula: see text] where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi10 and Phi3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k-2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k-2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Phi3.8 a weaker promoter relative to Phi10.
Pubmed: ABSTRACT Article (Jiang2003) Jiang, Y. & Hofstadler, S.A. A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry Anal Biochem, 2003, 316, 50-7 Abstract: In this work we present a rapid and fully automated method to purify and desalt PCR products prior to analysis by electrospray ionization mass spectrometry. The protocol employs a commercial pipette tip packed with an anion-exchange resin and comprises four primary steps: tip pretreatment, sample loading, rinsing, and sample elution. This tip-based purification/desalting protocol has two distinct advantages over previously published methods. First, the protocol can be performed either manually (1-12 samples at a time), using a standard p10 manual pipette, or in a fully automated microtiter plate format (96 samples at a time) employing standard laboratory robotics. Additionally, the entire protocol from crude PCR product to an ëlectrosprayableanalyte solution requires only 10 microl of crude product and takes less than 20 min. Using capillary gel electrophoresis, we demonstrate an overall recovery efficiency of approximately 80% and demonstrate the exquisite desalting efficiency with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Using an internal mass standard we demonstrate sub-ppm mass measurement error which provides an unambiguous base composition for a 120-mer PCR product.
Pubmed: ABSTRACT Article (Jilma2005) Jilma, B.; Marsik, C.; Kovar, F.; Wagner, O.F.; Stohlawetz, P.J. & Endler, G. The single nucleotide polymorphism Ser128Arg in the E-selectin gene is associated with enhanced coagulation during human endotoxemia. Blood, 2005, 105, 2380-2383 Abstract: The single nucleotide polymorphism (SNP) Ser128Arg in the E-selectin gene is overrepresented in certain patient groups with atherosclerosis or restenosis. We hypothesized and tested whether it may affect cytokine-induced levels of soluble (s) E-selectin, or be associated with proinflammatory or procoagulant properties in a well-standardized inflammation model. Healthy male volunteers (n = 157) received a lipopolysaccharide (LPS) infusion and were genotyped for the S128R SNP, and outcome parameters were measured by enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). The S128R SNP had no pronounced effects on basal or inducible sE-selectin levels, or levels of tumor necrosis factor or interleukin-6. However, carriers of the S128R SNP had 20% higher monocyte counts at 24 hours after LPS infusion. Importantly, the S128R allele enhanced thrombin generation by 50% to 80%, as measured by prothrombin fragment F(1+2) (P < .01), and hence fibrin formation (D-dimer) 2-fold (P = .01 to P = .002). However, tissue factor (TF) mRNA levels were not affected. The S128R E-selectin genotype is associated with procoagulant effects in a human model of endotoxin-induced, TF-triggered coagulation. This could contribute to its linkage with various thrombotic cardiovascular disorders.
Pubmed: ABSTRACT Article (Johnson2001) Johnson, G.C.; Esposito, L.; Barratt, B.J.; Smith, A.N.; Heward, J.; Di Genova, G.; Ueda, H.; Cordell, H.J.; Eaves, I.A.; Dudbridge, F.; Twells, R.C.; Payne, F.; Hughes, W.; Nutland, S.; Stevens, H.; Carr, P.; Tuomilehto-Wolf, E.; Tuomilehto, J.; Gough, S.C.; Clayton, D.G. & Todd, J.A. Haplotype tagging for the identification of common disease genes Nat Genet, 2001, 29, 233-7 Abstract: Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.
Pubmed: ABSTRACT Article (Johnson2004) Johnson, M.P.; Haupt, L.M. & Griffiths, L.R. Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR Nucleic Acids Res, 2004, 32, e55 Abstract: With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.
Pubmed: ABSTRACT Article (Jong2006) de Jong, J.; Lammertink, R.G.H. & Wessling, M. Membranes and microfluidics: a review. Lab Chip, 2006, 6, 1125-1139 Abstract: The integration of mass transport control by means of membrane functionality into microfluidic devices has shown substantial growth over the last 10 years. Many different examples of mass transport control have been reported, demonstrating the versatile use of membranes. This review provides an overview of the developments in this area of research. Furthermore, it aims to bridge the fields of microfabrication and membrane science from a membrane point-of-view. First the basic terminology of membrane science will be discussed. Then the integration of membrane characteristics on-chip will be categorized based on the used fabrication method. Subsequently, applications in various fields will be reviewed. Considerations for the use of membranes will be discussed and a checklist with selection criteria will be provided that can serve as a starting point for those researchers interested in applying membrane-technology on-chip. Finally, opportunities for microfluidics based on proven membrane technology will be outlined. A special focus in this review is made on the membrane properties of polydimethylsiloxane (PDMS), since this material is frequently used nowadays in master replication.
Pubmed: ABSTRACT Article (Jordan2002) Jordan, B.; Charest, A.; Dowd, J.F.; Blumenstiel, J.P.; Yeh Rf, R.F.; Osman, A.; Housman, D.E. & Landers, J.E. Genome complexity reduction for SNP genotyping analysis Proc Natl Acad Sci U S A, 2002, 99, 2942-7 Abstract: Efficient single nucleotide polymorphism (SNP) genotyping methods are necessary to accomplish many current gene discovery goals. A crucial element in large-scale SNP genotyping is the number of individual biochemical reactions that must be performed. An efficient method that can be used to simultaneously amplify a set of genetic loci across a genome with high reliability can provide a valuable tool for large-scale SNP genotyping studies. In this paper we describe and characterize a method that addresses this goal. We have developed a strategy for reducing genome complexity by using degenerate oligonucleotide primer (DOP)-PCR and applied this strategy to SNP genotyping in three complex eukaryotic genomes; human, mouse, and Arabidopsis thaliana. Using a single DOP-PCR primer, SNP loci spread throughout a genome can be amplified and accurately genotyped directly from a DOP-PCR product mixture. DOP-PCRs are extremely reproducible. The DOP-PCR method is transferable to many species of interest. Finally, we describe an in silico approach that can effectively predict the SNP loci amplified in a given DOP-PCR, permitting the design of an efficient set of reactions for large-scale, genome-wide SNP studies.
Pubmed: ABSTRACT Article (Jorde2000) Jorde, L.B.; Watkins, W.S.; Bamshad, M.J.; Dixon, M.E.; Ricker, C.E.; Seielstad, M.T. & Batzer, M.A. The distribution of human genetic diversity: a comparison of mitochondrial, autosomal, and Y-chromosome data. Am J Hum Genet, 2000, 66, 979-988 Abstract: We report a comparison of worldwide genetic variation among 255 individuals by using autosomal, mitochondrial, and Y-chromosome polymorphisms. Variation is assessed by use of 30 autosomal restriction-site polymorphisms (RSPs), 60 autosomal short-tandem-repeat polymorphisms (STRPs), 13 Alu-insertion polymorphisms and one LINE-1 element, 611 bp of mitochondrial control-region sequence, and 10 Y-chromosome polymorphisms. Analysis of these data reveals substantial congruity among this diverse array of genetic systems. With the exception of the autosomal RSPs, in which an ascertainment bias exists, all systems show greater gene diversity in Africans than in either Europeans or Asians. Africans also have the largest total number of alleles, as well as the largest number of unique alleles, for most systems. GST values are 11%-18% for the autosomal systems and are two to three times higher for the mtDNA sequence and Y-chromosome RSPs. This difference is expected because of the lower effective population size of mtDNA and Y chromosomes. A lower value is seen for Y-chromosome STRs, reflecting a relative lack of continental population structure, as a result of rapid mutation and genetic drift. Africa has higher GST values than does either Europe or Asia for all systems except the Y-chromosome STRs and Alus. All systems except the Y-chromosome STRs show less variation between populations within continents than between continents. These results are reassuring in their consistency and offer broad support for an African origin of modern human populations.
Pubmed: ABSTRACT Article (Juncker2005) Juncker, D.; Schmid, H. & Delamarche, E. Multipurpose microfluidic probe. Nat Mater, 2005, 4, 622-628 Abstract: Microfluidic systems allow (bio)chemical processes to be miniaturized with the benefit of shorter time-to-result, parallelism, reduced sample consumption, laminar flow, and increased control and efficiency. However, such miniaturization inherently limits the size of the solid objects that can be processed and entails new challenges such as the interfacing of macroscopic samples with microscopic conduits. Here, we report a microfluidic probe (MFP) that overcomes these problems by combining the concepts of 'microfluidics' and of 'scanning probes'. Here, liquid boundaries formed by hydrodynamic forces underneath the MFP confine a flow of processing solution and replace the solid walls of closed microchannels. The MFP is therefore mobile and can be used to process large surfaces and objects by scanning across them. We illustrate the versatility of this concept with several examples including protein microarraying, complex gradient-formation, multiphase laminar-flow patterning, erasing, localized staining of cells and the contact-free detachment of a single cell. Many constraints imposed by the monolithic construction of microfluidic channels can now be circumvented using an MFP, opening up new avenues for microfluidic processing.
Pubmed: ABSTRACT Article (Kaack2004) Kaack, R.; Jung, A.; Wenz, H.; Zengerle, R. & Daub, M. PCR-slide: a modular and cascadable platform for DNA sample processing with integrated nanolitre dosage Micro Electro Mechanical Systems, 2004. 17th IEEE International Conference on. (MEMS), 2004, 338- 342 Abstract: For the first time we present a novel DNA sample processing platform with an integrated nanolitre dosage unit for extraction of processed materials in nanolitre quantities. A unique feature of the new platform is the possibility to link several sample processing steps including dilution/titration without any need for external pipetting equipment except for sample loading. This has been demonstrated by using the oligonucleotide ligase assay (OLA) coupled to the polymerase chain reaction (PCR), a sequence which requires a 1:15 dilution of OLA in PCR.
Article (Kaderali2003) Kaderali, L.; Deshpande, A.; Nolan, J.P. & White, P.S. Primer-design for multiplexed genotyping Nucleic Acids Res, 2003, 31, 1796-802 Abstract: Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be emultiplexed Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.
Pubmed: ABSTRACT Article (Kainz2000) Kainz, P. The PCR plateau phase - towards an understanding of its limitations. Biochim Biophys Acta, 2000, 1494, 23-27 Abstract: The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 microl reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DNA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.
Pubmed: ABSTRACT Article (Kalinina1997) Kalinina, O.; Lebedeva, I.; Brown, J. & Silver, J. Nanoliter scale PCR with TaqMan detection Nucleic Acids Res, 1997, 25, 1999-2004 Abstract: We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.
Pubmed: ABSTRACT Article (Kane2000) Kane, M.D.; Jatkoe, T.A.; Stumpf, C.R.; Lu, J.; Thomas, J.D. & Madore, S.J. Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays Nucleic Acids Res, 2000, 28, 4552-7 Abstract: To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined as the ability to detect a 3-fold change in mRNA. No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of approximately 10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any 'non-target' transcripts (cDNAs) >75% similar over the 50 base target may show cross-hybridization. Thus non-target sequences which have >75-80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target region) will contribute to the overall signal intensity. In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results. Together these results validate the utility of oligonucleotide probe (50mer) glass microarrays.
Pubmed: ABSTRACT Article (Kane2007) Kane, R.S. & Stroock, A.D. Nanobiotechnology: protein-nanomaterial interactions. Biotechnol Prog, 2007, 23, 316-319 Abstract: We review recent research that involves the interaction of nanomaterials such as nanoparticles, nanowires, and carbon nanotubes with proteins. We begin with a focus on the fundamentals of the structure and function of proteins on nanomaterials. We then review work in three areas that exploit these interactions: (1) sensing, (2) assembly of nanomaterials by proteins and proteins by nanomaterials, and (3) interactions with cells. We conclude with the identification of challenges and opportunities for the future.
Pubmed: ABSTRACT Article (Kang2001) Kang, S.H.; Shortreed, M.R. & Yeung, E.S. Real-time dynamics of single-DNA molecules undergoing adsorption and desorption at liquid-solid interfaces. Anal Chem, 2001, 73, 1091-1099 Abstract: The conformational dynamics and adsorption/desorption behavior of individual lambda-DNA molecules at liquid-solid interfaces were monitored by imaging within the evanescent field layer using total internal reflection fluorescence microscopy. At a fused-silica surface, molecular conformation and adsorption behavior were found to depend on both pH and buffer composition. A histogram of individual lambda-DNA adsorption durations measured by hydrodynamically flowing molecules along the interface exhibited asymmetry nearly identical to that of the corresponding elution peaks found in capillary liquid chromatography and capillary electrophoresis. The accessibility of the surface to the molecules, which is proportional to the capillary surface area-to-volume ratio, can be correlated with the capacity factor and the relative adsorption factor. At a C18 surface, the dynamics of individual DNA molecules changed with the addition of organic solvent as well as with pH. Hydrophobic interaction rather than electrostatic interaction was the major driving force for adsorption of individual DNA molecules.
Pubmed: ABSTRACT Article (Karlen2007) Karlen, Y.; McNair, A.; Perseguers, S.; Mazza, C. & Mermod, N. Statistical Significance of quantitative PCR. BMC Bioinformatics, 2007, 8, 131 Abstract: ABSTRACT: BACKGROUND: PCR has the potential to detect and precisely quantify specific DNA sequences, but it is not yet often used as a fully quantitative method. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. In this study, we evaluated the performance of known methods, and compared them with newly developed data processing strategies in terms of sensitivity, precision and robustness. RESULTS: Our results indicate that simple methods that do not rely on the estimation of the efficiency of the PCR amplification may provide reproducible and sensitive data, but that they do not quantify DNA with precision. Other evaluated methods based on sigmoidal or exponential curve fitting were generally of both poor sensitivity and precision. A statistical analysis of the parameters that influence efficiency indicated that it depends mostly on the selected amplicon and to a lesser extent on the particular biological sample analyzed. Thus, we devised various strategies based on individual or averaged efficiency values, which were used to assess the regulated expression of several genes in response to a growth factor. CONCLUSIONS: Overall, qPCR data analysis methods differ significantly in their performance, and this analysis identifies methods that provide DNA quantification estimates of high precision, robustness and reliability. These methods allow reliable estimations of relative expression ratio of two-fold or higher, and our analysis provides an estimation of the number of biological samples that have to be analyzed to achieve a given precision.
Pubmed: ABSTRACT Article (Karolchik2003) Karolchik, D.; Baertsch, R.; Diekhans, M.; Furey, T.S.; Hinrichs, A.; Lu, Y.T.; Roskin, K.M.; Schwartz, M.; Sugnet, C.W.; Thomas, D.J.; Weber, R.J.; Haussler, D. & Kent, W.J. The UCSC Genome Browser Database Nucleic Acids Res, 2003, 31, 51-4 Abstract: The University of California Santa Cruz (UCSC) Genome Browser Database is an up to date source for genome sequence data integrated with a large collection of related annotations. The database is optimized to support fast interactive performance with the web-based UCSC Genome Browser, a tool built on top of the database for rapid visualization and querying of the data at many levels. The annotations for a given genome are displayed in the browser as a series of tracks aligned with the genomic sequence. Sequence data and annotations may also be viewed in a text-based tabular format or downloaded as tab-delimited flat files. The Genome Browser Database, browsing tools and downloadable data files can all be found on the UCSC Genome Bioinformatics website (http://genome.ucsc.edu), which also contains links to documentation and related technical information.
Pubmed: ABSTRACT Article (Karrer1995) Karrer, E.E.; Lincoln, J.E.; Hogenhout, S.; Bennett, A.B.; Bostock, R.M.; Martineau, B.; Lucas, W.J.; Gilchrist, D.G. & Alexander, D. In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. Proc Natl Acad Sci U S A, 1995, 92, 3814-3818 Abstract: A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.
Pubmed: ABSTRACT Article (Karsai2002) Karsai, A.; Muller, S.; Platz, S. & Hauser, M.T. Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression Biotechniques, 2002, 32, 790-2, 794-6 Abstract: Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScript reverse transcriptase from 2.5 microg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10-20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.
Pubmed: ABSTRACT Article (Kartalov2006) Kartalov, E.P.; Anderson, W.F. & Scherer, A. The analytical approach to polydimethylsiloxane microfluidic technology and its biological applications. J Nanosci Nanotechnol, 2006, 6, 2265-2277 Abstract: This review article discusses PDMS (polydimethylsiloxane) microfluidic devices and their biological applications. First, the already developed devices are classified from the viewpoints of underlying technology within a common logical framework comprising single-layer, multilayer, and integrated devices, as well as surface chemistry modifications of PDMS. Combinatorial techniques are applied to re-derive existing devices within this framework. Next, the relevant scales of both microfluidics and biology are compared, obtaining the promise and limitations of PDMS microfluidics. Finally, the body of work is reclassified in terms of addressed biological applications and compared to the standard methods in cellular and molecular biology, to offer insights for future devices and applications.
Pubmed: ABSTRACT Article (Kato2006) Kato, T.; Inagaki, H.; Yamada, K.; Kogo, H.; Ohye, T.; Kowa, H.; Nagaoka, K.; Taniguchi, M.; Emanuel, B.S. & Kurahashi, H. Genetic variation affects de novo translocation frequency. Science, 2006, 311, 971 Abstract: Translocation is one of the most frequently occurring human chromosomal aberrations. The constitutional t(11;22)(q23;q11), which is the only known recurrent non-Robertsonian translocation, represents a good model for studying translocations in humans. Here we demonstrate polymorphisms of the palindromic sequence at the t(11;22) breakpoint that affect the frequency of de novo translocations in sperm from normal males. A typical allele consists of a perfect palindrome, producing ~10-5 de novo t(11;22) translocations. Alleles with an asymmetric center do not form the t(11;22). Our data show the importance of genome sequence on chromosomal rearrangements, a class of human mutation that is thought to be random.
Pubmed: ABSTRACT Article (Kazarian2007) Kazarian, S. Enhancing high-throughput technology and microfluidics with FTIR spectroscopic imaging. Anal Bioanal Chem, 2007
Pubmed: ABSTRACT Article (Ke2001) Ke, X.; Collins, A. & Ye, S. PIRA PCR designer for restriction analysis of single nucleotide polymorphisms Bioinformatics, 2001, 17, 838-9 Abstract: Primer-introduced restriction analysis (PIRA-PCR) is widely used to detect Single Nucleotide Polymorphisms (SNPs). To create artificial Restriction Fragment Length Polymorphism (RFLP), a mismatch is usually introduced near the end of the primer that is close to the mutation of interest. We describe in this report a www-based computer program that screens for the suitable mismatches, designs the primers, lists the appropriate restriction enzymes and other related information. AVAILABILITY: The computer program, with related descriptions, is available at http://cedar.genetics.soton.ac.uk/public_html/primer2.html.
Pubmed: ABSTRACT Article (Kennedy2003) Kennedy, G.C.; Matsuzaki, H.; Dong, S.; min Liu, W.; Huang, J.; Liu, G.; Su, X.; Cao, M.; Chen, W.; Zhang, J.; Liu, W.; Yang, G.; Di, X.; Ryder, T.; He, Z.; Surti, U.; Phillips, M.S.; Jacino, M.T.B.; Fodor, S.P.A. & Jones, K.W. Large-scale genotyping of complex DNA. Nat Biotechnol, 2003, 21, 1233-1237 Abstract: Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.
Pubmed: ABSTRACT Article (Kent2002) Kent, W.J.; Sugnet, C.W.; Furey, T.S.; Roskin, K.M.; Pringle, T.H.; Zahler, A.M. & Haussler, D. The human genome browser at UCSC Genome Res, 2002, 12, 996-1006 Abstract: As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.
Pubmed: ABSTRACT Article (Kerby2006) Kerby, M.B.; Legge, R.S. & Tripathi, A. Measurements of kinetic parameters in a microfluidic reactor. Anal Chem, 2006, 78, 8273-8280 Abstract: Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, residence time, and bed resistance were measured with dye pulses. A simple criterion was derived to evaluate the importance of flow-dependent mass-transfer resistances when using microreactors for calculating kinetic rate constants. In the absence of mass-transfer resistances, the Michaelis-Menten kinetic parameters are shown to be flow independent and are appropriately predicted using low substrate conversion data. A comparison of the kinetic parameters with those obtained using solution-phase enzymatic reactions shows a significant decrease in enzyme activity in the immobilized conformation. The immobilized Km of AP is approximately 6 times greater while the kcat is reduced by approximately 28 times. Contradictions found in literature on the evaluation of Michaelis-Menten kinetic parameters for immobilized enzymes in microfluidic reactors are addressed. When product molecules occupy a significant number of enzymatic sites or modify the enzyme activity, the assumed Michaelis-Menten mechanism can no longer be valid. Under these conditions, the calculations of "apparent" kinetic rate constants, based on Michaelis-Menten kinetics, can superficially show a dependence on flow rate conditions even in the absence of mass-transfer resistances. High substrate conversions are shown to depend on flow rate. A kinetic model based on known mechanisms of the alkaline phosphatase enzyme reaction is tested to predict the measurements for high substrate conversion. The study provides a basis for appropriate use of mass-transfer and reaction arguments in successful application of enzymatic microreactors.
Pubmed: ABSTRACT Article (Kermekchiev2003) Kermekchiev, M.B.; Tzekov, A. & Barnes, W.M. Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR Nucleic Acids Res, 2003, 31, 6139-47 Abstract: Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.
Pubmed: ABSTRACT Article (Kerr2000) Kerr, M.K.; Martin, M. & Churchill, G.A. Analysis of variance for gene expression microarray data. J Comput Biol, 2000, 7, 819-837 Abstract: Spotted cDNA microarrays are emerging as a powerful and cost-effective tool for large-scale analysis of gene expression. Microarrays can be used to measure the relative quantities of specific mRNAs in two or more tissue samples for thousands of genes simultaneously. While the power of this technology has been recognized, many open questions remain about appropriate analysis of microarray data. One question is how to make valid estimates of the relative expression for genes that are not biased by ancillary sources of variation. Recognizing that there is inherent "noise" in microarray data, how does one estimate the error variation associated with an estimated change in expression, i.e., how does one construct the error bars? We demonstrate that ANOVA methods can be used to normalize microarray data and provide estimates of changes in gene expression that are corrected for potential confounding effects. This approach establishes a framework for the general analysis and interpretation of microarray data.
Pubmed: ABSTRACT Article (Khandurina2002) Khandurina, J. & Guttman, A. Bioanalysis in microfluidic devices J Chromatogr A, 2002, 943, 159-83 Abstract: Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) offer highly efficient platforms for simultaneous analysis of a large number of biologically important molecules, possessing great potential for genome, proteome and metabolome studies. Development and implementation of microfluidic-based bioanalytical tools involves both established and evolving technologies, including microlithography, micromachining, micro-electromechanical systems technology and nanotechnology. This article provides an overview of the latest developments in the key device subject areas and the basic interdisciplinary technologies. Important aspects of DNA and protein analysis, interfacing issues and system integration are all thoroughly discussed, along with applications for this novel ynergizedtechnology in high-throughput separations of biologically important molecules. This review also gives a better understanding of how to utilize these technologies as well as to provide appropriate technical solutions to problems perceived as being more fundamental.
Pubmed: ABSTRACT Article (Khandurina2000) Khandurina, J.; McKnight, T.E.; Jacobson, S.C.; Waters, L.C.; Foote, R.S. & Ramsey, J.M. Integrated system for rapid PCR-based DNA analysis in microfluidic devices. Anal Chem, 2000, 72, 2995-3000 Abstract: An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.
Pubmed: ABSTRACT Article (Kierzek2002) Kierzek, A.M. STOCKS: STOChastic Kinetic Simulations of biochemical systems with Gillespie algorithm Bioinformatics, 2002, 18, 470-81 Abstract: MOTIVATION: The availability of a huge amount of molecular data concerning various biochemical reactions provoked numerous attempts to study the dynamics of cellular processes by means of kinetic models and computer simulations. Biochemical processes frequently involve small numbers of molecules (e.g. a few molecules of a transcriptional regulator binding to one oleculeof a DNA regulatory region). Such reactions are subject to significant stochastic fluctuations. Monte Carlo methods must be employed to study the functional consequences of the fluctuations and simulate processes that cannot be modelled by continuous fluxes of matter. This provides the motivation to develop software dedicated to Monte Carlo simulations of cellular processes with the rigorously proven Gillespie algorithm. RESULTS: STOCKS, software for the stochastic kinetic simulation of biochemical processes is presented. The program uses a rigorously derived Gillespie algorithm that has been shown to be applicable to the study of prokaryotic gene expression. Features dedicated to the study of cellular processes are implemented, such as the possibility to study a process in the range of several cell generations with the application of a simple cell division model. Taking expression of Escherichia coli beta-galactosidase as an example, it is shown that the program is able to simulate systems composed of reactions varying in several orders of magnitude by means of reaction rates and the numbers of molecules involved. AVAILABILITY: The software is available at ftp://ibbrain.ibb.waw.pl/stocksand http://www.ibb.waw.pl/stocks. Supplementary information: Parameters of the model of prokaryotic gene expression are available in example files of software distribution.
Pubmed: ABSTRACT Article (Kim2003) Kim, S.; Ruparel, H.D.; Gilliam, T.C. & Ju, J. Digital genotyping using molecular affinity and mass spectrometry Nat Rev Genet, 2003, 4, 1001-8 Abstract: The goal of DNA sequencing and genotyping is to efficiently generate accurate high-throughput digital genetic information that unambiguously identifies sources of genetic variation and clearly distinguishes heterozygous from homozygous variants. Recent advances in mass-spectrometry-based DNA sequencing and genotyping bode well for meeting these criteria. Pilot studies show that these recently developed approaches allow unambiguous multiplex detection of heterozygous variants and the identification of deletion and insertion variants.
Pubmed: ABSTRACT Article (Kim2006) Kim, S.; Song, Y.; Skipper, P. & Han, J. Electrohydrodynamic Generation and Delivery of Monodisperse Picoliter Droplets Using a Poly(dimethylsiloxane) Microchip Analytical Chemistry, 2006, 78, 8011-8019
PDF: Kim2006.pdf Article (Kirk2002) Kirk, B.W.; Feinsod, M.; Favis, R.; Kliman, R.M. & Barany, F. Single nucleotide polymorphism seeking long term association with complex disease Nucleic Acids Res, 2002, 30, 3295-311 Abstract: Successful investigation of common diseases requires advances in our understanding of the organization of the genome. Linkage disequilibrium provides a theoretical basis for performing candidate gene or whole-genome association studies to analyze complex disease. However, to constructively interrogate SNPs for these studies, technologies with sufficient throughput and sensitivity are required. A plethora of suitable and reliable methods have been developed, each of which has its own unique advantage. The characteristics of the most promising genotyping and polymorphism scanning technologies are presented. These technologies are examined both in the context of complex disease investigation and in their capacity to face the unique physical and molecular challenges (allele amplification, loss of heterozygosity and stromal contamination) of solid tumor research.
Pubmed: ABSTRACT Article (Klerks2006) Klerks, M.M.; van Bruggen, A.H.C.; Zijlstra, C. & Donnikov, M. Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control. Appl Environ Microbiol, 2006, 72, 3879-3886 Abstract: This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.
Pubmed: ABSTRACT Article (Kling2006) Kling, J. Moving diagnostics from the bench to the bedside. Nat Biotechnol, 2006, 24, 891-893
Pubmed: ABSTRACT Article (Knoblauch2002) Knoblauch, H.; Bauerfeind, A.; Krahenbuhl, C.; Daury, A.; Rohde, K.; Bejanin, S.; Essioux, L.; Schuster, H.; Luft, F.C. & Reich, J.G. Common haplotypes in five genes influence genetic variance of LDL and HDL cholesterol in the general population Hum Mol Genet, 2002, 11, 1477-85 Abstract: We studied the association between common haplotypes in six relevant lipid metabolism genes with plasma lipid levels. We selected single-nucleotide polymorphisms (SNPs) in the cholesterol ester transfer protein (CETP), lipoprotein lipase (LPL), hepatic triglyceride lipase (HL), low-density lipoprotein cholesterol receptor (LDLR), apolipoprotein E (ApoE) and lecithin-cholesterol acyltransferase (LCAT) genes, and studied 732 individuals from 184 German families. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) were similar to those reported in other European and American populations. Haplotypes derived from SNP combinations resulted in more significance and of a higher degree than did single SNPs in the genotype-phenotype association analysis. Reduction of the polygenic variance attributable to haplotypes was estimated using variance components analysis. Under the biometrical genetic model, allelic association of haplotypes was highly significant for HDL, LDL and the LDL/HDL ratio. The residual kinship correlation was reduced accordingly. The ApoE gene had a strong effect on trait variation; however, the other genes also contributed substantially. An epistatic interaction could not be demonstrated in this sample. The data are consistent with the notion that common genetic variants influence common traits.
Pubmed: ABSTRACT Article (Koh2003) Koh, C.G.; Tan, W.; Zhao, M.Q.; Ricco, A.J. & Fan, Z.H. Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection Anal Chem, 2003, 75, 4591-8 Abstract: An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.
Pubmed: ABSTRACT Article (Kojima2005) Kojima, T.; Takei, Y.; Ohtsuka, M.; Kawarasaki, Y.; Yamane, T. & Nakano, H. PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets. Nucleic Acids Res, 2005, 33, e150 Abstract: We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.
Pubmed: ABSTRACT Article (Kokocinski2003) Kokocinski, F.; Wrobel, G.; Hahn, M. & Lichter, P. QuickLIMS: facilitating the data management for DNA-microarray fabrication Bioinformatics, 2003, 19, 283-4 Abstract: Summary: QuickLIMS is a Microsoft Access-based laboratory information and management system, capable of processing all information for microarray production. The program's operational flow is protocol-based, dynamically adapting to changes of the process. It interacts with the laboratory robot and with other database systems over the network, and it represents a complete solution for the management of the entire manufacturing process. Availability and supplementary information: http://www.dkfz-heidelberg.de/kompl_genome/Other/QuickLims/index.html Contact: f.kokocinski@dkfz.def.kokocinski@dkfz.de
Pubmed: ABSTRACT Article (Kokoris2000) Kokoris, M.; Dix, K.; Moynihan, K.; Mathis, J.; Erwin, B.; Grass, P.; Hines, B. & Duesterhoeft, A. High-throughput SNP genotyping with the Masscode system Mol Diagn, 2000, 5, 329-40 Abstract: QIAGEN Genomics, Inc, has developed the Masscode tagging system for DNA labeling and detection. In this application, the Masscode system is described as applied to high-throughput single-nucleotide polymorphism (SNP) genotyping. The labeling system is based on a small-molecular-weight tag that is covalently attached through a photocleavable linker to a DNA oligonucleotide. The tagged oligonucleotide is used as a primer in an allele-specific PCR SNP discrimination assay. The allele-specific amplicons are differentiated through their Masscode tag assignments. After a photolysis step to cleave the tags from the amplicon, the samples are introduced into a single quadrupole mass spectrometry detection system for analysis. Genotyping determinations are based on the relative proportions of the paired allele tags. The system has a lower limit of detection in the femtomolar range (10(-15) M). At present, 30 different Masscode tags may be used simultaneously in a multiplex fashion to routinely provide more than 40,000 SNP genotyping measurements daily. Further developments will allow for the simultaneous detection of several hundred tags.
Pubmed: ABSTRACT Article (Kopp1997) Kopp, M.U.; Crabtree, H.J. & Manz, A. Developments in technology and applications of microsystems Curr Opin Chem Biol, 1997, 1, 410-9 Abstract: In the past year, microchips as applied to miniaturised total analysis systems, or microTAS, have benefited from technological improvements in their fabrication and been applied to analysis in many different biological areas. From a technological perspective, salient work includes fast, cheap and easy micromachining in polymers and integrated optical detection. From the bioapplications perspective, advances in DNA and protein separations, cell manipulations, immunoassays and polymerase chain reaction using on-chip electrophoretic separation stand out.
Pubmed: ABSTRACT Article (Kopp1998) Kopp, M.U.; Mello, A.J. & Manz, A. Chemical amplification: continuous-flow PCR on a chip Science, 1998, 280, 1046-8 Abstract: A micromachined chemical amplifier was successfully used to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device is analogous to an electronic amplifier and relies on the movement of sample through thermostated temperature zones on a glass microchip. Input and output of material (DNA) is continuous, and amplification is independent of input concentration. A 20-cycle PCR amplification of a 176-base pair fragment from the DNA gyrase gene of Neisseria gonorrhoeae was performed at various flow rates, resulting in total reaction times of 90 seconds to 18.7 minutes.
Pubmed: ABSTRACT Article (Korn2003) Korn, K.; Gardellin, P.; Liao, B.; Amacker, M.; Bergstrom, A.; Bjorkman, H.; Camacho, A.; Dorhofer, S.; Dorre, K.; Enstrom, J.; Ericson, T.; Favez, T.; Gosch, M.; Honegger, A.; Jaccoud, S.; Lapczyna, M.; Litborn, E.; Thyberg, P.; Winter, H. & Rigler, R. Gene expression analysis using single molecule detection Nucleic Acids Res, 2003, 31, e89 Abstract: Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.
Pubmed: ABSTRACT Article (Kraus2006) Kraus, T.; Verpoorte, E.; Linder, V.; Franks, W.; Hierlemann, A.; Heer, F.; Hafizovic, S.; Fujii, T.; de Rooij, N.F. & Koster, S. Characterization of a microfluidic dispensing system for localised stimulation of cellular networks. Lab Chip, 2006, 6, 218-229 Abstract: We present a 3-D microfluidic device designed for localized drug delivery to cellular networks. The device features a flow cell comprising a main channel for nutrient delivery as well as multiple channels for drug delivery. This device is one key component of a larger, fully integrated system now under development, based upon a microelectrode array (MEA) with on-chip CMOS circuitry for recording and stimulation of electrogenic cells (e.g. neurons, cardiomyocytes). As a critical system unit, the microfluidics must be carefully designed and characterized to ensure that candidate drugs are delivered to specific regions of the culture at known concentrations. Furthermore, microfluidic design and functionality is dictated by the size, geometry, and material/electrical characteristics of the CMOS MEA. Therefore, this paper reports on the design considerations and fabrication of the flow cell, including theoretical and experimental analysis of the mass transfer properties of the nutrient and drug flows, which are in good agreement with one another. To demonstrate proof of concept, the flow cell was mounted on a dummy CMOS chip, which had been plated with HL-1 cardiomyocytes. A test chemical compound was delivered to the cell culture in a spatially resolved manner. Envisioned applications of this stand-alone system include simultaneous toxicological testing of multiple compounds and chemical stimulation of natural neural networks for neuroscience investigations.
Pubmed: ABSTRACT Article (Kricka2002) Kricka, L.J. Stains, labels and detection strategies for nucleic acids assays Ann Clin Biochem, 2002, 39, 114-29 Abstract: Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the final decade of the 20th century (1991-2000). During this period, important families of cyanine dyes were developed for sensitive detection of double-stranded DNA, single-stranded DNA, and oligo(deoxy)nucleotides in gels and in solution, and families of energy transfer primers were produced for DNA sequencing applications. The continuing quest for improved labels for hybridization assays has produced a series of candidate labels including genes encoding enzymes, microparticles (e.g. quantum dots, nanocrystals, phosphors), and new examples of the fluorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. firefly luciferase mutants). Label detection technologies for use in northern and southern blotting assays have focused on luminescent methods, particularly enhanced chemiluminescence for peroxidase labels and adamantyl 1,2-dioxetanes for alkaline phosphatase labels. Sets of labels have been selected to meet the demands of multicolour assays (e.g. four-colour sequencing and single nucleotide primer extension assays). Non-separation assay formats have emerged based on fluorescence polarization, fluorescence energy transfer (TaqMan, molecular beacons) and channelling principles. Microanalytical devices (microchips), high-throughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and fluorescent methods currently dominate in many of these applications.
Pubmed: ABSTRACT Article (Kricka2002a) Kricka, L.J.; Fortina, P.; Panaro, N.J.; Wilding, P.; Alonso-Amigo, G. & Becker, H. Fabrication of plastic microchips by hot embossing Lab Chip, 2002, 2, 1-4 Abstract: Plastic microchips with microchannels (100 microm wide, 40 microm deep) of varying designs have been fabricated in polymethylmethacrylate by a hot embossing process using an electroform tool produced starting with silicon chip masters. Hot-embossed chips were capped with a polymethylmethacrylate top using a proprietary solvent bonding process. Holes were drilled through the top of the chip to allow access to the channels. The chips were tested with fluid and shown to fill easily. The seal between the top of the chip and the hot embossed base was effective, and there was no leakage from the channels when fluid was pumped through the microchannels. The chips were also tested with a semen sample and the plastic chip performed identically to the previous silicon-glass and glass versions of the chip. This microfabrication technique offers a viable and potentially high-volume low cost production method for fabricating transparent microchips for analytical applications.
Pubmed: ABSTRACT Article (Kricka2003) Kricka, L.J. & Wilding, P. Microchip PCR Anal Bioanal Chem, 2003, 377, 820-5 Abstract: Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties
Pubmed: ABSTRACT Article (Krishnan2001) Krishnan, M.; Namasivayam, V.; Lin, R.; Pal, R. & Burns, M.A. Microfabricated reaction and separation systems Curr Opin Biotechnol, 2001, 12, 92-8 Abstract: Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated ab-on-a-chipsystems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.
Pubmed: ABSTRACT Other (Kroke2004) Kroke, A.; Hinrichs, S. & Dahl, A. P1.05: Extension of the internet-based assessment tool resource EPIDAS with instruments for the assessment of physical activity in children Biometrical Journal, 2004, 46, 98 Abstract: No Abstract
DOI: http://dx.doi.org/10.1002/bimj.200490153 Article (Kruglyak2001) Kruglyak, L. & Nickerson, D.A. Variation is the spice of life. Nat Genet, 2001, 27, 234-236
Pubmed: ABSTRACT Article (Kubista2006) Kubista, M.; Andrade, J.M.; Bengtsson, M.; Forootan, A.; Jonák, J.; Lind, K.; Sindelka, R.; Sjöback, R.; Sjögreen, B.; Strömbom, L.; Ståhlberg, A. & Zoric, N. The real-time polymerase chain reaction. Mol Aspects Med, 2006, 27, 95-125 Abstract: The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.
Pubmed: ABSTRACT Article (Kuimelis1997) Kuimelis, R.G.; Livak, K.J.; Mullah, B. & Andrus, A. Structural analogues of TaqMan probes for real-time quantitative PCR Nucleic Acids Symp Ser, 1997, 255-6 Abstract: We have prepared and evaluated a series of structural analogues of TaqMan PCR probes in an effort to identify second-generation probes with improved physical properties and performance. Modifications have included non-nucleosidic dye linkers, 2O-Me RNA substitutions, and pyrimidine C5-propyne substitutions.
Pubmed: ABSTRACT Article (Kunkel2000) Kunkel, T.A. & Bebenek, K. DNA replication fidelity Annu Rev Biochem, 2000, 69, 497-529 Abstract: DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.
Pubmed: ABSTRACT Article (Kwok2001) Kwok, P.Y. Methods for genotyping single nucleotide polymorphisms Annu Rev Genomics Hum Genet, 2001, 2, 235-58 Abstract: One of the fruits of the Human Genome Project is the discovery of millions of DNA sequence variants in the human genome. The majority of these variants are single nucleotide polymorphisms (SNPs). A dense set of SNP markers opens up the possibility of studying the genetic basis of complex diseases by population approaches. In all study designs, a large number of individuals must be genotyped with a large number of markers. In this review, the current status of SNP genotyping is discussed in terms of the mechanisms of allelic discrimination, the reaction formats, and the detection modalities. A number of genotyping methods currently in use are described to illustrate the approaches being taken. Although no single genotyping method is ideally suited for all applications, a number of good genotyping methods are available to meet the needs of many study designs. The challenges for SNP genotyping in the near future include increasing the speed of assay development, reducing the cost of the assays, and performing multiple assays in parallel. Judging from the accelerated pace of new method development, it is hopeful that an ideal SNP genotyping method will be developed soon.
Pubmed: ABSTRACT Article (Kwok1989) Kwok, S. & Higuchi, R. Avoiding false positives with PCR Nature, 1989, 339, 237-8 Abstract: The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.
Pubmed: ABSTRACT Article (Koehler2005) Köhler, J.M. & Henkel, T. Chip devices for miniaturized biotechnology. Appl Microbiol Biotechnol, 2005, 69, 113-125 Abstract: Chip devices were introduced in chemistry and molecular biology to improve the read-out of information from molecular systems by efficient analytical procedures and to organize automated experiments. Biochips and chip reactor systems are of interest for cellular processes, too, and can be regarded as components in interfaces for the information exchange between living nature and digital electronic systems. In this minireview, different types of chip reactors for biotechnological applications like nanotiterplates, chip thermocyclers and devices for segmented flow operations are discussed. Finally, an outlook is given on the application of chip reactor systems, which are promising tools for automated experiments with highly parallelized screening procedures, for artificial microcompartmentation, cell analogue systems, micro-ecological studies, investigations on modulated morphogenesis, and for a bioanalogue molecular nanotechnology.
Pubmed: ABSTRACT Article (Lagally2005) Lagally, E. & Soh, H. Integrated Genetic Analysis Microsystems Critical Reviews in Solid State and Material Sciences, 2005, 30, 207-233 Abstract: The advent of integrated microsystems for genetic analysis allows the acquisition of information at unprecedented length and time scales. The convergence of molecular biology, chemistry, physics, and materials science is required for their design and construction. The utility of the microsystems originates from increased analysis speed, lower analysis cost, and higher parallelism leading to increased assay throughput. In addition, when fully integrated, this technology will enable portable systems for high-speed in situ analyses, permitting a new standard in disciplines such as clinical chemistry, personalized medicine, forensics, biowarfare detection, and epidemiology. This article presents an overview of the recent history of integrated genetic analysis microsystems with an emphasis on materials aspects, and provides a perspective on current developments and future prospects.
PDF: Lagally2005.pdf Article (Lagally2001a) Lagally, E.T.; Emrich, C.A. & Mathies, R.A. Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis. Lab Chip, 2001, 1, 102-107 Abstract: A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.
Pubmed: ABSTRACT Article (Lagally2004a) Lagally, E.T. & Mathies, R.A. Integrated genetic analysis microsystems Journal of Physics D: Applied Physics, 2004, 37, R245-R261 Abstract: With the completion of the Human Genome Project and the ongoing DNA sequencing of the genomes of other animals, bacteria, plants and others, a wealth of new information about the genetic composition of organisms has become available. However, as the demand for sequence information grows, so does the workload required both to generate this sequence and to use it for targeted genetic analysis. Microfabricated genetic analysis systems are well poised to assist in the collection and use of these data through increased analysis speed, lower analysis cost and higher parallelism leading to increased assay throughput. In addition, such integrated microsystems may point the way to targeted genetic experiments on single cells and in other areas that are otherwise very difficult. Concomitant with these advantages, such systems, when fully integrated, should be capable of forming portable systems for high-speed in situ analyses, enabling a new standard in disciplines such as clinical chemistry, forensics, biowarfare detection and epidemiology. This review will discuss the various technologies available for genetic analysis on the microscale, and efforts to integrate them to form fully functional robust analysis devices.
PDF: Lagally2004a.pdf Article (Lagally2001) Lagally, E.T.; Medintz, I. & Mathies, R.A. Single-molecule DNA amplification and analysis in an integrated microfluidic device Anal Chem, 2001, 73, 565-70 Abstract: Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.
Pubmed: ABSTRACT Article (Lagally2004) Lagally, E.T.; Scherer, J.R.; Blazej, R.G.; Toriello, N.M.; Diep, B.A.; Ramchandani, M.; Sensabaugh, G.F.; Riley, L.W. & Mathies, R.A. Integrated portable genetic analysis microsystem for pathogen/infectious disease detection. Anal Chem, 2004, 76, 3162-3170 Abstract: An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.
Pubmed: ABSTRACT Article (Landegren1988) Landegren, U.; Kaiser, R.; Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science, 1988, 241, 1077-1080 Abstract: An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.
Pubmed: ABSTRACT Article (Lander1999) Lander, E.S. Array of hope. Nat Genet, 1999, 21, 3-4
Pubmed: ABSTRACT Article (Lander2001) Lander, E.S.; Linton, L.M.; Birren, B.; Nusbaum, C.; Zody, M.C.; Baldwin, J.; Devon, K.; Dewar, K.; Doyle, M.; FitzHugh, W.; Funke, R.; Gage, D.; Harris, K.; Heaford, A.; Howland, J.; Kann, L.; Lehoczky, J.; LeVine, R.; McEwan, P.; McKernan, K.; Meldrim, J.; Mesirov, J.P.; Miranda, C.; Morris, W.; Naylor, J.; Raymond, C.; Rosetti, M.; Santos, R.; Sheridan, A.; Sougnez, C.; Stange-Thomann, N.; Stojanovic, N.; Subramanian, A.; Wyman, D.; Rogers, J.; Sulston, J.; Ainscough, R.; Beck, S.; Bentley, D.; Burton, J.; Clee, C.; Carter, N.; Coulson, A.; Deadman, R.; Deloukas, P.; Dunham, A.; Dunham, I.; Durbin, R.; French, L.; Grafham, D.; Gregory, S.; Hubbard, T.; Humphray, S.; Hunt, A.; Jones, M.; Lloyd, C.; McMurray, A.; Matthews, L.; Mercer, S.; Milne, S.; Mullikin, J.C.; Mungall, A.; Plumb, R.; Ross, M.; Shownkeen, R.; Sims, S.; Waterston, R.H.; Wilson, R.K.; Hillier, L.W.; McPherson, J.D.; Marra, M.A.; Mardis, E.R.; Fulton, L.A.; Chinwalla, A.T.; Pepin, K.H.; Gish, W.R.; Chissoe, S.L.; Wendl, M.C.; Delehaunty, K.D.; Miner, T.L.; Delehaunty, A.; Kramer, J.B.; Cook, L.L.; Fulton, R.S.; Johnson, D.L.; Minx, P.J.; Clifton, S.W.; Hawkins, T.; Branscomb, E.; Predki, P.; Richardson, P.; Wenning, S.; Slezak, T.; Doggett, N.; Cheng, J.F.; Olsen, A.; Lucas, S.; Elkin, C.; Uberbacher, E.; Frazier, M. & others Initial sequencing and analysis of the human genome Nature, 2001, 409, 860-921 Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
Pubmed: ABSTRACT Article (Lao2000) Lao, A.I.K.; Lee, T.M.H.; Hsing, I. & Ip, N.Y. Precise temperature control of microfluidic chamber for gas and liquid phase reactions Sensors and Actuators A: Physical, 2000, 84, 11-17 Abstract: A silicon-based micromachined fluidic chamber with integrated platinum heaters and sensors has been developed and thermally well characterized. This device is highly applicable to both gas and liquid phase reactions that require excellent thermal control. The chamber is thermally isolated from the bulk silicon by a thin silicon nitride membrane resulting in low power consumption. The digitally feedback controlled device demonstrates the ability of precise temperature control, excellent temperature uniformity, rapid heating and cooling. These thermal characteristics are ascribable to the success of miniaturized reaction chamber and Micro Total Analysis Systems ([mu]TAS). In addition, gain scheduling control algorithm in conjunction with normal feedback proportional and integral (PI) scheme was implemented to provide better thermal cycling performance. 3D numerical simulation was also conducted to map the spatial temperature distribution within the miniaturized fluidic device. Simulation results and experimental data show good agreements.
PDF: Lao2000.pdf Article (Laser2004) Laser, D.J. & Santiago, J.G. TOPICAL REVIEW: A review of micropumps Journal of Micromechanics and Microengineering, 2004, 14, 35-+
Article (Latorra2003) Latorra, D.; Campbell, K.; Wolter, A. & Hurley, J.M. Enhanced allele-specific PCR discrimination in SNP genotyping using 3locked nucleic acid (LNA) primers Hum Mutat, 2003, 22, 79-85 Abstract: The specificity and reliability of locked nucleic acid (LNA) substitution at the 3position of allele-specific PCR (AS-PCR) primers for SNP detection was investigated in direct comparison to DNA primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3LNA mismatch combinations were tested in triplicate with the plasmid target. LNA primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3LNA primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3LNA primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.
Pubmed: ABSTRACT Article (Laval2000) Laval, J.M.; Mazeran, P.E. & Thomas, D. Nanobiotechnology and its role in the development of new analytical devices Analyst, 2000, 125, 29-33 Abstract: Physical methods of molecule observation and manipulation will prove useful, not only as research tools for investigating biomolecular structure and behavior, but also for the creation of nanostructures. Supramolecular and self-assembling structures are able to generate nanostructures, with many such systems being of biological origin. They form the interface between nanotechnology and biotechnology. Whereas biotechnological processes usually involve populations of cells or molecules, nanotechnological methods operate at the level of individual molecule manipulation. This article considers what advances have been made through cross-fertilisation between nanotechnology and biotechnology to develop for the next millennium new analytical tools at the microscale, using nanostructures as the sensitive part and with the ability to detect individual molecules.
Pubmed: ABSTRACT Article (Lay1997) Lay, M.J. & Wittwer, C.T. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR Clin Chem, 1997, 43, 2262-7 Abstract: A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5 in the presence of a 3fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 degrees C; if the single-base mutation is present, the probe melts at 57 degrees C. Concurrent amplification and analysis from genomic DNA takes 20-45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
Pubmed: ABSTRACT Article (Le2002) Le Hellard, S.; Ballereau, S.J.; Visscher, P.M.; Torrance, H.S.; Pinson, J.; Morris, S.W.; Thomson, M.L.; Semple, C.A.; Muir, W.J.; Blackwood, D.H.; Porteous, D.J. & Evans, K.L. SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis Nucleic Acids Res, 2002, 30, e74 Abstract: We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.
Pubmed: ABSTRACT Article (Le2001) Le Novere, N. MELTING, computing the melting temperature of nucleic acid duplex Bioinformatics, 2001, 17, 1226-7 Abstract: MELTING computes the enthalpy and entropy of an oligonucleotide duplex helix-coil transition, and then its melting temperature. The program uses the method of nearest-neighbours. The set of thermodynamic parameters can be easily customized. The program provides several correction methods for the concentration of salt. MELTING is a free program, available at no cost and open-source. Perl scripts are provided to show how MELTING can be used to construct more ambitious programs. AVAILABILITY: MELTING is available for several platforms (http://www.pasteur.fr/recherche/unites/neubiomol/meltinghome.html) and is accessible via a www server (http://bioweb.pasteur.fr/seqanal/interfaces/melting.html). CONTACT: nl223@cus.cam.ac.uk
Pubmed: ABSTRACT Article (Leamon2003) Leamon, J.H.; Lee, W.L.; Tartaro, K.R.; Lanza, J.R.; Sarkis, G.J.; deWinter, A.D.; Berka, J. & Lohman, K.L. A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions Electrophoresis, 2003, 24, 3769-77 Abstract: We demonstrate successful, simultaneous polymerase chain reaction (PCR) amplification of up to 300 000 discrete reactions in a novel platform, the PicoTiterPlate. In addition to elevated throughput, the PicoTiterPlate based amplifications (PTPCR) can be performed in extremely small volumes: individual reactions volumes are as low as 39.5 pL, with a total 15.3 microL reaction volume for the entire PicoTiterPlate. The bulk PTPCR product can be recovered and assayed with real-time PCR, or discrete PTPCR products can be driven to solid supports, enabling downstream applications such as translation/transcription or sequencing.
Pubmed: ABSTRACT Article (Lee2007) Lee, H.; Liu, Y.; Ham, D. & Westervelt, R.M. Integrated cell manipulation system--CMOS/microfluidic hybrid. Lab Chip, 2007, 7, 331-337 Abstract: Manipulation of biological cells using a CMOS/microfluidic hybrid system is demonstrated. The hybrid system starts with a custom-designed CMOS (complementary metal-oxide semiconductor) chip fabricated in a semiconductor foundry. A microfluidic channel is post-fabricated on top of the CMOS chip to provide biocompatible environments. The motion of individual biological cells that are tagged with magnetic beads is directly controlled by the CMOS chip that generates microscopic magnetic field patterns using an on-chip array of micro-electromagnets. Furthermore, the CMOS chip allows high-speed and programmable reconfiguration of the magnetic fields, substantially increasing the manipulation capability of the hybrid system. Extending from previous work that verified the concept of the hybrid system, this paper reports a set of manipulation experiments with biological cells, which further confirms the advantage of the hybrid approach. To enhance the biocompatibility of the system, the microfluidic channel is redesigned and the temperature of the device is monitored by on-chip sensors. Combining microelectronics and microfluidics, the CMOS/microfluidic hybrid system presents a new model for a cell manipulation platform in biological and biomedical applications.
Pubmed: ABSTRACT Article (Lee2004) Lee, I.; Dombkowski, A.A. & Athey, B.D. Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray Nucleic Acids Res, 2004, 32, 681-90 Abstract: Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all possible mismatch probes is impractical. Our study provides guidelines for designing non-perfectly matched DNA probes to target DNA sequences as desired throughout the genome. These guidelines are based on the analysis of hybridization data between perfectly matched and non-perfectly matched DNA sequences (single-point or double-point mutated) calculated in silico. Large changes in hybridization temperature predicted by these guidelines for non-matched oligonucleotides fit independent experimental data very well. Applying the guidelines to find oligonucleotide microarray probes for P450 genes, we confirmed the ability of our point mutation method to differentiate the individual genes in terms of thermodynamic calculations of hybridization and sequence similarity.
Pubmed: ABSTRACT Article (Lee1993) Lee, L.G.; Connell, C.R. & Bloch, W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res, 1993, 21, 3761-3766 Abstract: Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.
Pubmed: ABSTRACT Article (Lee2003) Lee, T.M.; Carles, M.C. & Hsing, I.M. Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection Lab Chip, 2003, 3, 100-5 Abstract: Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.
Pubmed: ABSTRACT Article (Legendre2006) Legendre, L.A.; Bienvenue, J.M.; Roper, M.G.; Ferrance, J.P. & Landers, J.P. A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples. Anal Chem, 2006, 78, 1444-1451 Abstract: A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.
Pubmed: ABSTRACT Article (Lekanne2002) Lekanne Deprez, R.H.; Fijnvandraat, A.C.; Ruijter, J.M. & Moorman, A.F. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions Anal Biochem, 2002, 307, 63-9 Abstract: The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using SYBR green I and the LightCycler that better permits accurate quantification of mRNAs. Omission of dithiothreitol from the cDNA synthesis reaction was found to be crucial. This resulted in a lower cycle number at which the cDNA level is determined (C(T) value), steeper amplification curves, and removal of background fluorescence in the subsequent PCR. In addition, the choice of the cDNA priming oligo can improve detection sensitivity even further. In contrast to hexamer primer usage, both gene-specific and oligo-dT(VN) priming were very efficient and accurate, with gene-specific priming being the most sensitive. Finally, accurate quantification of mRNAs by real-time PCR using SYBR green I requires verification of the specificity of PCR by both melting curve and gel analysis.
Pubmed: ABSTRACT Article (Lenstra1995) Lenstra, J.A. The applications of the polymerase chain reaction in the life sciences Cell Mol Biol (Noisy-le-grand), 1995, 41, 603-14 Abstract: The polymerase chain reaction (PCR) allows the in vitro amplification of DNA fragments starting with tiny amounts of biological sample and oligonucleotide primers derived from sequence data. Since the technique is fast and easy, PCR has taken the DNA-technology to the routine laboratoria. We present a survey of the following applications of PCR: 1) The amplification of gene fragments as fast alternative of cloning. 2) The modification of DNA fragments. 3) The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping. 4) DNA analysis of arachaeological specimens. 5) The detection of mutations relevant for inherited diseases, malignant transformation or tissue typing. 6) The analysis of genetic markers for forensic applications, for paternity testing and for the mapping of hereditary traits. 7) The species-specific amplification of DNA segments between interspersed-repeat elements. 8) The study of gene expression.
Pubmed: ABSTRACT Article (Leone1998) Leone, G.; van Schijndel, H.; van Gemen, B.; Kramer, F.R. & Schoen, C.D. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res, 1998, 26, 2150-2155 Abstract: Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities.
Pubmed: ABSTRACT Article (Lesage2002) Lesage, S.; Zouali, H.; Cezard, J.P.; Colombel, J.F.; Belaiche, J.; Almer, S.; Tysk, C.; C, O.'.; Gassull, M.; Binder, V.; Finkel, Y.; Modigliani, R.; Gower-Rousseau, C.; Macry, J.; Merlin, F.; Chamaillard, M.; Jannot, A.S.; Thomas, G. & Hugot, J.P. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease Am J Hum Genet, 2002, 70, 845-57 Abstract: CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.
Pubmed: ABSTRACT Article (Letondal2001) Letondal, C. A Web interface generator for molecular biology programs in Unix Bioinformatics, 2001, 17, 73-82 Abstract: MOTIVATION: Almost all users encounter problems using sequence analysis programs. Not only are they difficult to learn because of the parameters, syntax and semantic, but many are different. That is why we have developed a Web interface generator for more than 150 molecular biology command-line driven programs, including: phylogeny, gene prediction, alignment, RNA, DNA and protein analysis, motif discovery, structure analysis and database searching programs. The generator uses XML as a high-level description language of the legacy software parameters. Its aim is to provide users with the equivalent of a basic Unix environment, with program combination, customization and basic scripting through macro registration. RESULTS: The program has been used for three years by about 15000 users throughout the world; it has recently been installed on other sites and evaluated as a standard user interface for EMBOSS programs.
Pubmed: ABSTRACT Article (Leung2003) Leung, Y.F. & Cavalieri, D. Fundamentals of cDNA microarray data analysis. Trends Genet, 2003, 19, 649-659 Abstract: Microarray technology is a powerful approach for genomics research. The multi-step, data-intensive nature of this technology has created an unprecedented informatics and analytical challenge. It is important to understand the crucial steps that can affect the outcome of the analysis. In this review, we provide an overview of the contemporary trend on various main analysis steps in the microarray data analysis process, which includes experimental design, data standardization, image acquisition and analysis, normalization, statistical significance inference, exploratory data analysis, class prediction and pathway analysis, as well as various considerations relevant to their implementation.
Pubmed: ABSTRACT Article (Lewis1998) Lewis, J.K.; Krone, J.R. & Nelson, R.W. Mass spectrometric methods for evaluating point mutations Biotechniques, 1998, 24, 102, 104, 106, 108 passim Abstract: Two methods for internally calibrating spectra resulting from the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of partially digested proteins are described. Partial digestion of proteins results in a large number of ion signals present in the MALDI-TOF mass spectrum, which in turn represent a significant over-sampling of each amino acid present in the analyte. This over-sampling allows ion signals of undisputed origin to be used as internal calibrants for the evaluation of fragments suspected to contain point mutations. Correlated with the correct amino acid sequence, the mass values of all ion signals (calibrants and analytes) are observed to fall into a single low-error data set. Conversely, empirically derived data applied to an incorrect sequence split the data into subsets of different errors. The methods take advantage of the self-consistent nature of data generated during the enzymatic mass mapping of proteins using MALDI-TOF, and they aid in the rapid, sensitive and accurate evaluation of point mutations present in proteins.
Pubmed: ABSTRACT Article (Li2004) Li, B.; Kadura, I.; Fu, D.J. & Watson, D.E. Genotyping with TaqMAMA Genomics, 2004, 83, 311-20 Abstract: TaqMAMA combines the quantitative strengths of TaqMan with the allele-specific PCR of MAMA. In this article we develop TaqMAMA as a technique for screening human DNA samples for known genetic polymorphisms. In the first set of experiments, plasmids that model all types of genetic polymorphisms were used to understand the relationship between TaqMAMA primer/template mismatches and their strength of allelic discrimination. These data can be used to improve allelic discrimination of other primer extension genotyping methodologies through directed use of nucleotide mismatches. We used the data to derive a guide for TaqMAMA primer design and DNA strand selection for TaqMAMA genotyping assays. The guide was then used to develop assays for 11 known and novel human genetic polymorphisms. Genotypes were assigned quickly and accurately in all cases. TaqMAMA genotyping assays require minimal development time, have a high probability of success, produce reliable data that are straightforward to analyze, and are very cost-competitive.
Pubmed: ABSTRACT Article (Li2007) Li, L.; Boedicker, J.Q. & Ismagilov, R.F. Using a Multijunction Microfluidic Device To Inject Substrate into an Array of Preformed Plugs without Cross-Contamination: Comparing Theory and Experiments. Anal Chem, 2007, 79, 2756-2761 Abstract: In this paper we describe a multijunction microfluidic device for the injection of a substrate into an array of preformed plugs carried by an immiscible fluid in a microchannel. The device uses multiple junctions to inject substrate into preformed plugs without synchronization of the flow of substrate and the array of preformed plugs of reagent, which reduces cross-contamination of the plugs, eliminates formation of small droplets of substrate, and allows a greater range of injection ratios compared to that of a single T-junction. The device was based on a previously developed physical model for transport that was here adapted to describe injection and experimentally verified. After characterization, the device was applied to two biochemical assays, including evaluation of the enzymatic activity of thrombin and determination of the coagulation time of human blood plasma, which both provided reliable results. The reduction of cross-contamination and greater range of injection ratios achieved by this device may improve the processes that involve addition and titration of reagents into plugs, such as high-throughput screening of protein crystallization conditions.
Pubmed: ABSTRACT Article (Li2006) Li, S.; Fozdar, D.; Ali, M.; Li, H.; Shao, D.; Vykoukal, D.; Vykoukal, J.; Floriano, P.; Olsen, M.; McDevitt, J.; Gascoyne, P. & Chen, S. A continuous-flow polymerase chain reaction microchip with regional velocity control Microelectromechanical Systems, Journal of, 2006, 15, 223- 236
PDF: Li2006.pdf Article (Li2005) Li, Y.; Liu, H.; Lin, H. & Chen, S. Gold nanoparticles for microfluidics-based biosensing of PCR products by hybridization-induced fluorescence quenching. Electrophoresis, 2005, 26, 4743-4750 Abstract: Colloidal gold nanoparticles were used to develop a simple microfluidics-based bioassay that is able to recognize and detect specific DNA sequences via conformational change-induced fluorescence quenching. In this method, a self-assembled monolayer of gold nanoparticles was fabricated on the channel wall of a microfluidic chip, and DNA probes were bonded to the monolayer via thiol groups at one end and a fluorophore dye was attached to the other end of the probe. The created construct is spontaneously assembled into a constrained arch-like conformation on the particle surface and, under which, the fluorescence of fluorophores is quenched by gold nanoparticles. Hybridization of target DNAs results in a conformational change of the construct and then restores the fluorescence, which serves as a sensing method for the target genes. The nanocomposite constructed on the glass surface was characterized by UV absorbance measurement and the quenching efficiency for different fluorophores was evaluated by Stern-Volmer studies. The applicability of proposed assay was first demonstrated by the use of a pair of synthesized complementary and noncomplementary DNA sequences. The method was further applied for the detection of the PCR product of dengue virus with the use of enterovirus as the negative control, and results indicate that the assay is specific for the target gene. Moreover, using this approach, dehybridization, hybridization, and detection of the target genes can be performed in situ on the same microfluidic channel. Thus, this method could be regarded as one-pot reaction and it holds great promises for clinical diagnostics.
Pubmed: ABSTRACT Article (Li2003) Li, Z.; Bai, X.; Ruparel, H.; Kim, S.; Turro, N.J. & Ju, J. A photocleavable fluorescent nucleotide for DNA sequencing and analysis Proc Natl Acad Sci U S A, 2003, 100, 414-9 Abstract: DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3alpha,4alpha-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.
Pubmed: ABSTRACT Article (Liao2005) Liao, C.; Lee, G.; Liu, H.; Hsieh, T. & Luo, C. Miniature RT-PCR system for diagnosis of RNA-based viruses. Nucleic Acids Res, 2005, 33, e156 Abstract: This paper presents an innovative portable chip-based RT-PCR system for amplification of specific nucleic acid and detection of RNA-based viruses. The miniature RT-PCR chip is fabricated using MEMS (Micro-electro-mechanical-system) techniques, and comprises a micro temperature control module and a PDMS (polydimethylsiloxane)-based microfluidic control module. The heating and sensing elements of temperature control module are both made of platinum and are located within the reaction chambers in order to generate a rapid and uniform thermal cycling. The microfluidic control module is capable of automating testing process with minimum human intervention. In this paper, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses, namely dengue virus type-2 and enterovirus 71 (EV 71). The experimental data confirm the ability of the system to perform a two-step RT-PCR process. The developed miniature system provides a crucial tool for the diagnosis of RNA-based viruses.
Pubmed: ABSTRACT Article (Lin2000) Lin, Y.; Yang, C. & Huang, M. Simulation and experimental validation of micro polymerase chain reaction chips Sensors and Actuators B: Chemical, 2000, 71, 127-133 Abstract: This study used finite element analysis to simulate the temperature characteristics of a micro polymerase chain reaction (PCR) chip. The micro-PCR chip was fabricated on a silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. The main goal of this study was to analyze the temperature uniformity and distribution of the micro-PCR chip, the temperature distribution of the DNA sample, and the transient temperature difference between the heater and DNA sample. The finite element analysis results were also confirmed by one-dimensional theoretic analysis. The simulation results were used to improve the thermal cycling time of a rapid micro-PCR system, consisting of a rapid thermal cycling system and a micro-PCR chip. The improved thermal cycles of the rapid [mu]PCR system were verified using serum samples from patients with chronic hepatitis C. The hepatitis C virus (HCV) amplicon of the rapid [mu]PCR system was analyzed by slab gel electrophoresis with DNA marker separation in parallel.
PDF: Lin2000.pdf Article (Lionello2005) Lionello, A.; Josserand, J.; Jensen, H. & Girault, H.H. Protein adsorption in static microsystems: effect of the surface to volume ratio Lab Chip, 2005, 5, 254-60 Abstract: A numerical model for the adsorption kinetics of proteins on the walls of a microchannel has been developed using the finite element method (FEM) to address the coupling with diffusion phenomena in the restricted microchannel volume. Time evolutions of the concentration of one species are given, both in solution and on the microchannel walls. The model illustrates the adsorption limitation sometimes observed when the microdimensions of these systems induce a global depletion of the bulk solution. A new non-dimensional parameter is introduced to predict the final value of the coverage of any microsystem under static adsorption. A working curve and a criteria (h/K[Gamma](max) > 10) are provided in order to choose, for given adsorption characteristics, the value of the volume-to-surface ratio (i.e. the channel height h) avoiding depletion effects on the coverage (relative coverage greater than 90% of the theoretical one). Simulations were compared with confocal microscopy measurements of IgG antibody adsorption on the walls of a PET microchannel. The fit of the model to the experimental data show that the adsorption is under apparent kinetic control.
Pubmed: ABSTRACT Article (Lipshutz1995) Lipshutz, R.J.; Morris, D.; Chee, M.; Hubbell, E.; Kozal, M.J.; Shah, N.; Shen, N.; Yang, R. & Fodor, S.P. Using oligonucleotide probe arrays to access genetic diversity. Biotechniques, 1995, 19, 442-447 Abstract: As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms are found to access individual genetic variation. It is only through a greater understanding of genetic diversity that the true benefit of the Human Genome Project will be realized. One approach, hybridization to high-density arrays of oligonucleotides, is a fast and effective means of accessing this genetic variation. Light-directed chemical synthesis has been used to generate miniaturized, high-density arrays of oligonucleotide probes. Application-specific oligonucleotide probe array designs have been developed for the rapid screening of characterized genes. Dedicated instrumentation and software have been developed for array hybridization, fluorescence detection and data acquisition and analysis. In a specific and challenging application, oligonucleotide probe arrays have been used to screen the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome to explore genetic diversity and detect mutations conferring resistance to antiviral drugs. Results from this application strongly suggest that oligonucleotide probe arrays will be a powerful tool for rapid investigations in sequence checking, pathogen detection, expression monitoring and DNA molecular recognition.
Pubmed: ABSTRACT Article (Liss2002) Liss, B. Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Res, 2002, 30, e89 Abstract: The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT-PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.
Pubmed: ABSTRACT Article (Litborn2000) Litborn, E. & Roeraade, J. Liquid lid for biochemical reactions in chip-based nanovials J Chromatogr B Biomed Sci Appl, 2000, 745, 137-47 Abstract: The present paper describes a new technique to suppress evaporation of solvent from very small volumes. Vials (15 nl) on a silicon microchip were filled with water, and covered with a thin, flowing film of a volatile liquid (e.g., octane). Water evaporation was greatly reduced. At 37 degrees C, no appreciable loss of water could be observed over a period of 90 min. At 95 degrees C, most of the water sample was left in the vial for more than 3 min. The applicability of the method is demonstrated by a tryptic digest, where the resulting peptide fragments were analyzed by capillary electrophoresis. In addition, a discussion of the possibilities and limitations of some alternative methods is included in the paper, as well as an outlook on future developments of the liquid lid concept.
Pubmed: ABSTRACT Article (Liu2006) Liu, B.; Xu, B.; Zhang, G.; Du, W. & Luo, Q. Micro-separation toward systems biology. J Chromatogr A, 2006, 1106, 19-28 Abstract: Current biology is experiencing transformation in logic or philosophy that forces us to reevaluate the concept of cell, tissue or entire organism as a collection of individual components. Systems biology that aims at understanding biological system at the systems level is an emerging research area, which involves interdisciplinary collaborations of life sciences, computational and mathematical sciences, systems engineering, and analytical technology, etc. For analytical chemistry, developing innovative methods to meet the requirement of systems biology represents new challenges as also opportunities and responsibility. In this review, systems biology-oriented micro-separation technologies are introduced for comprehensive profiling of genome, proteome and metabolome, characterization of biomolecules interaction and single cell analysis such as capillary electrophoresis, ultra-thin layer gel electrophoresis, micro-column liquid chromatography, and their multidimensional combinations, parallel integrations, microfabricated formats, and nano technology involvement. Future challenges and directions are also suggested.
Pubmed: ABSTRACT Article (Liu2006a) Liu, C.N.; Toriello, N.M. & Mathies, R.A. Multichannel PCR-CE microdevice for genetic analysis. Anal Chem, 2006, 78, 5474-5479 Abstract: We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.
Pubmed: ABSTRACT Article (Liu2002) Liu, J.; Enzelberger, M. & Quake, S. A nanoliter rotary device for polymerase chain reaction Electrophoresis, 2002, 23, 1531-6 Abstract: Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.
Pubmed: ABSTRACT Article (Liu2003) Liu, J.; Hansen, C. & Quake, S.R. Solving the "world-to-chip" interface problem with a microfluidic matrix. Anal Chem, 2003, 75, 4718-4723 Abstract: We report an effective solution to the macroscopic/microfluidic interface issue and demonstrate how microfluidics can achieve impressive economies of scale in reducing the complexity of pipetting operations. Using an N x N microfluidic matrix with N = 20, we performed N2 = 400 distinct PCR reactions with only 2N + 1 = 41 pipetting steps, compared with the 3N2 = 1200 steps required with conventional fluid handling. Each vertex of the matrix has a 3-nL reactor, and a single 2-microL aliquot of polymerase is amortized over all 400 independent reactions, thus dramatically reducing sample overhead and minimizing reagent consumption. Beyond PCR, the matrix chip provides a general method to perform chemical and biological experiments with precious reagents in a highly automated fashion.
Pubmed: ABSTRACT Article (Liu2002a) Liu, W. & Saint, D.A. Validation of a quantitative method for real time PCR kinetics Biochem Biophys Res Commun, 2002, 294, 347-53 Abstract: Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the assumptions regarding amplification efficiency before one starts the real time PCR quantitation. Here we further develop and test the validity of a new mathematical model which dynamically fits real time PCR data with good correlation (R(2)=0.9995+/-0.002, n=50). The method is capable of measuring cycle-by-cycle PCR amplification efficiencies and demonstrates that these change dynamically. Validation of the method revealed the intrinsic relationship between the initial amount of gene transcript and kinetic parameters. A new quantitative method is proposed which represents a simple but accurate quantitative method.
Pubmed: ABSTRACT Article (Liu2002b) Liu, W. & Saint, D.A. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics Anal Biochem, 2002, 302, 52-9 Abstract: Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative C(T) method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative C(T) method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.
Pubmed: ABSTRACT Article (Liu2002c) Liu, Y.; Rauch, C.B.; Stevens, R.L.; Lenigk, R.; Yang, J.; Rhine, D.B. & Grodzinski, P. DNA amplification and hybridization assays in integrated plastic monolithic devices. Anal Chem, 2002, 74, 3063-3070 Abstract: PCR amplification, DNA hybridization, and a hybridization wash have been integrated in a disposable monolithic DNA device, containing all of the necessary fluidic channels and reservoirs. These integrated devices were fabricated in polycarbonate plastic material by CO2 laser machining and were assembled using a combination of thermal bonding and adhesive tape bonding. Pluronics polymer phase change valves were implemented in the devices to fulfill the valving requirements. Pluronics polymer material is PCR compatible, and 30% Pluronics polymer valves provide enough holding pressure to ensure a successful PCR amplification. By reducing the temperature locally, to approximately 5 degrees C, Pluronics valves were liquefied and easily opened. A hybridization channel was made functional by oligonucleotide deposition, using Motorola proprietary surface attachment chemistry. Reagent transport on the device was provided by syringe pumps, which were docked onto the device. Peltier thermal electrical devices powered the heating and cooling functionality of the device. Asymmetrical PCR amplification and subsequent hybridization detection of both Escherichia coli K-12 MG1655 and Enterococcus faecalis DNAE genes have been successfully demonstrated in these disposable monolithic devices.
Pubmed: ABSTRACT Article (Livak1999) Livak, K.J. Allelic discrimination using fluorogenic probes and the 5' nuclease assay Genet Anal, 1999, 14, 143-9 Abstract: Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.
Pubmed: ABSTRACT Article (Livak1995) Livak, K.J.; Flood, S.J.; Marmaro, J.; Giusti, W. & Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 1995, 4, 357-362 Abstract: The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.
Pubmed: ABSTRACT Article (Livak2001) Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods, 2001, 25, 402-8 Abstract: The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Pubmed: ABSTRACT Article (Lizardi1998) Lizardi, P.M.; Huang, X.; Zhu, Z.; Bray-Ward, P.; Thomas, D.C. & Ward, D.C. Mutation detection and single-molecule counting using isothermal rolling-circle amplification Nat Genet, 1998, 19, 225-32 Abstract: Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.
Pubmed: ABSTRACT Article (Longo1990) Longo, M.C.; Berninger, M.S. & Hartley, J.L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions Gene, 1990, 93, 125-8 Abstract: Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.
Pubmed: ABSTRACT Article (Loseke2003) Loseke, S.; Grage-Griebenow, E.; Wagner, A.; Gehlhar, K. & Bufe, A. Differential expression of IFN-alpha subtypes in human PBMC: evaluation of novel real-time PCR assays J Immunol Methods, 2003, 276, 207-22 Abstract: Studies of the human IFN-alpha subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-alpha subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-alpha mRNA for the subtypes alpha(2), alpha(6), alpha(8) and alpha(1/13) in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of beta-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-alpha subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-alpha mRNA and shows differences in the composition of constitutive IFN-alpha subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-alpha subtypes investigated but to different expression levels. Among the subtypes detected, IFN-alpha(13/1) and alpha(2) are the major transcripts followed by alpha(8), and finally alpha(6) as a minor transcribed subtype. Time-kinetics of IFN-alpha transcriptional activation also revealed variations in the course of IFN-alpha transcription between NDV, RSV or HSV. The data obtained from the real-time PCR assays correlated well with IFN-alpha(2) protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-alpha subtype expression.
Pubmed: ABSTRACT Article (Lovmar2003) Lovmar, L.; Fredriksson, M.; Liljedahl, U.; Sigurdsson, S. & Syvanen, A.C. Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA Nucleic Acids Res, 2003, 31, e129 Abstract: Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.
Pubmed: ABSTRACT Article (Lu2007) Lu, C.; Xie, Y.; Yang, Y.; Cheng, M.M.; Koh, C.; Bai, Y.; Lee, L.J. & Juang, Y. New valve and bonding designs for microfluidic biochips containing proteins. Anal Chem, 2007, 79, 994-1001 Abstract: Two major concerns in the design and fabrication of microfluidic biochips are protein binding on the channel surface and protein denaturing during device assembly. In this paper, we describe new methods to solve these problems. A "fishbone" microvalve design based on the concept of superhydrophobicity was developed to replace the capillary valve in applications where the chip surface requires protein blocking to prevent nonspecific binding. Our experimental results show that the valve functions well in a CD-like ELISA device. The packaging of biochips containing pre-loaded proteins is also a challenging task since conventional sealing methods often require the use of high temperatures, electric voltages, or organic solvents that are detrimental to the protein activity. Using CO2 gas to enhance the diffusion of polymer molecules near the device surface can result in good bonding at low temperatures and low pressure. This bonding method has little influence on the activity of the pre-loaded proteins after bonding.
Pubmed: ABSTRACT Article (Lyamichev1999) Lyamichev, V.; Mast, A.L.; Hall, J.G.; Prudent, J.R.; Kaiser, M.W.; Takova, T.; Kwiatkowski, R.W.; Sander, T.J.; de Arruda, M.; Arco, D.A.; Neri, B.P. & Brow, M.A. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat Biotechnol, 1999, 17, 292-296 Abstract: Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
Pubmed: ABSTRACT Article (Dyck1971) M. F. Dyck, P.R.W. Lytic effects of plastic surfaces on erythrocytes Journal of Biomedical Materials Research, 1971, 5, 207-223 Abstract: Twenty-one commercial plastics were exposed to 10% red blood cell suspension in buffered saline and their hemolytic effect was determined. The depression in surface tension of physiological saline solution exposed to the plastic surfaces was recorded and brought into relationship with their hemolytic behavior. It has been shown that both effects occur simultaneously. It is proposed to use the depression in surface tension of saline exposed to prospective plastic applicants as a bloodcindependent screening test for evaluation of plastics which are intended to be exposed to a cardiovascular environment.
PDF: Dyck1971.pdf Article (Madou2006) Madou, M.; Zoval, J.; Jia, G.; Kido, H.; Kim, J. & Kim, N. Lab on a cd. Annu Rev Biomed Eng, 2006, 8, 601-628 Abstract: In this paper, centrifuge-based microfluidic platforms are reviewed and compared with other popular microfluidic propulsion methods. The underlying physical principles of centrifugal pumping in microfluidic systems are presented and the various centrifuge fluidic functions, such as valving, decanting, calibration, mixing, metering, heating, sample splitting, and separation, are introduced. Those fluidic functions have been combined with analytical measurement techniques, such as optical imaging, absorbance, and fluorescence spectroscopy and mass spectrometry, to make the centrifugal platform a powerful solution for medical and clinical diagnostics and high throughput screening (HTS) in drug discovery. Applications of a compact disc (CD)-based centrifuge platform analyzed in this review include two-point calibration of an optode-based ion sensor, an automated immunoassay platform, multiple parallel screening assays, and cellular-based assays. The use of modified commercial CD drives for high-resolution optical imaging is discussed as well. From a broader perspective, we compare technical barriers involved in applying microfluidics for sensing and diagnostic use and applying such techniques to HTS. The latter poses less challenges and explains why HTS products based on a CD fluidic platform are already commercially available, whereas we might have to wait longer to see commercial CD-based diagnostics.
Pubmed: ABSTRACT Article (Makarenkov2006) Makarenkov, V.; Kevorkov, D.; Zentilli, P.; Gagarin, A.; Malo, N. & Nadon, R. HTS-Corrector: software for the statistical analysis and correction of experimental high-throughput screening data. Bioinformatics, 2006, 22, 1408-1409 Abstract: MOTIVATION: High-throughput screening (HTS) plays a central role in modern drug discovery, allowing for testing of >100,000 compounds per screen. The aim of our work was to develop and implement methods for minimizing the impact of systematic error in the analysis of HTS data. To the best of our knowledge, two new data correction methods included in HTS-Corrector are not available in any existing commercial software or freeware. RESULTS: This paper describes HTS-Corrector, a software application for the analysis of HTS data, detection and visualization of systematic error, and corresponding correction of HTS signals. Three new methods for the statistical analysis and correction of raw HTS data are included in HTS-Corrector: background evaluation, well correction and hit-sigma distribution procedures intended to minimize the impact of systematic errors. We discuss the main features of HTS-Corrector and demonstrate the benefits of the algorithms.
Pubmed: ABSTRACT Article (Malek2006) Malek, C.G.K. Laser processing for bio-microfluidics applications (part I). Anal Bioanal Chem, 2006, 385, 1351-1361 Abstract: This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips in particular for applications in which silicon is not the preferred material base. Part I of this review addresses applications and devices using UV lasers for laser ablation and surface treatment of microchannels, in particular in polymers.
Pubmed: ABSTRACT Article (Malek2006a) Malek, C.G.K. Laser processing for bio-microfluidics applications (part II). Anal Bioanal Chem, 2006, 385, 1362-1369 Abstract: This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips, in particular for applications in which silicon is not the preferred material base. This review addresses applications and devices based on ablation using femtosecond lasers, infrared lasers as well as laser-induced micro-joining, and the laser-assisted generation of micro-replication tools, for subsequent replication of polymeric chips with a technique like laser LIGA.
Pubmed: ABSTRACT Article (Malmstadt2006) Malmstadt, N.; Nash, M.; Purnell, R. & Schmidt, J. Automated Formation of Lipid-Bilayer Membranes in a Microfluidic Device Nano Lett., 2006, 6, 1961-1965
PDF: Malmstadt2006.pdf Article (Manz1990) Manz, A.; Graber, N. & Widmer, H. Miniaturized total chemical analysis systems: a novel concept for chemical sensing Sens. Actuators B, 1990, B1, 244-248
PDF: Manz1990.pdf Article (Marcus2006) Marcus, J.; Anderson, W. & Quake, S. Microfluidic Single-Cell mRNA Isolation and Analysis. Anal Chem, 2006, 78, 3084-3089 Abstract: Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.
Pubmed: ABSTRACT Article (Markoulatos2002) Markoulatos, P.; Siafakas, N. & Moncany, M. Multiplex polymerase chain reaction: a practical approach J Clin Lab Anal, 2002, 16, 47-51 Abstract: Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.
Pubmed: ABSTRACT Article (Marsh2002) Marsh, S.; Kwok, P. & McLeod, H.L. SNP databases and pharmacogenetics: great start, but a long way to go Hum Mutat, 2002, 20, 174-9 Abstract: With the recent publication of the human genome project there has been an explosion of data available for pharmacogenetic research. Web-based databases containing information on single nucleotide polymorphisms (SNPs) are readily accessible to researchers, but there has been little comment on their utility. We used seven major international databases to identify SNPs in 74 genes involved in drug pathways. Very little overlap was seen among the databases, with only eight out of a putative 893 SNPs (approximately 1%) common to the most commonly used databases. Problems with false positives, secondary to a high degree of homology in gene families, were also observed. These studies suggest researchers limiting their studies to one database would miss a great deal of information. Effort to update compilation databases, such as HGVbase, GeneSNP, PharmGKB, and HOWDY, and the aggressive removal of false positives from all databases is required if these resources are to facilitate the intended growth in pharmacogenetics research.
Pubmed: ABSTRACT Article (Marth2001) Marth, G.; Yeh, R.; Minton, M.; Donaldson, R.; Li, Q.; Duan, S.; Davenport, R.; Miller, R.D. & Kwok, P.Y. Single-nucleotide polymorphisms in the public domain: how useful are they? Nat Genet, 2001, 27, 371-2 Abstract: There is a concerted effort by a number of public and private groups to identify a large set of human single-nucleotide polymorphisms (SNPs). As of March 2001, 2.84 million SNPs have been deposited in the public database, dbSNP, at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/). The 2.84 million SNPs can be grouped into 1.65 million non-redundant SNPs. As part of the International SNP Map Working Group, we recently published a high-density SNP map of the human genome consisting of 1.42 million SNPs (ref. 3). In addition, numerous SNPs are maintained in proprietary databases. Our survey of more than 1,200 SNPs indicates that more than 80% of TSC and Washington University candidate SNPs are polymorphic and that approximately 50% of the candidate SNPs from these two sources are common SNPs (with minor allele frequency of > or =20%) in any given population.
Pubmed: ABSTRACT Article (Marth1999) Marth, G.T.; Korf, I.; Yandell, M.D.; Yeh, R.T.; Gu, Z.; Zakeri, H.; Stitziel, N.O.; Hillier, L.; Kwok, P.Y. & Gish, W.R. A general approach to single-nucleotide polymorphism discovery Nat Genet, 1999, 23, 452-6 Abstract: Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.
Pubmed: ABSTRACT Article (Martin2000) Martin, E.R.; Lai, E.H.; Gilbert, J.R.; Rogala, A.R.; Afshari, A.J.; Riley, J.; Finch, K.L.; Stevens, J.F.; Livak, K.J.; Slotterbeck, B.D.; Slifer, S.H.; Warren, L.L.; Conneally, P.M.; Schmechel, D.E.; Purvis, I.; Vance, M.A.P.; Roses, A.D. & Vance, J.M. SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease. Am J Hum Genet, 2000, 67, 383-394 Abstract: There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P
Pubmed: ABSTRACT Article (Martin1998) Martin, V.J.M.; Ho, E.T.; McKeown, S.R.; Johnston, S.R.; McCarthy, P.J.; Rajab, N.F. & Downes, C.S. Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis, 1998, 13, 1-8 Abstract: ABSTRACT I: Management of invasive transitional cell human bladder carcinoma. The two main treatment options for invasive transitional cell bladder carcinoma are radiotherapy or primary cystectomy with urinary diversion or bladder substitution. Approximately 50% of patients fail to respond to radiotherapy and such patients so treated are disadvantaged by the absence of predictive information regarding their radiosensitivity, since the tumour gains additional time for metastatic spread before cystectomy is performed. The SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, is regarded as a good measure of radiosensitivity. However, the assay is time consuming and provides results for only approximately 70% of human tumours. In this paper three bladder transitional cell carcinoma cell lines (HT1376, UMUC-3 and RT112) were exposed to X-irradiation (0-10 Gy). We have compared the responses obtained using a clonogenic assay and a more clinically feasible alkaline single cell gel electrophoresis (Comet) assay. A very good inverse correlation was obtained between cell survival (clonogenic assay) and mean tail moment (Comet assay) for the three cell lines, indicating that the Comet assay can be used to predict the radio-responsiveness of individual cell lines. The clinical usefulness of the assay for predicting response to radiotherapy in bladder cancer patients is currently being investigated. ABSTRACT II: Fluorescent in situ hybridization (FISH) Comets for the identification of damaged and repaired DNA sequences in individual cells. In mammalian cells the extent of DNA damage is partly and the rate of DNA repair very considerably dependent on DNA position and transcription. This has been established by biochemical techniques which are labour intensive and require large numbers of cells. The Comet assay for overall DNA damage and repair is relatively simple and allows individual cells to be examined. Here we present a protocol for combination of the Comet assay with fluorescent in situ hybridization (FISH) using a p53 gene probe which allows specific observation of p53 sequences within DNA comets. Chromosome-specific probes can also be used. Optimization of the FISH/Comet protocol to include automation of the analysis is currently underway to facilitate future application of the technique to study selective DNA damage and repair in defined sequences in single mammalian cells.
Pubmed: ABSTRACT Article (Matsubara2005) Matsubara, Y.; Kerman, K.; Kobayashi, M.; Yamamura, S.; Morita, Y. & Tamiya, E. Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes. Biosens Bioelectron, 2005, 20, 1482-1490 Abstract: A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.
Pubmed: ABSTRACT Article (Matsuda2002) Matsuda, K.; Tomozawa, S.; Fukusho, S.; Yoshino, T.; Murakami, T. & Mitsuhashi, M. Gene expression analysis from nuclear Poly(A) RNA Biotechniques, 2002, 32, 1014-6, 1018, 1020 Abstract: Quantitation of the level of specific mRNA involves the isolation of total RNA or poly(A)+ RNA as a starting materiaL Thus, this result is the sum of the transcription and degradation of mRNA. Here we report a rapid, sensitive, and high-throughput methodology for gene expression analysis from nuclear poly(A)+ RNA via the reduction of the cytosolic components. The cells were first trapped on the glass fiber membranes of 96-well filter plates and subsequently exposed to non-ionic detergent to achieve cell membrane permeation. The cytosolic components, which contain preexisting mRNA, were removed by washing with the appropriate buffer, while nuclei remained in the filter plates. Lysis buffer was then used to release nuclear mRNA, which was collected on oligo(dT)-immobilized PCR plates for the capture of poly(A)+ RNA, on which RT-PCR was performed. The reduction of the cytosolic components and the preservation of the nuclear components were confirmed by electron microscopy, agarose gel electrophoresis, PCR of mtDNA, and RT-PCR of pre-splicing immature beta-actin poly(A)+ RNA. Using this method, we clearly identified UVC-induced p21 gene expression that is not detectable with conventional whole cell methods.
Pubmed: ABSTRACT Article (Matsuzaki2004a) Matsuzaki, H.; Dong, S.; Loi, H.; Di, X.; Liu, G.; Hubbell, E.; Law, J.; Berntsen, T.; Chadha, M.; Hui, H.; Yang, G.; Kennedy, G.C.; Webster, T.A.; Cawley, S.; Walsh, P.S.; Jones, K.W.; Fodor, S.P.A. & Mei, R. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods, 2004, 1, 109-111 Abstract: We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.
Pubmed: ABSTRACT Article (Matsuzaki2004) Matsuzaki, H.; Loi, H.; Dong, S.; Tsai, Y.; Fang, J.; Law, J.; Di, X.; Liu, W.; Yang, G.; Liu, G.; Huang, J.; Kennedy, G.C.; Ryder, T.B.; Marcus, G.A.; Walsh, P.S.; Shriver, M.D.; Puck, J.M.; Jones, K.W. & Mei, R. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res, 2004, 14, 414-425 Abstract: The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized to investigate the genetic causes of complex human diseases. Here we present a high-throughput genotyping platform that uses a one-primer assay to genotype over 10,000 SNPs per individual on a single oligonucleotide array. This approach uses restriction digestion to fractionate the genome, followed by amplification of a specific fractionated subset of the genome. The resulting reduction in genome complexity enables allele-specific hybridization to the array. The selection of SNPs was primarily determined by computer-predicted lengths of restriction fragments containing the SNPs, and was further driven by strict empirical measurements of accuracy, reproducibility, and average call rate, which we estimate to be >99.5%, >99.9%, and>95%, respectively [corrected]. With average heterozygosity of 0.38 and genome scan resolution of 0.31 cM, the SNP array is a viable alternative to panels of microsatellites (STRs). As a demonstration of the utility of the genotyping platform in whole-genome scans, we have replicated and refined a linkage region on chromosome 2p for chronic mucocutaneous candidiasis and thyroid disease, previously identified using a panel of microsatellite (STR) markers.
Pubmed: ABSTRACT Article (Mattick2001) Mattick, J.S. & Gagen, M.J. The evolution of controlled multitasked gene networks: the role of introns and other noncoding RNAs in the development of complex organisms. Mol Biol Evol, 2001, 18, 1611-1630 Abstract: Eukaryotic phenotypic diversity arises from multitasking of a core proteome of limited size. Multitasking is routine in computers, as well as in other sophisticated information systems, and requires multiple inputs and outputs to control and integrate network activity. Higher eukaryotes have a mosaic gene structure with a dual output, mRNA (protein-coding) sequences and introns, which are released from the pre-mRNA by posttranscriptional processing. Introns have been enormously successful as a class of sequences and comprise up to 95% of the primary transcripts of protein-coding genes in mammals. In addition, many other transcripts (perhaps more than half) do not encode proteins at all, but appear both to be developmentally regulated and to have genetic function. We suggest that these RNAs (eRNAs) have evolved to function as endogenous network control molecules which enable direct gene-gene communication and multitasking of eukaryotic genomes. Analysis of a range of complex genetic phenomena in which RNA is involved or implicated, including co-suppression, transgene silencing, RNA interference, imprinting, methylation, and transvection, suggests that a higher-order regulatory system based on RNA signals operates in the higher eukaryotes and involves chromatin remodeling as well as other RNA-DNA, RNA-RNA, and RNA-protein interactions. The evolution of densely connected gene networks would be expected to result in a relatively stable core proteome due to the multiple reuse of components, implying that cellular differentiation and phenotypic variation in the higher eukaryotes results primarily from variation in the control architecture. Thus, network integration and multitasking using trans-acting RNA molecules produced in parallel with protein-coding sequences may underpin both the evolution of developmentally sophisticated multicellular organisms and the rapid expansion of phenotypic complexity into uncontested environments such as those initiated in the Cambrian radiation and those seen after major extinction events.
Pubmed: ABSTRACT Article (McCarrey1992) McCarrey, J.R.; Dilworth, D.D. & Sharp, R.M. Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products. Genet Anal Tech Appl, 1992, 9, 117-123 Abstract: We have used analysis of ethidium-bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products to assess the effects of X-chromosome inactivation during spermatogenesis in the mouse. RT-PCR was performed on total RNA from eight different spermatogenic cell types, including premeiotic spermatogonia, meiotic spermatocytes, and postmeiotic spermatids, to detect transcripts from five different X-linked structural genes (Pgk-1, Zfx, Pdha-1, Hprt, and Phka) and two autosomal genes (Pgk-2 and beta-actin). Relative intensities of ethidium-bromide-stained RT-PCR products representing transcripts from each gene in each cell type were analyzed by densitometry using the Image program (version 1.4, NIH), and normalized against beta-actin values. These results suggest a coordinate inactivation of the X-linked loci at the onset of meiosis, followed by variable rates of decline of corresponding transcript levels reflecting differential mRNA stabilities and/or leaky expression after inactivation. Technically, these results indicate that analysis of ethidium-bromide-stained RT-PCR products can be used to provide a "semiquantitative" indication of relative levels of specific transcripts in a developing cell lineage without using radioactive probes to quantitate these products.
Pubmed: ABSTRACT Article (McCarthy2000) McCarthy, J.J. & Hilfiker, R. The use of single-nucleotide polymorphism maps in pharmacogenomics Nat Biotechnol, 2000, 18, 505-8 Abstract: Single-nucleotide polymorphisms (SNPs), common variations among the DNA of individuals, are being uncovered and assembled into large SNP databases that promise to enable the dissection of the genetic basis of disease and drug response (i.e., pharmacogenomics). Although great strides have been made in understanding the diversity of the human genome, such as the frequency, distribution, and type of genetic variation that exists, the feasibility of applying this information to uncover useful pharmacogenomic markers is uncertain. The health care industry is clamoring for access to SNP databases for use in research in the hope of revolutionizing the drug development process. As the reality of using SNPs to uncover drug response markers is rarely addressed, this review discusses practical issues, such as patient sample size, SNP density and genome coverage, and data interpretation, that will be important for determining the applicability of pharmacogenomic information to medical practice.
Pubmed: ABSTRACT Article (McManus2002) McManus, M.T.; Haines, B.B.; Dillon, C.P.; Whitehurst, C.E.; van Parijs, L.; Chen, J. & Sharp, P.A. Small interfering RNA-mediated gene silencing in T lymphocytes J Immunol, 2002, 169, 5754-60 Abstract: Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.
Pubmed: ABSTRACT Article (McManus2002a) McManus, M.T.; Petersen, C.P.; Haines, B.B.; Chen, J. & Sharp, P.A. Gene silencing using micro-RNA designed hairpins Rna, 2002, 8, 842-50 Abstract: During RNA interference (RNAi), long dsRNA is processed to approximately 21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are approximately 21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.
Pubmed: ABSTRACT Article (McManus2002b) McManus, M.T. & Sharp, P.A. Gene silencing in mammals by small interfering RNAs Nat Rev Genet, 2002, 3, 737-47 Abstract: Among the 3 billion base pairs of the human genome, there are approximately 30,000-40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.
Pubmed: ABSTRACT Article (McVean2004) McVean, G.A.; Myers, S.R.; Hunt, S.; Deloukas, P.; Bentley, D.R. & Donnelly, P. The fine-scale structure of recombination rate variation in the human genome Science, 2004, 304, 581-4 Abstract: The nature and scale of recombination rate variation are largely unknown for most species. In humans, pedigree analysis has documented variation at the chromosomal level, and sperm studies have identified specific hotspots in which crossing-over events cluster. To address whether this picture is representative of the genome as a whole, we have developed and validated a method for estimating recombination rates from patterns of genetic variation. From extensive single-nucleotide polymorphism surveys in European and African populations, we find evidence for extreme local rate variation spanning four orders in magnitude, in which 50% of all recombination events take place in less than 10% of the sequence. We demonstrate that recombination hotspots are a ubiquitous feature of the human genome, occurring on average every 200 kilobases or less, but recombination occurs preferentially outside genes.
Pubmed: ABSTRACT Article (Meldrum2002) Meldrum, D.R. & Holl, M.R. Tech.Sight. Microfluidics. Microscale bioanalytical systems. Science, 2002, 297, 1197-1198
Pubmed: ABSTRACT Article (Mello2002) de Mello, A. Plastic fantastic? Lab Chip, 2002, 2, 31N-36N
Pubmed: ABSTRACT Article (Mir1999) Mir, K.U. & Southern, E.M. Determining the influence of structure on hybridization using oligonucleotide arrays. Nat Biotechnol, 1999, 17, 788-792 Abstract: We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.
Pubmed: ABSTRACT Article (Mitchell2001) Mitchell, P. Microfluidics--downsizing large-scale biology. Nat Biotechnol, 2001, 19, 717-721
Pubmed: ABSTRACT Article (Mitra2003) Mitra, R.D.; Butty, V.L.; Shendure, J.; Williams, B.R.; Housman, D.E. & Church, G.M. Digital genotyping and haplotyping with polymerase colonies Proc Natl Acad Sci U S A, 2003, 100, 5926-31 Abstract: Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.
Pubmed: ABSTRACT Article (Mitra1999) Mitra, R.D. & Church, G.M. In situ localized amplification and contact replication of many individual DNA molecules Nucleic Acids Res, 1999, 27, e34 Abstract: We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or olonies have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.
Pubmed: ABSTRACT Article (Mitra2003a) Mitra, R.D.; Shendure, J.; Olejnik, J.; Edyta Krzymanska, O. & Church, G.M. Fluorescent in situ sequencing on polymerase colonies Anal Biochem, 2003, 320, 55-65 Abstract: Integration of DNA isolation, amplification, and sequencing can be achieved by the use of polymerase colonies (polonies) and cycles of fluorescent dNTP incorporation. In this paper, we present four advances that bring us closer to sequencing genomes cost-effectively using the polony technology. First, a polymerase trapping technique enables efficient nucleotide extension by DNA polymerase in a polyacrylamide matrix and eliminates loss of enzyme during sequencing cycles. Next, we present two novel types of reversibly dye-labeled nucleotide analogues, show that DNA polymerase can incorporate these analogues, and demonstrate that the dyes can be removed by thiol reduction or light exposure. Using these nucleotides, we have sequenced multiple polonies in parallel. In addition, we have found that a high density of polonies can be achieved with minimal overlap between adjacent polonies by limiting the concentration of free primer in the polony amplification reactions. Finally, we have developed software for automated image alignment and sequence calling.
Pubmed: ABSTRACT Article (Miyazaki2003) Miyazaki, S.; Sugawara, H.; Gojobori, T. & Tateno, Y. DNA Data Bank of Japan (DDBJ) in XML Nucleic Acids Res, 2003, 31, 13-6 Abstract: The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has collected and released more entries and bases than last year. This is mainly due to large-scale submissions from Japanese sequencing teams on mouse, rice, chimpanzee, nematoda and other organisms. The contributions of DDBJ over the past year are 17.3% (entries) and 10.3% (bases) of the combined outputs of the International Nucleotide Sequence Databases (INSD). Our complete genome sequence database, Genome Information Broker (GIB), has been improved by incorporating XML. It is now possible to perform a more sophisticated database search against the new GIB than the ordinary BLAST or FASTA search.
Pubmed: ABSTRACT Article (Mogensen2004) Mogensen, K.B.; Klank, H. & Kutter, J.P. Recent developments in detection for microfluidic systems. Electrophoresis, 2004, 25, 3498-3512 Abstract: Microfluidic systems have become more and more important in the field of analytical chemistry. Detection methods on these microsystems are essential for the identification and quantification of chemical species that are being analyzed. This review concentrates on the latest developments of optical detection methods and mass spectrometry in conjunction with microfluidic systems. Electrochemical methods are discussed in another review in the same issue of this journal. Within the optical detection section, topics such as multiplexed detection and the use of waveguides are discussed. Within the discussion of mass spectrometry, the main focus is on electrospray emitters as interfaces between microsystem and spectrometer. Apart from optical detection and mass spectrometry, other techniques such as flame ionization and nuclear magnetic resonance are also mentioned.
Pubmed: ABSTRACT Article (Moore1996) Moore, M.J. Gene expression. When the junk isn't junk. Nature, 1996, 379, 402-403
Pubmed: ABSTRACT Article (Morrison2006) Morrison, T.; Hurley, J.; Garcia, J.; Yoder, K.; Katz, A.; Roberts, D.; Cho, J.; Kanigan, T.; Ilyin, S.E.; Horowitz, D.; Dixon, J.M. & Brenan, C.J.H. Nanoliter high throughput quantitative PCR. Nucleic Acids Res, 2006 Abstract: Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.
Pubmed: ABSTRACT Article (Morrison1998) Morrison, T.B.; Weis, J.J. & Wittwer, C.T. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification Biotechniques, 1998, 24, 954-8, 960, 962 Abstract: Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number. A simple and generic method for monitoring product synthesis with the double-stranded DNA dye, SYBR Green I provides initial template copy number estimation limited only by stochastic effects. To reach this degree of sensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and therefore, specific product formation was monitored by fluorescence acquisition at temperatures at which only specific products are double-stranded. Second, anti-Taq antibodies were used to reduce nonspecific product generation. The log-linear portion of the fluorescence vs. cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained. These fractional cycle numbers were plotted against the log of starting template copies to give linear standard curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reaction could be distinguished from the absence of template, although stochastic effects increased the variance of concentration estimates below 10 copies. Above 10 copies per reaction, typical replicate coefficients of variation were 6%-37%, with better precision at higher copy numbers.
Pubmed: ABSTRACT Article (Mott2006) Mott, D.R.; Howell, P.B.; Golden, J.P.; Kaplan, C.R.; Ligler, F.S. & Oran, E.S. Toolbox for the design of optimized microfluidic components. Lab Chip, 2006, 6, 540-549 Abstract: A computational "toolbox" for the a priori design of optimized microfluidic components is presented. These components consist of a microchannel under low-Reynolds number, pressure-driven flow, with an arrangement of grooves cut into the top and bottom to generate a tailored cross-channel flow. An advection map for each feature (i.e., groove of a particular shape and orientation) predicts the lateral transport of fluid within the channel due to that feature. We show that applying these maps in sequence generates an excellent representation of the outflow distribution for complex designs that combine these basic features. The effect of the complex three-dimensional flow field can therefore be predicted without solving the governing flow equations through the composite geometry, and the resulting distribution of fluids in the channel is used to evaluate how well a component performs a specified task. The generation and use of advection maps is described, and the toolbox is applied to determine optimal combinations of features for specified mixer sizes and mixing metrics.
Pubmed: ABSTRACT Article (Muller2002) Muller, P.Y.; Janovjak, H.; Miserez, A.R. & Dobbie, Z. Processing of gene expression data generated by quantitative real-time RT-PCR Biotechniques, 2002, 32, 1372-4, 1376, 1378-9 Abstract: Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.
Pubmed: ABSTRACT Article (Mullis1992) Mullis, K.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G. & Erlich, H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. 1986 Biotechnology, 1992, 24, 17-27
Pubmed: ABSTRACT Article (Mullis1986) Mullis, K.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G. & Erlich, H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction Cold Spring Harb Symp Quant Biol, 1986, 51 Pt 1, 263-73
Pubmed: ABSTRACT Article (Musyanovych2005) Musyanovych, A.; Mailänder, V. & Landfester, K. Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction. Biomacromolecules, 2005, 6, 1824-1828 Abstract: Polymerase chain reaction (PCR) was successfully carried out inside stable and narrowly distributed water-in-oil nanodroplets with a size of 100-300 nm in diameter. The droplets were obtained by the miniemulsion process. Each aqueous droplet serves as a single nanoreactor for the PCR. It was found that the size of the droplets highly depends on the sonication parameters (i.e., time and amplitude) and that these parameters have a great influence on the final concentration of the PCR product. The parameters were chosen that way that conditions for single molecule chemistry were obtained, since the 3D-space is compartimentalized in small nanoreactors in each of which the same reaction takes place in a highly parallel fashion on every single DNA molecule.
Pubmed: ABSTRACT Article (Myer2001) Myer, S.E. & Day, D.J. Synthesis and application of circularizable ligation probes Biotechniques, 2001, 30, 584-8, 590, 592-3 Abstract: We describe a PCR-based approach for the synthesis of circularizable ligation probes (CLiPs). CLiPs are single-stranded probes that consist of target-specific ends separated by a noncomplementary "linker" sequence. When hybridized to a target, the CLiP forms a nicked circle that may be sealed by DNA ligase only if the 5' and 3' ends show perfect Watson-Crick base pairing, thus enabling the discrimination of single nucleotide polymorphisms. Primers incorporating target sequence at their 5' end and plasmid sequence at the 3' end were used in a PCR amplification. In addition, the antisense primer was 5' labeled with biotin, and the amplification was performed in the presence of fluorescently labeled dUTP. The resulting PCR product was captured with streptavidin-coated paramagnetic beads, and the top strand, which forms the CLiP, was alkali eluted. This PCR-based method has allowed the synthesis of CLiPs that are larger and more highly labeled than has previously been possible, with ligation efficiencies similar to those of the purest chemically synthesized padlock probes. Ligations performed in the presence of cognate or mismatched sequence were analyzed by denaturing PAGE using a fluorescent DNA sequencer. Genotyping using target immobilized to nylon membranes was also performed. The CLiPs were readily able to distinguish between mutant and wild-type alleles for the common genetic disorder, 21-hydroxylase deficiency. Additionally, CLiPs of different lengths were synthesized and compared.
Pubmed: ABSTRACT Article (Naef2003) Naef, F.; Socci, N.D. & Magnasco, M. A study of accuracy and precision in oligonucleotide arrays: extracting more signal at large concentrations. Bioinformatics, 2003, 19, 178-184 Abstract: MOTIVATION: Despite the success and popularity of oligonucleotide arrays as a high-throughput technique for measuring mRNA expression levels, quantitative calibration studies have until now been limited. The main reason is that suitable data was not available. However, calibration data recently produced by Affymetrix now permits detailed studies of the intensity dependent sensitivity. Given a certain transcript concentration, it is of particular interest to know whether current analysis methods are capable of detecting differential expression ratios of 2 or higher. RESULTS: Using the calibration data, we demonstrate that while current techniques are capable of detecting changes in the low to mid concentration range, the situation is noticeably worse for high concentrations. In this regime, expression changes as large as 4 fold are severely biased, and changes of 2 are often undetectable. Such effects are mainly the consequence of the sequence specific binding properties of probes, and not the result of optical saturation in the fluorescence measurements. GeneChips are manufactured such that each transcript is probed by a set of sequences with a wide affinity range. We show that this property can be used to design a method capable of reducing the high intensity bias. The idea behind our methods is to transfer the weight of a measurement to a subset of probes with optimal linear response at a given concentration, which can be achieved using local embedding techniques. AVAILABILITY: Program source code will be sent electronically upon request.
Pubmed: ABSTRACT Article (Nagai2001) Nagai, H.; Murakami, Y.; Morita, Y.; Yokoyama, K. & Tamiya, E. Development of a microchamber array for picoliter PCR Anal Chem, 2001, 73, 1043-7 Abstract: A microchamber array for PCR was developed by semiconductor microfabrication technology. The microchambers were designed to be of picoliter quantity. To optimize fluid retention, the surface states of the substrate and the inner walls were examine for four different types of microchamber. The substrate was silicon, while silicon dioxide was selected for the inner walls. PCR was performed in the microchamber array, and the amplification of DNA was detected using a technique based on the energy transfer of fluorescent dyes. The lower volume limit for PCR was investigated using various sizes of microchambers. Microchambers with volume greater than 86 pL gave successful PCR. In addition, the system was improved in order to take up the PCR product. To prevent mixing of the samples, the samples were dried after PCR using a membrane that permeates only vapor.
Pubmed: ABSTRACT Article (Nakano1994) Nakano, H.; Matsuda, K.; Yohda, M.; Nagamune, T.; Endo, I. & Yamane, T. High speed polymerase chain reaction in constant flow. Biosci Biotechnol Biochem, 1994, 58, 349-352 Abstract: A new simple reactor of the tubing type was developed for polymerase chain reaction (PCR). A thin Teflon capillary tube was used as a tubing reactor in which the reaction mixture of PCR was driven by a pump at a constant flow rate. The sample was treated with three successive thermal stages for denaturation, annealing, and elongation of DNA and primers as a function of the position in the tube. The amplification yield was about a half of that obtained by a commercial thermocycler. Moreover, the total reaction time from 12 to 18 min, which was one-tenth of the time generally required by conventional thermocyclers using metal blocks, assured substaintial amplification of a DNA fragment. In addition, this reactor could be also used for rapid cycle-sequences. This new device will be easily incorporated into automated and rapid DNA analysis systems for DNA sequencing.
Pubmed: ABSTRACT Article (Nakano2005) Nakano, M.; Komatsu, J.; Kurita, H.; Yasuda, H.; Katsura, S. & Mizuno, A. Adaptor polymerase chain reaction for single molecule amplification. J Biosci Bioeng, 2005, 100, 216-218 Abstract: The adaptor polymerase chain reaction (PCR) permits the amplification of DNA fragments with arbitrary sequences. In this paper, we describe the successful amplification of plasmid-derived single molecule DNAs digested by a restriction enzyme. By using adaptors made of short and long oligonucleotides, nonspecific interactions during PCR were suppressed. The method will be applicable to the detection of single molecule DNA fragments even if their sequence is unknown.
Pubmed: ABSTRACT Article (Naqvi2002) Naqvi, A.; Nahar, P. & Gandhi, R.P. Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes. Anal Biochem, 2002, 306, 74-78 Abstract: Polypropylene and polyethylene surfaces are activated by introducing an active functional group through 1-fluoro-2 nitro-4-azidobenzene by UV irradiation. Horseradish peroxidase and glucose oxidase are immobilized onto the activated surfaces, simply by incubating the enzymes at 37 degrees C. When untreated surfaces are used, insignificant immobilization of the enzymes is observed.
Pubmed: ABSTRACT Article (Nath2000) Nath, K.; Sarosy, J.W.; Hahn, J. & Di Como, C.J. Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems J Biochem Biophys Methods, 2000, 42, 15-29 Abstract: In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.
Pubmed: ABSTRACT Article (Nazarenko1997) Nazarenko, I.A.; Bhatnagar, S.K. & Hohman, R.J. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res, 1997, 25, 2516-2521 Abstract: A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.
Pubmed: ABSTRACT Article (Neely2006) Neely, L.A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.; Nadel, M.; Harris, J.; Gullans, S. & Rooke, J. A single-molecule method for the quantitation of microRNA gene expression. Nat Methods, 2006, 3, 41-46 Abstract: MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps. The Direct miRNA assay hybridizes two spectrally distinguishable fluorescent locked nucleic acid (LNA)-DNA oligonucleotide probes to the miRNA of interest, and then tagged molecules are directly counted on a single-molecule detection instrument. In this study, we show the assay is sensitive to femtomolar concentrations of miRNA (500 fM), has a three-log linear dynamic range and is capable of distinguishing among miRNA family members. Using this technology, we quantified expression of 45 human miRNAs within 16 different tissues, yielding a quantitative differential expression profile that correlates and expands upon published results.
Pubmed: ABSTRACT Article (Nellaaker2006) Nellåker, C.; Wållgren, U. & Karlsson, H. Molecular Beacon-Based Temperature Control and Automated Analyses for Improved Resolution of Melting Temperature Analysis Using SYBR I Green Chemistry. Clin Chem, 2006 Abstract: Introduction:Melting temperature analysis of products amplified with SYBR I Green chemistry is a cheap and effective method for identification of sequence differences. When used in conventional quantitative real-time PCR instruments (qPCR), this method is limited by temperature variations over the heating block and low numbers of fluorescence measurements during the dissociation step, which hamper the ability of most instruments to report accurate and precise melting temperatures. METHODS: We designed a molecular beacon-based temperature indicator probe (Tm-probe) to control for variations in temperatures over the heating block of the instrument. In addition, we wrote an automated curve-fit analysis algorithm of dissociation data to use multiple data points with a gaussian curve fit to extrapolate precise melting temperatures. RESULTS: Use of the Tm-probe in conjuction with the analysis algorithm and multiple dissociations improved SDs of melting temperatures over a 96-well plate from 0.19 to 0.06 degrees C CONCLUSIONS: Melting temperature analyses with SYBR I Green chemistry on conventional qPCR instruments can be improved by the use of a Tm-probe in conjunction with curve-fit analysis of data. Resolution improvement up to 3-fold is possible and allows additional melting temperatures to be identified.
Pubmed: ABSTRACT Article (Neuzil2006) Neuzil, P.; Zhang, C.; Pipper, J.; Oh, S. & Zhuo, L. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Res, 2006, 34, e77 Abstract: We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175 degrees C s(-1) and a cooling rate of -125 degrees C s(-1). A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.
Pubmed: ABSTRACT Article (Newton1989) Newton, C.R.; Graham, A.; Heptinstall, L.E.; Powell, S.J.; Summers, C.; Kalsheker, N.; Smith, J.C. & Markham, A.F. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) Nucleic Acids Res, 1989, 17, 2503-16 Abstract: We have improved the olymerase chain reaction(PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.
Pubmed: ABSTRACT Article (Nogva2004) Nogva, H.K. & Rudi, K. Potential influence of the first PCR cycles in real-time comparative gene quantifications. Biotechniques, 2004, 37, 246-8, 250-3 Abstract: There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.
Pubmed: ABSTRACT Article (Nolan2006) Nolan, T.; Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat Protoc, 2006, 1, 1559-1582 Abstract: The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.
Pubmed: ABSTRACT Article (Northrup1998) Northrup, M.A.; Benett, B.; Hadley, D.; Landre, P.; Lehew, S.; Richards, J. & Stratton, P. A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers Anal Chem, 1998, 70, 918-22 Abstract: In this paper, we describe a miniature analytical thermal cycling instrument (MATCI) to amplify and detect DNA via the polymerase chain reaction in real-time. The MATCI is an integrated, miniaturized analytical system that uses silicon-based, high-efficiency reaction chambers with integrated heaters and simple, inexpensive electronics to precisely control the reaction temperatures. Optical windows in the silicon and solid-state, diode-based detection components are employed to perform real-time fluorescence monitoring of product DNA production. The entire system fits into a briefcase and runs on rechargeable batteries. The applications of this miniaturized nucleic acid analysis system include clinical, research, environmental, and agricultural analyses as well as others which require rapid, portable, and accurate analysis of biological samples for nucleic acids. This paper describes the MATCI and presents results from ultrafast thermal cycling and real-time PCR detection. Examples include human genes and pathogenic viruses and bacteria.
Pubmed: ABSTRACT Article (Nygaard2006) Nygaard, V. & Hovig, E. Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling Nucleic Acids Res, 2006, 34, 996-1014 Abstract: The possibility of performing microarray analysis on limited material has been demonstrated in a number of publications. In this review we approach the technical aspects of mRNA amplification and several important implicit consequences, for both linear and exponential procedures. Amplification efficiencies clearly allow profiling of extremely small samples. The conservation of transcript abundance is the most important issue regarding the use of sample amplification in combination with microarray analysis, and this aspect has generally been found to be acceptable, although demonstrated to decrease in highly diluted samples. The fact that variability and discrepancies in microarray profiles increase with minute sample sizes has been clearly documented, but for many studies this does appear to have affected the biological conclusions. We suggest that this is due to the data analysis approach applied, and the consequence is the chance of presenting misleading results. We discuss the issue of amplification sensitivity limits in the light of reports on fidelity, published data from reviewed articles and data analysis approaches. These are important considerations to be reflected in the design of future studies and when evaluating biological conclusions from published microarray studies based on extremely low input RNA quantities.
Pubmed: ABSTRACT Article (Obeid2003) Obeid, P.J.; Christopoulo, T.K.; Crabtree, H.J. & Backhouse, C.J. Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection Anal Chem, 2003, 75, 288-95 Abstract: We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.
Pubmed: ABSTRACT Article (Oda1998) Oda, R.P.; Strausbauch, M.A.; Huhmer, A.F.; Borson, N.; Jurrens, S.R.; Craighead, J.; Wettstein, P.J.; Eckloff, B.; Kline, B. & Landers, J.P. Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA Anal Chem, 1998, 70, 4361-8 Abstract: Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.
Pubmed: ABSTRACT Article (Olejnik1998) Olejnik, J.; Krzymanska-Olejnik, E. & Rothschild, K.J. Photocleavable affinity tags for isolation and detection of biomolecules Methods Enzymol, 1998, 291, 135-54
Pubmed: ABSTRACT Article (Olejnik1998a) Olejnik, J.; Krzymanska-Olejnik, E. & Rothschild, K.J. Photocleavable aminotag phosphoramidites for 5termini DNA/RNA labeling Nucleic Acids Res, 1998, 26, 3572-6 Abstract: We report the design and evaluation of two non-nucleosidic photocleavable aminotag phosphor-amidites. These reagents introduce a photocleavable amino group on the 5terminal phosphate of synthetic oligonucleotides. The 5photocleavable amino group enables introduction of a variety of amine-reactive markers onto synthetic oligonucleotides as well as immobilization on activated solid supports. The photocleavable bond on the 5phosphate can then be selectively cleaved by near-UV illumination, thereby enabling release of the marker or detachment of the oligonucleotide from a solid support. The preparation of photocleavable conjugates with biotin, digoxigenin and tetramethylrhodamine are described. In the case of biotin, a conjugate was used in a high sensitivity hybridization assay as a photocleavable probe for a complementary sequence immobilized on beads. It is also demonstrated that the 5PC-amino group can be used as an affinity tag for photocleavage-mediated affinity purification and phosphorylation of synthetic oligonucleotides in conjunction with activated supports. Such 5PC-amino labeled oligonucleotides should be useful in a variety of applications in molecular biology including multiple non-radioactive probing of DNA/RNA blots, affinity isolation and purification of nucleic acids binding proteins, diagnostic assays requiring release of the probe-target complex or specific marker, cassette mutagenesis and PCR. They will also enable the spatially-addressable photorelease of the probe-target complexes or marker molecules for diagnostic purposes.
Pubmed: ABSTRACT Article (Olivier2002) Olivier, M.; Chuang, L.M.; Chang, M.S.; Chen, Y.T.; Pei, D.; Ranade, K.; de Witte, A.; Allen, J.; Tran, N.; Curb, D.; Pratt, R.; Neefs, H.; de Arruda Indig, M.; Law, S.; Neri, B.; Wang, L. & Cox, D.R. High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology Nucleic Acids Res, 2002, 30, e53 Abstract: The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1beta) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.
Pubmed: ABSTRACT Article (Ott2003) Ott, J. & Hoh, J. Set association analysis of SNP case-control and microarray data J Comput Biol, 2003, 10, 569-74 Abstract: Common heritable diseases (omplex traits are assumed to be due to multiple underlying susceptibility genes. While genetic mapping methods for Mendelian disorders have been very successful, the search for genes underlying complex traits has been difficult and often disappointing. One of the reasons may be that most current gene-mapping approaches are still based on conventional methodology of testing one or a few SNPs at a time. Here, we demonstrate a simple strategy that allows for the joint analysis of multiple disease-associated SNPs in different genomic regions. Our set-association method combines information over SNPs by forming sums of relevant single-marker statistics. As previously hypothesized, we show here that this approach successfully addresses the urse of dimensionalityproblem--too many variables should be estimated with a comparatively small number of observations. We also report results of simulation studies showing that our method furnishes unbiased and accurate significance levels. Power calculations demonstrate good power even in the presence of large numbers of nondisease associated SNPs. We extended our method to microarray expression data, where expression levels for large numbers of genes should be compared between two tissue types. In applications to such data, our approach turned out to be highly efficient.
Pubmed: ABSTRACT Article (Ottesen2006) Ottesen, E.A.; Hong, J.W.; Quake, S.R. & Leadbetter, J.R. Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria Science, 2006, 314, 1464-1467 Abstract: Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.
PDF: Ottesen2006.pdf Article (Ovstebo2003) Ovstebo, R.; Haug, K.B.; Lande, K. & Kierulf, P. PCR-based calibration curves for studies of quantitative gene expression in human monocytes: development and evaluation Clin Chem, 2003, 49, 425-32 Abstract: BACKGROUND: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol. METHODS: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, beta-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected. RESULTS: Within- and between-run variations (CVs) of cDNA controls (TF and beta-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4-10% (within) and 15-38% (between) using both daily and rand meancalibration curves. CVs for the beta-actin mRNA controls were 12% (within) and 19-28% (between). Estimates of each step contribution to the total variation were as follows: CV(RT-PCR), 28%; CV(PCR), 15%; CV(RT), 23% (difference between CV(RT-PCR) and CV(PCR)). PCR efficiencies were as follows: beta-actin calibrator/target, 1.96/1.95; TF calibrator/target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8). CONCLUSIONS: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system total variance, power analysis may be used to disclose differences that can be reliably detected at a specified power.
Pubmed: ABSTRACT Article (Paez2004) Paez, J.G.; Lin, M.; Beroukhim, R.; Lee, J.C.; Zhao, X.; Richter, D.J.; Gabriel, S.; Herman, P.; Sasaki, H.; Altshuler, D.; Li, C.; Meyerson, M. & Sellers, W.R. Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification Nucleic Acids Res, 2004, 32, e71 Abstract: Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.
Pubmed: ABSTRACT Article (Pal2004) Pal, R.; Yang, M.; Johnson, B.N.; Burke, D.T. & Burns, M.A. Phase change microvalve for integrated devices. Anal Chem, 2004, 76, 3740-3748 Abstract: An active microvalve that uses a meltable piston in place of a conventional solid material to obstruct fluid flow in a microfluidic channel has been developed. This phase change valve is simple to operate and requires no additional fabrication steps. The valve is inherently latched, reusable, and leak-proof (to at least 250 psi) and can be electronically addressed using resistive heaters. The valve has been characterized for a range of operational parameters that will serve as a design guide. For the designs tested, piston displacements of 5 mm or more in 1 s have been achieved. Valves 1.4 mm in length in a 50 microm x 200 microm channel have been integrated on a biochemical reaction device, and successful DNA amplification using PCR has been achieved. The phase change valve can be easily implemented in an array format that can be used to realize complex microfluidic circuits.
Pubmed: ABSTRACT Article (Paliwal2007) Paliwal, S.; Iglesias, P.A.; Campbell, K.; Hilioti, Z.; Groisman, A. & Levchenko, A. MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast. Nature, 2007, 446, 46-51 Abstract: The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.
Pubmed: ABSTRACT Article (Palkova2004) Palková, Z.; Váchová, L.; Valer, M. & Preckel, T. Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system. Cytometry A, 2004, 59, 246-253 Abstract: BACKGROUND: Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types. METHODS: We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection. RESULTS: We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations. Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells. In addition, we have characterized the infection state of Magnaportae grisea fungal spores. CONCLUSIONS: Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types.
Pubmed: ABSTRACT Article (Papp2003) Papp, A.C.; Pinsonneault, J.K.; Cooke, G. & Sadee, W. Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves Biotechniques, 2003, 34, 1068-72 Abstract: We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3end of the forward or reverse primer, and additionally incorporating a 3mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.
Pubmed: ABSTRACT Article (Park2005) Park, J.; Kim, B.; Choi, S.K.; Hong, S.; Lee, S.H. & Lee, K. An efficient cell separation system using 3D-asymmetric microelectrodes. Lab Chip, 2005, 5, 1264-1270 Abstract: An efficient 3D-asymmetric microelectrode system for high-throughput was designed and fabricated to enhance sorting sensitivities to the dielectric properties-size, morphology, conductivity, and permittivity-of living cells. The principle of the present system is based on the use of the relative strengths of negative dielectrophoretic and drag forces, as in a conventional 3D-microelectrode system. Whereas the typical 3D-microelectrode system has a constant electric field magnitude due to the constant width of the microelectrodes and a fixed gap between face-to-face microelectrodes, the present 3D-asymmetric microelectrode system has electric fields of continuously varying magnitudes along the transverse direction of a channel owing to the changing widths of the electrodes in the half-circular shaped cross section of the microchannel. Thus, varying dielectric forces are generated, leading to increased sorting sensitivity through differentially induced forces to definitely distinct cell types. Numerical analysis verified the improved sensitivity of the present system for sorting living cells. The feasibility of using the newly fabricated system under experimental conditions was tested by demonstrating that a mixed population of mouse P19 embryonic carcinoma (EC) and red blood cells (RBCs) was effectively sorted to different wells depending on their respective relative physical properties.
Pubmed: ABSTRACT Article (Park2006) Park, M.C.; Hur, J.Y.; Kwon, K.W.; Park, S. & Suh, K.Y. Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus. Lab Chip, 2006, 6, 988-994 Abstract: We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.
Pubmed: ABSTRACT Article (Pastinen1997) Pastinen, T.; Kurg, A.; Metspalu, A.; Peltonen, L. & Syvänen, A.C. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res, 1997, 7, 606-614 Abstract: We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.
Pubmed: ABSTRACT Article (Patil2001) Patil, N.; Berno, A.J.; Hinds, D.A.; Barrett, W.A.; Doshi, J.M.; Hacker, C.R.; Kautzer, C.R.; Lee, D.H.; Marjoribanks, C.; McDonough, D.P.; Nguyen, B.T.; Norris, M.C.; Sheehan, J.B.; Shen, N.; Stern, D.; Stokowski, R.P.; Thomas, D.J.; Trulson, M.O.; Vyas, K.R.; Frazer, K.A.; Fodor, S.P. & Cox, D.R. Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21 Science, 2001, 294, 1719-23 Abstract: Global patterns of human DNA sequence variation (haplotypes) defined by common single nucleotide polymorphisms (SNPs) have important implications for identifying disease associations and human traits. We have used high-density oligonucleotide arrays, in combination with somatic cell genetics, to identify a large fraction of all common human chromosome 21 SNPs and to directly observe the haplotype structure defined by these SNPs. This structure reveals blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes.
Pubmed: ABSTRACT Article (Pattyn2003) Pattyn, F.; Speleman, F.; De Paepe, A. & Vandesompele, J. RTPrimerDB: the Real-Time PCR primer and probe database Nucleic Acids Res, 2003, 31, 122-3 Abstract: The real-time polymerase chain reaction (PCR) methodology has become increasingly popular for nucleic acids detection and/or quantification. As primer/probe design and experimental evaluation is time-consuming, we developed a public database application for the storage and retrieval of validated real-time PCR primer and probe sequence records. The integrity and accuracy of the data are maintained by linking to and querying other reference databases. RTPrimerDB provides free public access through the Web to perform queries and submit user based information. Primer/probe records can be searched for by official gene symbol, nucleotide sequence, type of application, detection chemistry, LocusLink or Single Nucleotide Polymorphism (SNP) identifier, and submitter's name. Each record is directly linked to LocusLink, dbSNP and/or PubMed to retrieve additional information on the gene/SNP for which the primers/probes are designed. Currently, the database contains primer/probe records for human, mouse, rat, fruit fly and zebrafish, and all current detection chemistries such as intercalating dyes (SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons. Real-time PCR primer/probe records are available at http://www.realtimeprimerdatabase.ht.st.
Pubmed: ABSTRACT Article (Peccoud1996) Peccoud, J. & Jacob, C. Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys J, 1996, 71, 101-108 Abstract: Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplication has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.
Pubmed: ABSTRACT Article (Peirson2007) Peirson, S.N. & Butler, J.N. Quantitative polymerase chain reaction. Methods Mol Biol, 2007, 362, 349-362 Abstract: Quantitative PCR (qPCR) has entered widespread use with the increasing availability of real-time PCR. By the incorporation of fluorescent dyes in the reaction mixture, increases in amplification products can be monitored throughout the reaction, enabling measurements to be taken in the exponential phase of the reaction, before the reaction plateau. Whatever the platform or chemistry involved, the starting point of a real-time assay is a tissue-specific RNA and the end point of a real-time reaction is an amplification plot. As such, rather than focusing on specific platforms or chemistries, herein we address the basic principles that underlie sample preparation, experimental design, use of internal controls, assay considerations, and approaches to data analysis. The advent of real-time PCR has enabled high-throughput analysis of multiple transcripts from small tissue samples, with an unparalleled dynamic range and sensitivity. However, to new users, this technique may seem to require extensive optimization and troubleshooting to obtain reliable data; this is further compounded by the mass of technical variations present throughout the literature. The aim of this article is to provide the necessary basics to get a quantitative real-time PCR assay up and running, and to address some of the problems that may arise and how these may be resolved.
Pubmed: ABSTRACT Article (Peirson2003) Peirson, S.N.; Butler, J.N. & Foster, R.G. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis Nucleic Acids Res, 2003, 31, e73 Abstract: Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.
Pubmed: ABSTRACT Article (Petersen2003) Petersen, M. & Wengel, J. LNA: a versatile tool for therapeutics and genomics Trends Biotechnol, 2003, 21, 74-81 Abstract: Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes (NAzyme. Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.
Pubmed: ABSTRACT Article (Petes2001) Petes, T.D. Meiotic recombination hot spots and cold spots Nat Rev Genet, 2001, 2, 360-9 Abstract: Meiotic recombination events are distributed unevenly throughout eukaryotic genomes. This inhomogeneity leads to distortions of genetic maps that can hinder the ability of geneticists to identify genes by map-based techniques. Various lines of evidence, particularly from studies of yeast, indicate that the distribution of recombination events might reflect, at least in part, global features of chromosome structure, such as the distribution of modified nucleosomes.
Pubmed: ABSTRACT Article (Pettersson2003) Pettersson, M.; Bylund, M. & Alderborn, A. Molecular haplotype determination using allele-specific PCR and pyrosequencing technology Genomics, 2003, 82, 390-6 Abstract: Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.
Pubmed: ABSTRACT Article (Pfaffl2001) Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res, 2001, 29, e45 Abstract: Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Pubmed: ABSTRACT Article (Pfaffl2002) Pfaffl, M.W.; Horgan, G.W. & Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR Nucleic Acids Res, 2002, 30, e36 Abstract: Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST is explained and the usefulness of relative expression in real-time PCR using REST is discussed. The latest software version of REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
Pubmed: ABSTRACT Article (Phillips2003) Phillips, M.S.; Lawrence, R.; Sachidanandam, R.; Morris, A.P.; Balding, D.J.; Donaldson, M.A.; Studebaker, J.F.; Ankener, W.M.; Alfisi, S.V.; Kuo, F.S.; Camisa, A.L.; Pazorov, V.; Scott, K.E.; Carey, B.J.; Faith, J.; Katari, G.; Bhatti, H.A.; Cyr, J.M.; Derohannessian, V.; Elosua, C.; Forman, A.M.; Grecco, N.M.; Hock, C.R.; Kuebler, J.M.; Lathrop, J.A.; Mockler, M.A.; Nachtman, E.P.; Restine, S.L.; Varde, S.A.; Hozza, M.J.; Gelfand, C.A.; Broxholme, J.; Abecasis, G.R.; Boyce-Jacino, M.T. & Cardon, L.R. Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots Nat Genet, 2003, 33, 382-7 Abstract: Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved (aplotype blocks refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.
Pubmed: ABSTRACT Article (Pickering2002) Pickering, J.; Bamford, A.; Godbole, V.; Briggs, J.; Scozzafava, G.; Roe, P.; Wheeler, C.; Ghouze, F. & Cuss, S. Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms Nucleic Acids Res, 2002, 30, e60 Abstract: Association studies using common sequence variants or single nucleotide polymorphisms (SNPs) may provide a powerful approach to dissect the genetic inheritance of common complex traits. Such studies necessitate the development of cost-effective, high throughput technologies for scoring SNPs. The method described in this paper for the co-detection of both alleles of a SNP in a single homogeneous reaction combines the specificity of a high fidelity DNA ligation step with the power of rolling circle amplification. The incorporation of Amplifluor energy transfer primers enables signal detection in a homogeneous format, making this approach highly amenable to automation. The adaptation of the genotyping method for high throughput screening using conventional liquid handling systems is described.
Pubmed: ABSTRACT Article (Pierre1994) Pierre, B.S.; Neustock, P.; Schramm, U.; Wilhelm, D.; Kirchner, H. & Bein, G. Seasonal breakdown of polymerase chain reaction. Lancet, 1994, 343, 673
Pubmed: ABSTRACT Article (Ponomarenko2002) Ponomarenko, J.V.; Orlova, G.V.; Merkulova, T.I.; Gorshkova, E.V.; Fokin, O.N.; Vasiliev, G.V.; Frolov, A.S. & Ponomarenko, M.P. rSNP_Guide: an integrated database-tools system for studying SNPs and site-directed mutations in transcription factor binding sites Hum Mutat, 2002, 20, 239-48 Abstract: Since the human genome was sequenced in draft, single nucleotide polymorphism (SNP) analysis has become one of the keynote fields of bioinformatics. We have developed an integrated database-tools system, rSNP_Guide (http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/), devoted to prediction of transcription factor (TF) binding sites, alterations of which could be associated with disease phenotype. By inputting data on alterations in DNA sequence and in DNA binding pattern of an unknown TF, rSNP_Guide searches for a known TF with alterations in the recognition score calculated on the basis of TF site's sequence and consistent with the input alterations in DNA binding to the unknown TF. Our system has been tested on many relationships between known TF sites and diseases, as well as on site-directed mutagenesis data. Experimental verification of rSNP_Guide system was made on functionally important SNPs in human TDO2and mouse K-ras genes. Additional examples of analysis are reported involving variants in the human gammaA-globin (HBG1), hsp70(HSPA1A), and Factor IX (F9) gene promoters.
Pubmed: ABSTRACT Article (Pregibon2007) Pregibon, D.C.; Toner, M. & Doyle, P.S. Multifunctional encoded particles for high-throughput biomolecule analysis. Science, 2007, 315, 1393-1396 Abstract: High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.
Pubmed: ABSTRACT Article (Prince2001) Prince, J.A.; Feuk, L.; Howell, W.M.; Jobs, M.; Emahazion, T.; Blennow, K. & Brookes, A.J. Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation Genome Res, 2001, 11, 152-62 Abstract: We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.
Pubmed: ABSTRACT Article (Pruitt2001) Pruitt, K.D. & Maglott, D.R. RefSeq and LocusLink: NCBI gene-centered resources Nucleic Acids Res, 2001, 29, 137-40 Abstract: Thousands of genes have been painstakingly identified and characterized a few genes at a time. Many thousands more are being predicted by large scale cDNA and genomic sequencing projects, with levels of evidence ranging from supporting mRNA sequence and comparative genomics to computing ab initio models. This, coupled with the burgeoning scientific literature, makes it critical to have a comprehensive directory for genes and reference sequences for key genomes. The NCBI provides two resources, LocusLink and RefSeq, to meet these needs. LocusLink organizes information around genes to generate a central hub for accessing gene-specific information for fruit fly, human, mouse, rat and zebrafish. RefSeq provides reference sequence standards for genomes, transcripts and proteins; human, mouse and rat mRNA RefSeqs, and their corresponding proteins, are discussed here. Together, RefSeq and LocusLink provide a non-redundant view of genes and other loci to support research on genes and gene families, variation, gene expression and genome annotation. Additional information about LocusLink and RefSeq is available at http://www.ncbi.nlm.nih.gov/LocusLink/.
Pubmed: ABSTRACT Article (Psaltis2006) Psaltis, D.; Quake, S.R. & Yang, C. Developing optofluidic technology through the fusion of microfluidics and optics. Nature, 2006, 442, 381-386 Abstract: We describe devices in which optics and fluidics are used synergistically to synthesize novel functionalities. Fluidic replacement or modification leads to reconfigurable optical systems, whereas the implementation of optics through the microfluidic toolkit gives highly compact and integrated devices. We categorize optofluidics according to three broad categories of interactions: fluid-solid interfaces, purely fluidic interfaces and colloidal suspensions. We describe examples of optofluidic devices in each category.
Pubmed: ABSTRACT Article (Qin2006) Qin, L.; Beyer, R.P.; Hudson, F.N.; Linford, N.J.; Morris, D.E. & Kerr, K.F. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR. BMC Bioinformatics, 2006, 7, 23 Abstract: BACKGROUND: There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. RESULTS: We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch) and a sequence-based method of background adjustment (as in gcRMA) as the most important factors in methods' performances. However, we found poor reliability for methods using mismatch probes for low-intensity genes, which is in agreement with previous studies. CONCLUSION: We advocate use of sequence-based background adjustment in lieu of mismatch adjustment to achieve the best results across the intensity spectrum. No method of normalization or probeset summary showed any consistent advantages.
Pubmed: ABSTRACT Misc (R-Project.org) R.D.C. Team The R Project for Statistical Computing [www.R-Project.org]
Article (Rajeevan2003) Rajeevan, H.; Osier, M.V.; Cheung, K.H.; Deng, H.; Druskin, L.; Heinzen, R.; Kidd, J.R.; Stein, S.; Pakstis, A.J.; Tosches, N.P.; Yeh, C.C.; Miller, P.L. & Kidd, K.K. ALFRED: the ALelle FREquency Database. Update Nucleic Acids Res, 2003, 31, 270-1 Abstract: Elaboration of ALFRED (http://alfred.med.yale.edu) is being continued in two directions. One of which is developing tools for efficiently annotating the entries and checking the integrity of the data already in the database while the other is to increase the quantity and accessibility of data. Information contained in ALFRED such as, polymorphic sites, number of populations and frequency tables (one sample typed for one site) has significantly increased.
Pubmed: ABSTRACT Article (Ramensky2002) Ramensky, V.; Bork, P. & Sunyaev, S. Human non-synonymous SNPs: server and survey Nucleic Acids Res, 2002, 30, 3894-900 Abstract: Human single nucleotide polymorphisms (SNPs) represent the most frequent type of human population DNA variation. One of the main goals of SNP research is to understand the genetics of the human phenotype variation and especially the genetic basis of human complex diseases. Non-synonymous coding SNPs (nsSNPs) comprise a group of SNPs that, together with SNPs in regulatory regions, are believed to have the highest impact on phenotype. Here we present a World Wide Web server to predict the effect of an nsSNP on protein structure and function. The prediction method enabled analysis of the publicly available SNP database HGVbase, which gave rise to a dataset of nsSNPs with predicted functionality. The dataset was further used to compare the effect of various structural and functional characteristics of amino acid substitutions responsible for phenotypic display of nsSNPs. We also studied the dependence of selective pressure on the structural and functional properties of proteins. We found that in our dataset the selection pressure against deleterious SNPs depends on the molecular function of the protein, although it is insensitive to several other protein features considered. The strongest selective pressure was detected for proteins involved in transcription regulation.
Pubmed: ABSTRACT Article (Ranade2001) Ranade, K.; Chang, M.S.; Ting, C.T.; Pei, D.; Hsiao, C.F.; Olivier, M.; Pesich, R.; Hebert, J.; Chen, Y.D.; Dzau, V.J.; Curb, D.; Olshen, R.; Risch, N.; Cox, D.R. & Botstein, D. High-throughput genotyping with single nucleotide polymorphisms. Genome Res, 2001, 11, 1262-1268 Abstract: To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.
Pubmed: ABSTRACT Article (Rao2003) Rao, K.V.; Stevens, P.W.; Hall, J.G.; Lyamichev, V.; Neri, B.P. & Kelso, D.M. Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres Nucleic Acids Res, 2003, 31, e66 Abstract: Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.
Pubmed: ABSTRACT Article (Rawat2006) Rawat, M.; Singh, D.; Saraf, S. & Saraf, S. Nanocarriers: promising vehicle for bioactive drugs. Biol Pharm Bull, 2006, 29, 1790-1798 Abstract: Development of new delivery systems that deliver the potential drug specifically to the target site in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field nanobiotechnology which involves the combination of two most promising technologies of 21st century-biotechnology and nanotechnology. Nanobiotechnology encompasses a wide array of different techniques to improve the delivery of biotech drugs, and nanoparticles offer the most suitable form whose properties can be tailored by chemical methods. This review highlights the different types of nanoparticulate delivery systems employed for biotech drugs in the field of molecular medicine with a short overlook at its applications and the probable associated drawbacks.
Pubmed: ABSTRACT Article (Rehman1999) Rehman, F.N.; Audeh, M.; Abrams, E.S.; Hammond, P.W.; Kenney, M. & Boles, T.C. Immobilization of acrylamide-modified oligonucleotides by co-polymerization Nucleic Acids Res, 1999, 27, 649-55 Abstract: A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.
Pubmed: ABSTRACT Article (Reich2001) Reich, D.E.; Cargill, M.; Bolk, S.; Ireland, J.; Sabeti, P.C.; Richter, D.J.; Lavery, T.; Kouyoumjian, R.; Farhadian, S.F.; Ward, R. & Lander, E.S. Linkage disequilibrium in the human genome Nature, 2001, 411, 199-204 Abstract: With the availability of a dense genome-wide map of single nucleotide polymorphisms (SNPs), a central issue in human genetics is whether it is now possible to use linkage disequilibrium (LD) to map genes that cause disease. LD refers to correlations among neighbouring alleles, reflecting 'haplotypes' descended from single, ancestral chromosomes. The size of LD blocks has been the subject of considerable debate. Computer simulations and empirical data have suggested that LD extends only a few kilobases (kb) around common SNPs, whereas other data have suggested that it can extend much further, in some cases greater than 100 kb. It has been difficult to obtain a systematic picture of LD because past studies have been based on only a few (1-3) loci and different populations. Here, we report a large-scale experiment using a uniform protocol to examine 19 randomly selected genomic regions. LD in a United States population of north-European descent typically extends 60 kb from common alleles, implying that LD mapping is likely to be practical in this population. By contrast, LD in a Nigerian population extends markedly less far. The results illuminate human history, suggesting that LD in northern Europeans is shaped by a marked demographic event about 27,000-53,000 years ago.
Pubmed: ABSTRACT Article (Reyes2002) Reyes, D.R.; Iossifidis, D.; Auroux, P. & Manz, A. Micro total analysis systems. 1. Introduction, theory, and technology. Anal Chem, 2002, 74, 2623-2636
Pubmed: ABSTRACT Article (Rickert2004) Rickert, A.M.; Borodina, T.A.; Kuhn, E.J.; Lehrach, H. & Sperling, S. Refinement of single-nucleotide polymorphism genotyping methods on human genomic DNA: amplifluor allele-specific polymerase chain reaction versus ligation detection reaction-TaqMan Anal Biochem, 2004, 330, 288-97 Abstract: Single-nucleotide polymorphisms (SNPs) have proven to be powerful genetic markers for a variety of genetic applications, e.g., association studies leading to dissection of both monogenetic and complex diseases. However, no single SNP genotyping method has been broadly accepted. In the present study, we compared and refined two promising methods with potential for research and for diagnostic SNP genotyping: Amplifluor allele-specific polymerase chain reaction (PCR) and ligation detection reaction (LDR)-TaqMan. The methods are based on allele-specific primer extension and allele-specific ligation, respectively. Since LDR-TaqMan had previously been tested on just Arabidopsis thaliana, we adjusted the method for the more complex human genome. Amplifluor allele-specific PCR has a single-step and closed-tube format, whereas the LDR-TaqMan assay comprises two simple steps. Contrary to the primer-extension-based method, the ligation-based method can be multiplexed. Refining the LDR-TaqMan technique, we successfully replaced a previously suggested three-step multiplexing procedure with a less laborious two-step approach. Comparing refined LDR-TaqMan with Amplifluor allele-specific PCR in a family-based study, both techniques appeared similar with respect to high robustness and accuracy. As both approaches utilize primers with common tails, all SNPs can be assayed with the same couple of fluorescence reporting reagents, ensuring low establishing and running expenses.
Pubmed: ABSTRACT Article (Rinnova2000) Rinnova, M.; Novakova, M.; Kasicka, V. & Jiracek, J. Side reactions during photochemical cleavage of an alpha-methyl-6-nitroveratryl-based photolabile linker J Pept Sci, 2000, 6, 355-65 Abstract: The mechanisms of reactions causing irreversible inhibition of the activity of enzymes when irradiated in the presence of the recently developed alpha-methyl-6-nitroveratryl-based photolinker [Holmes CP. J. Org. Chem. 1997; 62: 2370-2380] have been investigated. Several experiments based on the interaction of the photolinker with model peptides or n-butylamine have been accomplished. A complexity of products, resulting from the side reactions competing with the ormalphotocleavage of the linker, have been found. The amino and thiol groups of the molecules present in the solvents upon irradiation were recognized as having a major influence on the course of photolysis. Some of these side products resulting from the interaction with amines were identified and the mechanisms by which they can be generated are discussed. The mechanism of the interaction of the thiol groups present in peptides or proteins with the photolinker is unclear and it remains to be further elucidated. It was found that the undesirable effects are favored by a basic pH and are largely reduced by a slightly acidic pH, together with the presence of dithiothreitol. Significant positive effects of dithiothreitol have been observed on the rate as well as the yield of the photocleavage. These results demonstrate that the use of photolabile linkers in biological media can be accompanied by undesired effects, which can be largely reduced by choosing appropriate conditions and additives.
Pubmed: ABSTRACT Article (Ririe1997) Ririe, K.M.; Rasmussen, R.P. & Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction Anal Biochem, 1997, 245, 154-60 Abstract: A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.
Pubmed: ABSTRACT Article (Risch2000) Risch, N.J. Searching for genetic determinants in the new millennium Nature, 2000, 405, 847-56 Abstract: Human genetics is now at a critical juncture. The molecular methods used successfully to identify the genes underlying rare mendelian syndromes are failing to find the numerous genes causing more common, familial, non-mendelian diseases. With the human genome sequence nearing completion, new opportunities are being presented for unravelling the complex genetic basis of non-mendelian disorders based on large-scale genome-wide studies. Considerable debate has arisen regarding the best approach to take. In this review I discuss these issues, together with suggestions for optimal post-genome strategies.
Pubmed: ABSTRACT Article (Riva2002) Riva, A. & Kohane, I.S. SNPper: retrieval and analysis of human SNPs Bioinformatics, 2002, 18, 1681-5 Abstract: Motivation: Single Nucleotide Polymorphisms (SNPs) are an increasingly important tool for the study of the human genome. SNPs can be used as markers to create high-density genetic maps, as causal candidates for diseases, or to reconstruct the history of our genome. SNP-based studies rely on the availability of large numbers of validated, high-frequency SNPs whose position on the chromosomes is known with precision. Although large collections of SNPs exist in public databases, researchers need tools to effectively retrieve and manipulate them. Results: We describe the implementation and usage of SNPper, a web-based application to automate the tasks of extracting SNPs from public databases, analyzing them and exporting them in formats suitable for subsequent use. Our application is oriented toward the needs of candidate-gene, whole-genome and fine-mapping studies, and provides several flexible ways to present and export the data. The application has been publicly available for over a year, and has received positive user feedback and high usage levels. Availability: SNPper is freely available at http://snpper.chip.org/. Registration is optional and provides access to some advanced features. Contact: Alberto.Riva@TCH.Harvard.edu
Pubmed: ABSTRACT Article (Riva2001) Riva, A. & Kohane, I.S. A web-based tool to retrieve human genome polymorphisms from public databases Proc AMIA Symp, 2001, 558-62 Abstract: Single Nucleotide Polymorphisms (SNPs) are the most important source of variation in our genome, and an invaluable tool in the hands of researchers who investigate genetic diseases. Databases of SNPs are growing at a very fast rate, and the ability to perform large-scale, high-resolution association studies is quickly becoming a reality. In this paper we describe SNPper, a web-based tool to search for SNPs in public databases. The system allows searching for all SNPs in a given set of genes (for candidate gene studies) or in a specified region of a chromosome. The information displayed for each gene or each SNP is fully annotated and linked to the leading bioinformatics web sites. The first release of SNPper is available on the web, and has received positive feedback from the genetic and bioinformatics community.
Pubmed: ABSTRACT Article (Rodriguez2003) Rodriguez, I.; Lesaicherre, M.; Tie, Y.; Zou, Q.; Yu, C.; Singh, J.; Meng, L.T.; Uppili, S.; Li, S.F.; Gopalakrishnakone, P. & Selvanayagam, Z.E. Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis Electrophoresis, 2003, 24, 172-8 Abstract: An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel oint-heatingscheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.
Pubmed: ABSTRACT Article (Rohde2001) Rohde, K. & Fuerst, R. Haplotyping and estimation of haplotype frequencies for closely linked biallelic multilocus genetic phenotypes including nuclear family information Hum Mutat, 2001, 17, 289-95 Abstract: With the discovery of single nucleotide polymorphisms (SNP) along the genome, genotyping of large samples of biallelic multilocus genetic phenotypes for (fine) mapping of disease genes or for population studies has become standard practice. A genetic trait, however, is mainly caused by an underlying defective haplotype, and populations are best characterized by their haplotype frequencies. Therefore, it is essential to infer from the phase-unknown genetic phenotypes in a sample drawn from a population the haplotype frequencies in the population and the underlying haplotype pairs in the sample in order to find disease predisposing genes by some association or haplotype sharing algorithm. Haplotype frequencies and haplotype pairs are estimated via a maximum likelihood approach by a well-known expectation maximization (EM) algorithm, adapting it to a large number (up to 30) of biallelic loci (SNP), and including nuclear family information, if available, into the analysis. Parents are treated as an independent sample from the population. Their genotyped offspring reduces the number of potential haplotype pairs for both parents, resulting in a higher accuracy of the estimation, and may also reduce computation time. In a series of simulations our approach of including nuclear family information has been tested against both the EM algorithm without nuclear family information and an alternative approach using GENEHUNTER for the haplotyping of the families, using the locus-by-locus allele counts of the sample. Our new approach is more precise in haplotyping in cases of a high number of heterozygous loci, whereas for a moderate number of heterozygous positions in the sample all three different approaches gave the same perfect results.
Pubmed: ABSTRACT Article (Romeo2007) Romeo, S.; Pennacchio, L.A.; Fu, Y.; Boerwinkle, E.; Hansen, A.T.; Hobbs, H.H. & Cohen, J.C. Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL. Nat Genet, 2007 Abstract: Resequencing genes provides the opportunity to assess the full spectrum of variants that influence complex traits. Here we report the first application of resequencing to a large population (n = 3,551) to examine the role of the adipokine ANGPTL4 in lipid metabolism. Nonsynonymous variants in ANGPTL4 were more prevalent in individuals with triglyceride levels in the lowest quartile than in individuals with levels in the highest quartile (P = 0.016). One variant (E40K), present in approximately 3% of European Americans, was associated with significantly lower plasma levels of triglyceride and higher levels of high-density lipoprotein cholesterol in European Americans from the Atherosclerosis Risk in Communities Study and in Danes from the Copenhagen City Heart Study. The ratio of nonsynonymous to synonymous variants was higher in European Americans than in African Americans (4:1 versus 1.3:1), suggesting population-specific relaxation of purifying selection. Thus, resequencing of ANGPTL4 in a multiethnic population allowed analysis of the phenotypic effects of both rare and common variants while taking advantage of genetic variation arising from ethnic differences in population history.
Pubmed: ABSTRACT Article (Roper2005) Roper, M.G.; Easley, C.J. & Landers, J.P. Advances in polymerase chain reaction on microfluidic chips. Anal Chem, 2005, 77, 3887-3893
Pubmed: ABSTRACT Article (Rosa2005) Rosa, M.; Dias, R.; da Graça Miguel, M. & Lindman, B. DNA-cationic surfactant interactions are different for double- and single-stranded DNA. Biomacromolecules, 2005, 6, 2164-2171 Abstract: The stability of DNA in solution and the phase behavior in mixtures with dodecyltrimethylammonium bromide (DTAB) were investigated. By means of circular dichroism, UV absorption, and differential scanning calorimetry, we found that for dilute solutions of DNA with no addition of salt the DNA molecules are in the single-stranded conformation, whereas the addition of a small amount of NaBr, 1 mM, is sufficient to stabilize the DNA double-helix. Furthermore, at higher DNA concentrations, native DNA becomes the most stable structure, which is due to a self-screening effect. By phase diagram determinations of the DNA-surfactant system, we found that the effect of salt on phase behavior mainly relates to a difference in interaction of the amphiphile between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The difference in association between ss and dsDNA with surfactants of different chain lengths can be interpreted in terms of an interplay between hydrophobic and electrostatic interactions, the latter being influenced by polymer flexibility. In this way, a nonmonotonic variation can be rationalized. A crossing of the phase separation lines with DNA concentration can be rationalized in terms of a change in relative stability of ss and dsDNA. The fact that ssDNA phase separates earlier than dsDNA in association with DTAB, may serve as a basis for a method of easily separating dsDNA from ssDNA by the addition of surfactant; this is verified as monitored by circular dichroism measurements.
Pubmed: ABSTRACT Article (Rouchka2002) Rouchka, E.C.; Gish, W. & States, D.J. Comparison of whole genome assemblies of the human genome Nucleic Acids Res, 2002, 30, 5004-14 Abstract: A fundamental problem in the human genome project is uncovering the correct assembly of the human genome. Many studies, including transcriptional analysis, SNP detection and characterization, gene finding and EST clustering, use genome assemblies as templates so it is important to determine the consistency among the various whole genome assemblies. A comparison of the order and orientation of the GenBank entries used to construct the NCBI and UCSC Goldenpath assemblies was made. In addition, a sequence level comparison was performed using MULTI, an efficient database search tool developed to make whole genome comparisons possible. The resulting comparisons show significant discrepancies in the sequence as well as in the order and orientation of GenBank entries used in constructing the NCBI and UCSC assemblies.
Pubmed: ABSTRACT Article (Ruano1990) Ruano, G.; Kidd, K.K. & Stephens, J.C. Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules Proc Natl Acad Sci U S A, 1990, 87, 6296-300 Abstract: We have developed a reliable method for the direct resolution of haplotypes or linkage phase from individuals who are multiply heterozygous in a given genomic region. The method is based on single-molecule dilution (SMD) of genomic template and amplification via biphasic polymerase chain reaction (booster PCR). We have verified the feasibility of the SMD method for a highly polymorphic region within the beta-globin cluster by analysis of triply heterozygous individuals of known haplotype. This approach should be useful in many studies in population or evolutionary genetics and in a variety of clinical settings.
Pubmed: ABSTRACT Article (Rubina2003) Rubina, A.Y.; Dementieva, E.I.; Stomakhin, A.A.; Darii, E.L.; Panov, S.V.; Barsky, V.E.; Ivanov, S.M.; Konovalova, E.V. & Mirzabekov, A.D. Hydrogel-based protein microchips: manufacturing, properties, and applications Biotechniques, 2003, 34, 1008-14, 1016-20, 1022 Abstract: Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.
Pubmed: ABSTRACT Article (Rubina2004) Rubina, A.Y.; Panov, S.V.; Dementieva, E.I.; Penov, D.N.; Butygin, A.V.; Vasiliskov, V.A.; Chudinov, A.V.; Mikheikin, A.L.; Mikhailovich, V.M. & Mirzabekov, A.D. Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production Anal Biochem, 2004, 325, 92-106 Abstract: Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.
Pubmed: ABSTRACT Article (Rudi2003) Rudi, K.; Rud, I. & Holck, A. A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed Nucleic Acids Res, 2003, 31, e62 Abstract: We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5EADregion and a 3region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.
Pubmed: ABSTRACT Article (Russom2003) Russom, A.; Ahmadian, A.; Andersson, H.; Nilsson, P. & Stemme, G. Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device Electrophoresis, 2003, 24, 158-61 Abstract: We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.
Pubmed: ABSTRACT Article (Russom2004) Russom, A.; Haasl, S.; Ohlander, A.; Mayr, T.; Brookes, A.J.; Andersson, H. & Stemme, G. Genotyping by dynamic heating of monolayered beads on a microheated surface. Electrophoresis, 2004, 25, 3712-3719 Abstract: A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for single-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4 degrees C-6 degrees C/min compared to earlier reported values of 2 degrees C-3 degrees C/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 microm was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.
Pubmed: ABSTRACT Article (Rutledge2003) Rutledge, R.G. & Cote, C. Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res., 2003, 31, e93 Abstract: Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.
PDF: Rutledge2003.pdf Article (Rutledge2003a) Rutledge, R.G. & Cote, C. Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res, 2003, 31, e93 Abstract: Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.
Pubmed: ABSTRACT Article (Ryley2006) Ryley, J. & Smith, O.M.P. Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae. Yeast, 2006, 23, 1065-1073 Abstract: We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.
Pubmed: ABSTRACT Article (Saal2002) Saal, L.H.; Troein, C.; Vallon-Christersson, J.; Gruvberger, S.; Borg, A. & Peterson, C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data Genome Biol, 2002, 3, SOFTWARE0003 Abstract: The microarray technique requires the organization and analysis of vast amounts of data. These data include information about the samples hybridized, the hybridization images and their extracted data matrices, and information about the physical array, the features and reporter molecules. We present a web-based customizable bioinformatics solution called BioArray Software Environment (BASE) for the management and analysis of all areas of microarray experimentation. All software necessary to run a local server is freely available.
Pubmed: ABSTRACT Article (Sachidanandam2001) Sachidanandam, R.; Weissman, D.; Schmidt, S.C.; Kakol, J.M.; Stein, L.D.; Marth, G.; Sherry, S.; Mullikin, J.C.; Mortimore, B.J.; Willey, D.L.; Hunt, S.E.; Cole, C.G.; Coggill, P.C.; Rice, C.M.; Ning, Z.; Rogers, J.; Bentley, D.R.; Kwok, P.Y.; Mardis, E.R.; Yeh, R.T.; Schultz, B.; Cook, L.; Davenport, R.; Dante, M.; Fulton, L.; Hillier, L.; Waterston, R.H.; McPherson, J.D.; Gilman, B.; Schaffner, S.; Etten, W.J.V.; Reich, D.; Higgins, J.; Daly, M.J.; Blumenstiel, B.; Baldwin, J.; Thomann, N.S.; Zody, M.C.; Linton, L.; Lander, E.S.; Altshuler, D. & Group, I.S.M.W. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature, 2001, 409, 928-933 Abstract: We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.
Pubmed: ABSTRACT Article (Safran2003) Safran, M.; Chalifa-Caspi, V.; Shmueli, O.; Olender, T.; Lapidot, M.; Rosen, N.; Shmoish, M.; Peter, Y.; Glusman, G.; Feldmesser, E.; Adato, A.; Peter, I.; Khen, M.; Atarot, T.; Groner, Y. & Lancet, D. Human Gene-Centric Databases at the Weizmann Institute of Science: GeneCards, UDB, CroW 21 and HORDE Nucleic Acids Res, 2003, 31, 142-6 Abstract: Recent enhancements and current research in the GeneCards (GC) (http://bioinfo.weizmann.ac.il/cards/) project are described, including the addition of gene expression profiles and integrated gene locations. Also highlighted are the contributions of specialized associated human gene-centric databases developed at the Weizmann Institute. These include the Unified Database (UDB) (http://bioinfo.weizmann.ac.il/udb) for human genome mapping, the human Chromosome 21 database at the Weizmann Insti-tute (CroW 21) (http://bioinfo.weizmann.ac.il/crow21), and the Human Olfactory Receptor Data Explora-torium (HORDE) (http://bioinfo.weizmann.ac.il/HORDE). The synergistic relationships amongst these efforts have positively impacted the quality, quantity and usefulness of the GeneCards gene compendium.
Pubmed: ABSTRACT Article (Saiki1985) Saiki, R.K.; Scharf, S.; Faloona, F.; Mullis, K.B.; Horn, G.T.; Erlich, H.A. & Arnheim, N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science, 1985, 230, 1350-4 Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
Pubmed: ABSTRACT Article (Salzberg2005) Salzberg, S.L. & Yorke, J.A. Beware of mis-assembled genomes. Bioinformatics, 2005, 21, 4320-4321
Pubmed: ABSTRACT Book (Sambrook1989) Sambrook, J.; Fritsch, E. & Maniatis, T. Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory Press, 1989
Article (Sano1992) Sano, T.; Smith, C.L. & Cantor, C.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science, 1992, 258, 120-122 Abstract: An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 x 10(-22) moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately x 10(5)) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.
Pubmed: ABSTRACT Article (Sant2006) Sant, H.J. & Gale, B.K. Geometric scaling effects on instrumental plate height in field flow fractionation. J Chromatogr A, 2006, 1104, 282-290 Abstract: This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (mu-TAS).
Pubmed: ABSTRACT Article (SantaLucia1998) SantaLucia, J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics Proc Natl Acad Sci U S A, 1998, 95, 1460-5 Abstract: A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodynamics is presented. DNA NN DeltaG degrees 37 parameters from seven laboratories are presented in the same format so that careful comparisons can be made. The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used different methods of data analysis; used different salt concentrations; and presented the NN thermodynamics in different formats. As a result of these differences, there has been much confusion regarding the NN thermodynamics of DNA polymers and oligomers. Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies remain. Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodynamics. Empirical salt dependencies are also derived for oligonucleotides and polymers.
Pubmed: ABSTRACT Article (SantaLucia1996) SantaLucia, J.; Allawi, H.T. & Seneviratne, P.A. Improved nearest-neighbor parameters for predicting DNA duplex stability Biochemistry, 1996, 35, 3555-62 Abstract: Thermodynamic data were determined from UV absorbance vs temperature profiles of 23 oligonucleotides. These data were combined with data from the literature for 21 sequences to derive improved parameters for the 10 Watson-Crick nearest neighbors. The observed trend in nearest-neighbor stabilities at 37 degrees C is GC > CG > GG > GA approximately GT approximately CA > CT > AA > AT > TA (where only the top strand is shown for each nearest neighbor). This trend suggests that both sequence and base composition are important determinants of DNA duplex stability. On average, the improved parameters predict deltaG degrees(37), deltaH degrees, deltaS degrees, and T(m) within 4%, 7%, 8%, and 2 degrees C, respectively. The parameters are optimized for the prediction of oligonucleotides dissolved in 1 M NaC1.
Pubmed: ABSTRACT Article (Sasaki1997) Sasaki, N.; Izawa, M.; Shimojo, M.; Shibata, K.; Akiyama, J.; Itoh, M.; Nagaoka, S.; Carninci, P.; Okazaki, Y.; Moriuchi, T.; Muramatsu, M.; Watanabe, S. & Hayashizaki, Y. A novel control system for polymerase chain reaction using a RIKEN GS384 thermalcycler DNA Res, 1997, 4, 387-91 Abstract: We have developed a novel high-throughput thermalcycler, the RIKEN GS384, which has a maximum of 1536 wells and whose temperature can be controlled accurately and simultaneously for a very small volume of a reaction mixture. In practice, the reaction is carried out using four 384-well (3.5 mm in diameter) plate formats which can be automatically moved using a robotic arm. To achieve accurate temperature control with high thermo-conductivity, we adopted Teflon-coated aluminum well plates closely sandwiched between silicon sheet-covered lids on top and a graphite sheet below. The lids were kept at a higher temperature (2 to 5 degrees C) than the reaction wells. The temperature of the 1536 sample wells was controlled accurately without temperature variability among the wells or evaporation, even for samples of very small volume (minimum 2 microliters). We also developed a new type of plate format which is similar to the 384-well place in terms of plate size, shape, and material, but which differs in the number (1536) and size (1.6 mm in diameter) of the wells. Since the amplification reactions could be done precisely as well, a total of 6144 reactions can potentially be carried out simultaneously using the GS384 thermalcycler. This is very promising for DNA microfabrication technology. This thermalcycler offers the advantage of high-throughput DNA analysis which should be useful for DNA diagnoses or for the human genome project.
Pubmed: ABSTRACT Article (Sauer2002) Sauer, S.; Gelfand, D.H.; Boussicault, F.; Bauer, K.; Reichert, F. & Gut, I.G. Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry Nucleic Acids Res, 2002, 30, e22 Abstract: In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.
Pubmed: ABSTRACT Article (Sauer2003) Sauer, S. & Gut, I.G. Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry Rapid Commun Mass Spectrom, 2003, 17, 1265-72 Abstract: Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed harge-tagging The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.
Pubmed: ABSTRACT Article (Sauer2002a) Sauer, S. & Gut, I.G. Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry J Chromatogr B Analyt Technol Biomed Life Sci, 2002, 782, 73-87 Abstract: In recent years matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) has emerged as a very powerful method for genotyping single nucleotide polymorphisms. The accuracy, speed of data accumulation, and data structure are the major features of MALDI. Several SNP genotyping methods have been implemented with a high degree of automation and are being applied for large-scale association studies. Most methods for SNP genotyping using MALDI mass spectrometric detection and their potential application for high-throughput are reviewed here.
Pubmed: ABSTRACT Article (Sauer2004) Sauer, S.; Kepper, P.; Smyra, A.; Dahl, A.; Ferse, F.T.; Lehrach, H. & Reinhardt, R. Automated solid-phase extraction for purification of single nucleotide polymorphism genotyping products prior to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometric analysis J Chromatogr A, 2004, 1049, 9-16 Abstract: In this article, we describe the application of a novel micro elution solid-phase extraction method for purification of short stretches of DNA products of single nucleotide polymorphisms (SNPs) prior to MALDI mass spectrometry analysis. An important feature of our method is that the purification columns containing a copolymer of divinylbenzene and N-vinylpyrrolidone can be used several times thereby significantly reducing costs. We implemented this DNA purification technology into a fully automated procedure including molecular biology, MALDI sample preparation, automated mass spectrometric analysis and SNP allele calling by software. Due to the facile purification protocol, the methodology shown could furthermore be used for other applications where efficient medium to high-throughput purification of nucleic acids is required.
Pubmed: ABSTRACT Article (Sauer2005) Sauer, S.; Lange, B.M.; Gobom, J.; Nyarsik, L.; Seitz, H. & Lehrach, H. Miniaturization in functional genomics and proteomics Nat Rev Genet, 2005, 6, 465-76 Abstract: Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.
Pubmed: ABSTRACT Article (Sauer2000) Sauer, S.; Lechner, D.; Berlin, K.; Lehrach, H.; Escary, J.L.; Fox, N. & Gut, I.G. A novel procedure for efficient genotyping of single nucleotide polymorphisms Nucleic Acids Res, 2000, 28, E13 Abstract: Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the OOD Assayfor SNP analysis.
Pubmed: ABSTRACT Article (Sauer2000a) Sauer, S.; Lechner, D.; Berlin, K.; Plancon, C.; Heuermann, A.; Lehrach, H. & Gut, I.G. Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay Nucleic Acids Res, 2000, 28, E100 Abstract: Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.
Pubmed: ABSTRACT Article (Sauer2003a) Sauer, S.; Lehrach, H. & Reinhardt, R. MALDI mass spectrometry analysis of single nucleotide polymorphisms by photocleavage and charge-tagging Nucleic Acids Res, 2003, 31, e63 Abstract: High-throughput procedures are an important requirement for future large-scale genetic studies such as genotyping of single nucleotide polymorphisms (SNPs). Matrix-assisted laser desorption/ ionisation mass spectrometry (MALDI-MS) has revolutionised the analysis of biomolecules and, in particular, provides a very attractive solution for the rapid typing of DNA. The analysis of DNA by MALDI can be significantly facilitated by a procedure termed harge-tagging We show here a novel approach for the generation of charge-tagged DNA using a photocleavable linker and its implementation in a molecular biological procedure for SNP genotyping consisting of PCR, primer extension, photocleavage and a chemical reaction prior to MALDI target preparation and analysis. The reaction sequence is amenable to liquid handling automation and requires no stringent purification procedures. We demonstrate this new method on SNPs in two genes involved in complex traits.
Pubmed: ABSTRACT Article (Saussoy2004) Saussoy, P.; Vaerman, J.; Straetmans, N.; Deneys, V.; Cornu, G.; Ferrant, A. & Latinne, D. Differentiation of acute myeloid leukemia from B- and T-lineage acute lymphoid leukemias by real-time quantitative reverse transcription-PCR of lineage marker mRNAs. Clin Chem, 2004, 50, 1165-1173 Abstract: BACKGROUND: Flow cytometry of lineage markers is useful in the classification of leukemias. Our aim was to assess whether the study of lineage genes at the RNA level would enable differentiation of acute myeloid leukemias (AMLs) from B-and T-lineage acute lymphoid leukemias (ALLs). METHODS: We measured mRNA of four lineage markers [CD19, CD79a, CD3e, and myeloperoxidase (MPO)] by reverse transcription followed by real-time quantitative (RTQ)-PCR. We investigated 72 acute leukemias (40 AMLs with 23-93% blast cells plus 27 B-lineage ALLs and 5 T-lineage ALLs) defined by morphologic criteria at diagnosis. RTQ-PCR analysis was performed on bone marrow without cell sorting. The expression of each gene was calculated as the difference in the threshold cycle [DeltaCT; CT value of target gene minus CT value of housekeeping gene (Abelson)]. RESULTS: Three patterns of expression were detected. In the first, CD19, CD79a, and MPO mRNAs were less abundant than CD3e. In the second pattern, MPO mRNA was more abundant than the other three mRNAs. In the third, CD19 or CD79a was more highly expressed than CD3e and MPO. The three patterns corresponded to T-ALL, AML, and B-ALL, respectively. The use of cutoffs to establish qualitatively the pattern of coexpression of the four lineage markers provided the same information as the comparison among the four DeltaCT values. Prospective use of the scoring system correctly classified each of 13 additional cases (8 AML, 4 B-lineage ALL, and 1 T-lineage ALL). CONCLUSION: Study of lineage markers at diagnosis by RTQ-PCR allows differentiation of AML from B-ALL or T-ALL without cell sorting, even when the bone marrow contains few blast cells.
Pubmed: ABSTRACT Conference (Schabmueller2000) Schabmueller, C.G.J.; Evans, A.G.R.; Brunnschweiler, A.; Ensell, G.J.; Leslie, D.L. & Lee, M.A. Courtois, B.; Crary, S.B.; Gabriel, K.J.; Karam, J.M.; Markus, K.W. & Tay, A.A.O. (ed.) Design, fabrication, and packaging of closed-chamber PCR chips for DNA amplification Design, Test, Integration, and Packaging of MEMS/MOEMS, SPIE, 2000, 4019, 362-369
DOI: http://link.aip.org/link/?PSI/4019/362/1 Article (Schifreen2002) Schifreen, R.S.; Storts, D.R. & Buller, A.M. The challenge of using SNPs in the understanding and treatment of disease Biotechniques, 2002, Suppl, 14-6, 18, 20-1
Pubmed: ABSTRACT Article (Schmittgen2001) Schmittgen, T.D. Real-time quantitative PCR Methods, 2001, 25, 383-5
Pubmed: ABSTRACT Article (Schmittgen2000) Schmittgen, T.D. & Zakrajsek, B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR J Biochem Biophys Methods, 2000, 46, 69-81 Abstract: The effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for beta-actin, beta-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of beta-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription (P<0.01). The expression of 18S rRNA increased with serum-stimulation when the cDNA synthesized from non-normalized, total RNA was assayed (P<0. 01) but not when the reverse transcriptions were normalized to RNA content (P>0.05). The expression of beta-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. beta-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.
Pubmed: ABSTRACT Article (Schmittgen2000a) Schmittgen, T.D.; Zakrajsek, B.A.; Mills, A.G.; Gorn, V.; Singer, M.J. & Reed, M.W. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods Anal Biochem, 2000, 285, 194-204 Abstract: Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.
Pubmed: ABSTRACT Article (Schneegass2001a) Schneegass, I.; Brautigam, R. & Kohler, J.M. Miniaturized flow-through PCR with different template types in a silicon chip thermocycler. Lab Chip, 2001, 1, 42-49 Abstract: Flow-through chip thermocyclers can be used in miniaturized rapid polymerase chain reaction (PCR) despite their high surface to volume ratio of samples. We demonstrated that a thermocycler made of silicon and glass chips and containing thin film transducers for heating and temperature control can be adapted to the amplification of various DNA templates of different sources and properties. Therefore, the concept of serial flow in a liquid/liquid two-phase system was combined with a surface management of inner side walls of the microchannel and an adaptation of PCR mixture composition. In addition, the process temperatures and the flow rates were optimized. Thus, a synthetic template originating from investigations on nucleic acid evolution with 106 base pairs [cooperative amplification of templates by cross hybridization (CATCH)], a house keeping gene with 379 base pairs [glutaraldehyde 3-phosphate dehydrogenase (GAPDH)] and a zinc finger protein relevant in human pathogenesis with 700 base pairs [Myc-interacting zinc finger protein-1, knock-out (Miz1-KO)] were amplified successfully. In all three cases the selectivity of priming and amplification could be shown by gel electrophoresis. The typical amplification time was 1 min per temperature cycle. So, the typical residence time of a sample volume inside the 25 cycle device amounts to less then half an hour. The energy consumption of the PCR chip for a 35 min PCR process amounts to less than 0.012 kW h.
Pubmed: ABSTRACT Article (Schneegass2001b) Schneegass, I. & Köhler, J.M. Flow-through polymerase chain reactions in chip thermocyclers. J Biotechnol, 2001, 82, 101-121 Abstract: The miniaturization of analytical devices by micromachining technology is destined to have a major impact on medical and bioanalytical fields. To meet the current demands for rapid DNA amplification, various instruments and innovative technologies have been introduced by several groups in recent years. The development of the devices was extended in different directions and adapted to corresponding applications. In this review the development of a variety of devices and components for performing DNA amplification as well as the comparison of batch-process thermocyclers with reaction chambers and flow-through devices for different purposes are discussed. The main attention is turned to a flow device concept for thermocycling using microfabricated elements for local heat flow management, for which simulations and considerations for further improvement regarding design, material choice and applied technology were performed. The present review article mainly discusses and compares thermocycling devices for rapid thermocycling made of silicon or of silicon and glass with a short excursion to the possibility of plastic chip devices. In order to perform polymerase chain reactions (PCRs) in the microreactors, special attention must be paid to the conditions of the internal surfaces. For microchips, surface effects are generally pronounced because the surface to volume ratio increases upon miniaturization. Solutions for solving this problem are presented. We propose an overview of layouts for batch-process thermocyclers with different parallelization of reaction chambers and also of different designs of continuous flow thermocycling chips, paying particular attention to the parameters which influence the efficiency of such chip devices. Finally we point out some recent issues for applications in the field of clinical diagnostics.
Pubmed: ABSTRACT Article (Schneider2005) Schneider, P.; Wolters, L.; Schoone, G.; Schallig, H.; Sillekens, P.; Hermsen, R. & Sauerwein, R. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum J Clin Microbiol, 2005, 43, 402-5 Abstract: Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 microl of venous blood to achieve this sensitivity, compared to 50 microl of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.
Pubmed: ABSTRACT Article (Schober2004) Schober, A.; Schlingloff, G.; Groß, A.; Henkel, T.; Albert, J.; Mayer, G.; Wurziger, H.; Döring, M. & Tietz, H. Miniaturisation of synthesis and screening in nanotiterplates: the concept of NanoSynTest TM Microsystem Technologies, 2004, V10, 281-292
PDF: Schober2004.pdf Article (Schork2000) Schork, N.J.; Fallin, D. & Lanchbury, J.S. Single nucleotide polymorphisms and the future of genetic epidemiology Clin Genet, 2000, 58, 250-64 Abstract: In this review, we consider the motivation behind contemporary single nucleotide polymorphism (SNP) initiatives. Many of these initiatives are projected to involve large, population-based surveys. We therefore emphasize the utility of SNPs for genetic epidemiology studies. We start by offering an overview of genetic polymorphism and discuss the historical use of polymorphism in the identification of disease-predisposing genes via meiotic mapping. We next consider some of the unique aspects of SNPs, and their relative advantages and disadvantages in human population-based analyses. In this context, we describe and critique the following six different areas of application for SNP technologies: Gene discovery and mapping. Association-based candidate polymorphism testing. Diagnostics and risk profiling. Prediction of response to environmental stimuli, xenobiotics and diet. Homogeneity testing and epidemiological study design. Physiologic genomics. We focus on key issues within each of these areas in an effort to point out potential problems that might plague the use of SNPs (or other forms of polymorphism) within them. However, we make no claim that our list of considerations are exhaustive. Rather, we believe that they may provide a starting point for further dialog about the ultimate utility of SNP technologies. In addition, although our emphasis is placed on applications of SNPs to the understanding of human phenotypes, we acknowledge that SNP maps and technologies applied to other species (e.g. the mouse genome, pathogen genomes, plant genomes, etc.) are also of tremendous interest.
Pubmed: ABSTRACT Article (Schulze2001) Schulze, A. & Downward, J. Navigating gene expression using microarrays--a technology review. Nat Cell Biol, 2001, 3, E190-E195 Abstract: Parallel quantification of large numbers of messenger RNA transcripts using microarray technology promises to provide detailed insight into cellular processes involved in the regulation of gene expression. This should allow new understanding of signalling networks that operate in the cell and of the molecular basis and classification of disease. But can the technology deliver such far-reaching promises?
Pubmed: ABSTRACT Article (Semple2000) Semple, C.A.; Morris, S.W.; Porteous, D.J. & Evans, K.L. In silico identification of transcripts and SNPs from a region of 4p linked with bipolar affective disorder Bioinformatics, 2000, 16, 735-8
Pubmed: ABSTRACT Article (Setterquist1996) Setterquist, R.A. & Smith, G.K. Ready to use agarose encapsulated PCR reagents Nucleic Acids Res, 1996, 24, 1580-1
Pubmed: ABSTRACT Article (Shandrick2002) Shandrick, S.; Ronai, Z. & Guttman, A. Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA Electrophoresis, 2002, 23, 591-5 Abstract: Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by ïn migratiocomplexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.
Pubmed: ABSTRACT Article (Sharan2004) Sharan, R.; Ben-Dor, A. & Yakhini, Z. Multiplexing schemes for generic SNP genotyping assays Pac Symp Biocomput, 2004, 140-51 Abstract: A generic genotyping assay utilizes a fixed set of reagents, which is independent of the actual target sample, to determine all present alleles. An example is the interrogation of several amplicons spanning polymorphic sites using an all k-mer array. Due to the high cost associated with a genotyping experiment, it is desirable to design a set of experiments, which maximizes the number of SNPs that can be genotyped in parallel per assay. In this study we investigate algorithmic approaches for optimally multiplexing SNP genotyping using generic assays. We devise a graph theoretic formulation of the problem and use it to derive an approximation algorithm for the problem, and several practical heuristics. We apply our methods to simulated and real data, for evaluating the multiplexing rates afforded by generic techniques. The results on real human data show the practicality of generic approaches for genotyping, allowing, e.g., the genotyping of 5000 SNPs using four all 7-mer arrays.
Pubmed: ABSTRACT Article (Shendure2005) Shendure, J.; Porreca, G.J.; Reppas, N.B.; Lin, X.; McCutcheon, J.P.; Rosenbaum, A.M.; Wang, M.D.; Zhang, K.; Mitra, R.D. & Church, G.M. Accurate multiplex polony sequencing of an evolved bacterial genome. Science, 2005, 309, 1728-1732 Abstract: We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.
Pubmed: ABSTRACT Article (Sherlock2001) Sherlock, G.; Hernandez-Boussard, T.; Kasarskis, A.; Binkley, G.; Matese, J.C.; Dwight, S.S.; Kaloper, M.; Weng, S.; Jin, H.; Ball, C.A.; Eisen, M.B.; Spellman, P.T.; Brown, P.O.; Botstein, D. & Cherry, J.M. The Stanford Microarray Database Nucleic Acids Res, 2001, 29, 152-5 Abstract: The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.
Pubmed: ABSTRACT Article (Sherry2001) Sherry, S.T.; Ward, M.H.; Kholodov, M.; Baker, J.; Phan, L.; Smigielski, E.M. & Sirotkin, K. dbSNP: the NCBI database of genetic variation Nucleic Acids Res, 2001, 29, 308-11 Abstract: In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Center for Biotechnology Information (NCBI) has established the dbSNP database [S.T.Sherry, M.Ward and K.Sirotkin (1999) Genome Res., 9, 677-679]. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. The complete contents of dbSNP can also be downloaded in multiple formats via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/.
Pubmed: ABSTRACT Article (Shi2006) Shi, L.; Reid, L.H.; Jones, W.D.; Shippy, R.; Warrington, J.A.; Baker, S.C.; Collins, P.J.; de Longueville, F.; Kawasaki, E.S.; Lee, K.Y.; Luo, Y.; Sun, Y.A.; Willey, J.M.; Setterquist, R.A.; Fischer, G.M.; Tong, W.; Dragan, Y.P.; Dix, D.J.; Frueh, F.W.; Goodsaid, F.M.; Herman, D.; Jensen, R.V.; Johnson, C.D.; Lobenhofer, E.K.; Puri, R.K.; Schrf, U.; Mieg, J.T.; Wang, C.; Wilson, M.; Wolber, P.K.; Zhang, L.; Slikker, W.; Shi, L.; Reid, L.H. & Consortium, M.A.Q.C. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol, 2006, 24, 1151-1161 Abstract: Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.
Pubmed: ABSTRACT Article (Shigemori2005) Shigemori, Y.; Mikawa, T.; Shibata, T. & Oishi, M. Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products. Nucleic Acids Res, 2005, 33, e126 Abstract: In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.
Pubmed: ABSTRACT Article (Shin2003) Shin, Y.; Cho, K.; Lim, S.; Chung, S.P.; Chung, C.; C., H.D. & Chang, J. PDMS-based micro PCR chip with Parylene coating J. Micromech. Microeng., 2003, 13, 768-774
PDF: Shin2003.pdf Article (Shippy2006) Shippy, R.; Smentek, S.F.; Jensen, R.V.; Jones, W.D.; Wolber, P.K.; Johnson, C.D.; Pine, P.S.; Boysen, C.; Guo, X.; Chudin, E.; Sun, Y.A.; Willey, J.C.; Mieg, J.T.; Mieg, D.T.; Setterquist, R.A.; Wilson, M.; Lucas, A.B.; Novoradovskaya, N.; Papallo, A.; Turpaz, Y.; Baker, S.C.; Warrington, J.A.; Shi, L. & Herman, D. Using RNA sample titrations to assess microarray platform performance and normalization techniques. Nat Biotechnol, 2006, 24, 1123-1131 Abstract: We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.
Pubmed: ABSTRACT Article (Shoffner1996) Shoffner, M.A.; Cheng, J.; Hvichia, G.E.; Kricka, L.J. & Wilding, P. Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR Nucleic Acids Res, 1996, 24, 375-9 Abstract: The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify CR friendlysurfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.
Pubmed: ABSTRACT Article (Silander2005) Silander, K.; Komulainen, K.; Ellonen, P.; Jussila, M.; Alanne, M.; Levander, M.; Tainola, P.; Kuulasmaa, K.; Salomaa, V.; Perola, M.; Peltonen, L. & Saarela, J. Evaluating whole genome amplification via multiply-primed rolling circle amplification for SNP genotyping of samples with low DNA yield. Twin Res Hum Genet, 2005, 8, 368-375 Abstract: The amount of available DNA is often a limiting factor in pursuing genetic analyses of large-scale population cohorts. An association between higher DNA yield from blood and several phenotypes associated with inflammatory states has recently been demonstrated, suggesting that exclusion of samples with very low DNA yield may lead to biased results in statistical analyses. Whole genome amplification (WGA) could present a solution to the DNA concentration-dependent sample selection. The aim was to thoroughly assess WGA for samples with low DNA yield, using the multiply-primed rolling circle amplification method. Fifty-nine samples were selected with the lowest DNA yield (less than 7.5 microg) among 799 samples obtained for one population cohort. The genotypes obtained from two replicate WGA samples and the original genomic DNA were compared by typing 24 single nucleotide polymorphisms (SNPs). Multiple genotype discrepancies were identified for 13 of the 59 samples. The largest portion of discrepancies was due to allele dropout in heterozygous genotypes in WGA samples. Pooling the WGA DNA replicates prior to genotyping markedly improved genotyping reproducibility for the samples, with only 7 discrepancies identified in 4 samples. The nature of discrepancies was mostly homozygote genotypes in the genomic DNA and heterozygote genotypes in the WGA sample, suggesting possible allele dropout in the genomic DNA sample due to very low amounts of DNA template. Thus, WGA is applicable for low DNA yield samples, especially if using pooled WGA samples. A higher rate of genotyping errors requires that increased attention be paid to genotyping quality control, and caution when interpreting results.
Pubmed: ABSTRACT Article (Simon2003) Simon, P. Q-Gene: processing quantitative real-time RT-PCR data Bioinformatics, 2003, 19, 1439-40 Abstract: Summary: Q-Gene is an application for the processing of quantitative real-time RT-PCR data. It offers the user the possibility to freely choose between two principally different procedures to calculate normalized gene expressions as either means of Normalized Expressions or Mean Normalized Expressions. In this contribution it will be shown that the calculation of Mean Normalized Expressions has to be used for processing simplex PCR data, while multiplex PCR data should preferably be processed by calculating Normalized Expressions. The two procedures, which are currently in widespread use and regarded as more or less equivalent alternatives, should therefore specifically be applied according to the quantification procedure used. Availability: Web access to this program is provided at http://www.biotechniques.com/softlib/qgene.html Contact: perikles@uni-tuebingen.de
Pubmed: ABSTRACT Article (Singh1988) Singh, N.P.; McCoy, M.T.; Tice, R.R. & Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res, 1988, 175, 184-191 Abstract: Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
Pubmed: ABSTRACT Article (Situma2006) Situma, C.; Hashimoto, M. & Soper, S.A. Merging microfluidics with microarray-based bioassays. Biomol Eng, 2006 Abstract: Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.
Pubmed: ABSTRACT Article (Sleytr2007) Sleytr, U.B.; Huber, C.; Ilk, N.; Pum, D.; Schuster, B. & Egelseer, E.M. S-layers as a tool kit for nanobiotechnological applications. FEMS Microbiol Lett, 2007, 267, 131-144 Abstract: Crystalline bacterial cell surface layers (S-layers) have been identified in a great number of different species of bacteria and represent an almost universal feature of archaea. Isolated native S-layer proteins and S-layer fusion proteins incorporating functional sequences self-assemble into monomolecular crystalline arrays in suspension, on a great variety of solid substrates and on various lipid structures including planar membranes and liposomes. S-layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological molecules (proteins, lipids, glycans, nucleic acids and combinations of them) enabling innovative approaches for the controlled 'bottom-up' assembly of functional supramolecular structures and devices. Here, we review the basic principles of S-layer proteins and the application potential of S-layers in nanobiotechnology and biomimetics including life and nonlife sciences.
Pubmed: ABSTRACT Article (Smigielski2000) Smigielski, E.M.; Sirotkin, K.; Ward, M. & Sherry, S.T. dbSNP: a database of single nucleotide polymorphisms Nucleic Acids Res, 2000, 28, 352-5 Abstract: In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Cancer for Biotechnology Information (NCBI) has established the dbSNP database. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. Submitted SNPs can also be downloaded via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/
Pubmed: ABSTRACT Article (Smith2007) Smith, C. Tools for drug discovery: tools of the trade. Nature, 2007, 446, 219-222
Pubmed: ABSTRACT Misc (Somack2005) Somack, R.; Hodges, S. & Pandey, V. Simple Qualitative and Quantitative Methods for Measuring Residual Activity of “Hot-Start” Enzyme Preparations 2005 Abstract: PCR precision, specificity, sensitivity and yield of “Hot-Start” DNA polymerases are often directly related to the effectiveness of the method used to impart the “Hot Start” feature as well as the stability of the resulting product. For example, “Hot-Start” enzymes containing elevated levels of residual (not rendered dormant by chemical modification) polymerase activity (RPA) have been implicated in failures in some low copy and multiplex assays. Standard methods for quantifying RPA present in “Hot-Start” DNA polymerases or “Hot-Start” DNA polymerase Master Mixes involve tedious and time-consuming protocols based on direct measurement of ?-P32 dNTP incorporation. Measuring fractions of a percent of residual activity in a Master Mix formulation is rather challenging, given the dilution of enzyme, even with a radiolabel. We have developed a set of non-radioactive, PCR-based assays useful for the detection and quantification of a wide range of RPA levels present in “hot start” DNA polymerase preparations and Master Mixes.
One-hundred fifty real-time assays were designed to have variation in performance. These assays were screened for sensitivity to AmpliTaq Gold® DNA Polymerase RPA using master mixes containing either AmpliTaq® DNA polymerase or AmpliTaq Gold® DNA polymerase and comparing agarose gel electrophoresis band patterns. Assays demonstrating higher amplicon yields with diminished side products due to the “Hot-Start” were further characterized by titrating AmpliTaq Gold Universal PCR Master Mix with increasing amounts of AmpliTaq DNA Polymerase. Three assays showed clear-cut gel patterns that are useful for estimating incremental RPA in the 0-10% ranges. Another assay demonstrated high incremental sensitivity to RPA in the 0-1% range. SYBR® Green PCR end-point melting curve analyses of 0% RPA (no added AmpliTaq® DNA polymerase) samples showed a major amplicon peak (Tm=81°C) and minor dimer peak (Tm=74°C). At 1% AmpliTaq® DNA polymerase addition the dimer peak increased while the amplicon peak completely disappeared. This phenomenon was exploited to develop a highly reproducible, quantitative SYBR Green assay method for RPA detection in the 0-1% range that can be performed on Applied Biosystems Sequence Detection platforms.
Article (Song2006a) Song, H.; Chen, D.L. & Ismagilov, R.F. Reactions in Droplets in Microfluidic Channels. Angew Chem Int Ed Engl, 2006, 45, 7336-7356 Abstract: Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.
Pubmed: ABSTRACT Article (Song2006) Song, J.M.; Yang, M. & Kwan, H.T. Development of a novel DNA chip based on a bipolar semiconductor microchip system. Biosens Bioelectron, 2006 Abstract: We have applied an integrated circuit photodiode array (PDA) chip system to a DNA chip. The PDA chip system, constructed using conventional bipolar semiconductor technology, acts as a solid transducer surface as well as a two-dimensional photodetector. DNA hybridization was performed directly on the PDA chip. The target DNA, the Bacillus subtilis sspE gene, was amplified by polymerase chain reaction (PCR). The 340-bp PCR product was labeled using digoxigenin (DIG). A silicon nitride layer on the photodiode was treated with poly-l-lysine to immobilize the DNA on the surface of the photodiode detection elements. Consequently, the surface of the photodiode detector became positively charged. An anti-DIG-alkaline phosphatase conjugate was reacted with the hybridized DIG-labeled DNA. A color reaction was performed based on the enzymatic reaction between nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining solution and a DNA complex containing antibodies. A blue precipitate was formed on the surfaces of the photodiode detection elements. Successful quantitative analysis of the hybridized PCR products was achieved from the light absorption properties of the blue enzymatic reaction product that was produced after a series of reaction processes. Our DNA chip system avoids the complicated optical alignments and light-collecting optical components that are usually required for an optical DNA chip device. As a result, a simple, compact, portable and low-cost DNA chip is achieved. This system has great potential as an alternative system to the conventional DNA reader.
Pubmed: ABSTRACT Article (Southern1992) Southern, E.M.; Maskos, U. & Elder, J.K. Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics, 1992, 13, 1008-1017 Abstract: An efficient method was developed for making complete sets of oligonucleotides of defined length, covalently attached to the surface of a glass plate, by synthesizing them in situ. A device carrying all octapurine sequences was used to explore factors affecting molecular hybridization of the tethered oligonucleotides, to develop computer-aided methods for analyzing the data, and to test the feasibility of using the method for sequence analysis. Further development is needed before the method can be used routinely, but our work shows that it has a number of potential advantages over gel-based methods: it should be easy to automate; the quality of the sequence results can be evaluated statistically; it provides a powerful way of comparing related sequences and detecting mutation; it can be applied to both DNA and RNA; and specific motifs can be incorporated into all sequences of the array to focus analysis on sequences of biological interest.
Pubmed: ABSTRACT Article (Spellman2002) Spellman, P.T.; Miller, M.; Stewart, J.; Troup, C.; Sarkans, U.; Chervitz, S.; Bernhart, D.; Sherlock, G.; Ball, C.; Lepage, M.; Swiatek, M.; Marks, W.L.; Goncalves, J.; Markel, S.; Iordan, D.; Shojatalab, M.; Pizarro, A.; White, J.; Hubley, R.; Deutsch, E.; Senger, M.; Aronow, B.J.; Robinson, A.; Bassett, D.; Stoeckert, C.J. & Brazma, A. Design and implementation of microarray gene expression markup language (MAGE-ML) Genome Biol, 2002, 3, RESEARCH0046 Abstract: BACKGROUND: Meaningful exchange of microarray data is currently difficult because it is rare that published data provide sufficient information depth or are even in the same format from one publication to another. Only when data can be easily exchanged will the entire biological community be able to derive the full benefit from such microarray studies. RESULTS: To this end we have developed three key ingredients towards standardizing the storage and exchange of microarray data. First, we have created a minimal information for the annotation of a microarray experiment (MIAME)-compliant conceptualization of microarray experiments modeled using the unified modeling language (UML) named MAGE-OM (microarray gene expression object model). Second, we have translated MAGE-OM into an XML-based data format, MAGE-ML, to facilitate the exchange of data. Third, some of us are now using MAGE (or its progenitors) in data production settings. Finally, we have developed a freely available software tool kit (MAGE-STK) that eases the integration of MAGE-ML into end users' systems. CONCLUSIONS: MAGE will help microarray data producers and users to exchange information by providing a common platform for data exchange, and MAGE-STK will make the adoption of MAGE easier.
Pubmed: ABSTRACT Article (Spira2007) Spira, A.; Beane, J.E.; Shah, V.; Steiling, K.; Liu, G.; Schembri, F.; Gilman, S.; Dumas, Y.; Calner, P.; Sebastiani, P.; Sridhar, S.; Beamis, J.; Lamb, C.; Anderson, T.; Gerry, N.; Keane, J.; Lenburg, M.E. & Brody, J.S. Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med, 2007, 13, 361-366 Abstract: Lung cancer is the leading cause of death from cancer in the US and the world. The high mortality rate (80-85% within 5 years) results, in part, from a lack of effective tools to diagnose the disease at an early stage. Given that cigarette smoke creates a field of injury throughout the airway, we sought to determine if gene expression in histologically normal large-airway epithelial cells obtained at bronchoscopy from smokers with suspicion of lung cancer could be used as a lung cancer biomarker. Using a training set (n = 77) and gene-expression profiles from Affymetrix HG-U133A microarrays, we identified an 80-gene biomarker that distinguishes smokers with and without lung cancer. We tested the biomarker on an independent test set (n = 52), with an accuracy of 83% (80% sensitive, 84% specific), and on an additional validation set independently obtained from five medical centers (n = 35). Our biomarker had approximately 90% sensitivity for stage 1 cancer across all subjects. Combining cytopathology of lower airway cells obtained at bronchoscopy with the biomarker yielded 95% sensitivity and a 95% negative predictive value. These findings indicate that gene expression in cytologically normal large-airway epithelial cells can serve as a lung cancer biomarker, potentially owing to a cancer-specific airway-wide response to cigarette smoke.
Pubmed: ABSTRACT Article (Stajich2002) Stajich, J.E.; Block, D.; Boulez, K.; Brenner, S.E.; Chervitz, S.A.; Dagdigian, C.; Fuellen, G.; Gilbert, J.G.; Korf, I.; Lapp, H.; Lehvaslaiho, H.; Matsalla, C.; Mungall, C.J.; Osborne, B.I.; Pocock, M.R.; Schattner, P.; Senger, M.; Stein, L.D.; Stupka, E.; Wilkinson, M.D. & Birney, E. The Bioperl toolkit: Perl modules for the life sciences Genome Res, 2002, 12, 1611-8 Abstract: The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.
Pubmed: ABSTRACT Article (Stanley2005) Stanley, K.K. & Szewczuk, E. Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection. Nucleic Acids Res, 2005, 33, e180 Abstract: Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce 'primer dimer' formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.
Pubmed: ABSTRACT Article (Steemers2006) Steemers, F.J.; Chang, W.; Lee, G.; Barker, D.L.; Shen, R. & Gunderson, K.L. Whole-genome genotyping with the single-base extension assay. Nat Methods, 2006, 3, 31-33 Abstract: We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.
Pubmed: ABSTRACT Article (Stein2002) Stein, L. Creating a bioinformatics nation Nature, 2002, 417, 119-20
Pubmed: ABSTRACT Article (Steitz1998) Steitz, T.A. A mechanism for all polymerases Nature, 1998, 391, 231-2
Pubmed: ABSTRACT Article (Stenman2001) Stenman, J. & Orpana, A. Accuracy in amplification. Nat Biotechnol, 2001, 19, 1011-1012
Pubmed: ABSTRACT Article (Stephens1990) Stephens, J.C.; Rogers, J. & Ruano, G. Theoretical underpinning of the single-molecule-dilution (SMD) method of direct haplotype resolution Am J Hum Genet, 1990, 46, 1149-55 Abstract: In a recent paper we have shown that DNA haplotypes of multiply heterozygous individuals can be resolved directly by polymerase-chain-reaction (PCR) amplification of a single molecule of genomic template. Our method (the single-molecule-dilution [SMD] method) relies on the stochastic separation of maternal and paternal alleles at high dilution. The stochasticity of separation and the potential for DNA shearing (which could separate the loci of interest) are two factors that can compromise the results of the experiment. This paper explores the consequences of these two factors and shows that the SMD method can be expected to work very reliably even in the presence of a moderate amount of DNA shearing.
Pubmed: ABSTRACT Article (Stocher2002) Stocher, M.; Leb, V.; Holzl, G. & Berg, J. A simple approach to the generation of heterologous competitive internal controls for real-time PCR assays on the LightCycler J Clin Virol, 2002, 25 Suppl 3, S47-53 Abstract: BACKGROUND: The real-time PCR technology allows convenient detection and quantification of virus derived DNA. This approach is used in many PCR based assays in clinical laboratories. Detection and quantification of virus derived DNA is usually performed against external controls or external standards. Thus, adequacy within a clinical sample is not monitored for. This can be achieved using internal controls that are co-amplified with the specific target within the same reaction vessel. OBJECTIVES: We describe a convenient way to prepare heterologous internal controls as competitors for real-time PCR based assays. STUDY DESIGN: The internal controls were devised as competitors in real-time PCR, e.g. LightCycler-PCR. The bacterial neomycin phosphotransferase gene (neo) was used as source for heterologous DNA. Within the neo gene a box was chosen containing sequences for four differently spaced forward primers, one reverse primer, and a pair of neo specific hybridization probes. Pairs of primers were constructed to compose of virus-specific primer sequences and neo box specific primer sequences. Using those composite primers in conventional preparative PCR four types of internal controls were amplified from the neo box and subsequently cloned. RESULTS: A panel of the four differently sized internal controls was generated and tested by LightCycler PCR using their virus-specific primers. All four different PCR products were detected with the single pair of neo specific FRET-hybridization probes. CONCLUSION: The presented approach to generate competitive internal controls for use in LightCycler PCR assays proved convenient und rapid. The obtained internal controls match most PCR product sizes used in clinical routine molecular assays and will assist to discriminate true from false negative results.
Pubmed: ABSTRACT Article (Stoesser2003) Stoesser, G.; Baker, W.; Van Den Broek, A.; Garcia-Pastor, M.; Kanz, C.; Kulikova, T.; Leinonen, R.; Lin, Q.; Lombard, V.; Lopez, R.; Mancuso, R.; Nardone, F.; Stoehr, P.; Tuli, M.A.; Tzouvara, K. & Vaughan, R. The EMBL Nucleotide Sequence Database: major new developments Nucleic Acids Res, 2003, 31, 17-22 Abstract: The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.
Pubmed: ABSTRACT Article (Strizhkov2000) Strizhkov, B.N.; Drobyshev, A.L.; Mikhailovich, V.M. & Mirzabekov, A.D. PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations Biotechniques, 2000, 29, 844-8, 850-2, 854 passim Abstract: PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.
Pubmed: ABSTRACT Article (Stryer1978) Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem, 1978, 47, 819-846
Pubmed: ABSTRACT Article (Su2002) Su, A.I.; Cooke, M.P.; Ching, K.A.; Hakak, Y.; Walker, J.R.; Wiltshire, T.; Orth, A.P.; Vega, R.G.; Sapinoso, L.M.; Moqrich, A.; Patapoutian, A.; Hampton, G.M.; Schultz, P.G. & Hogenesch, J.B. Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci U S A, 2002, 99, 4465-4470 Abstract: High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (http://expression.gnf.org) that integrates data visualization and curation of current gene annotations.
Pubmed: ABSTRACT Article (Su2004) Su, X. & Smith, L.M. Demonstration of a universal surface DNA computer Nucleic Acids Res, 2004, 32, 3115-23 Abstract: A fundamental concept in computer science is that of the universal Turing machine, which is an abstract definition of a general purpose computer. A general purpose (universal) computer is defined as one which can compute anything that is computable. It has been shown that any computer which is able to simulate Boolean logic circuits of any complexity is such a general purpose computer. The field of DNA computing was founded in 1994 by Adleman solution of a 7-bit instance of the Hamiltonian path problem. This work, as well as most of the subsequent experimental and theoretical investigations in the area, focused primarily upon the solution of NP-complete problems, which are a subset of the larger universal class of problems. In the present work a surface DNA computer capable of simulating Boolean logic circuits is demonstrated. This was done by constructing NOR and OR gates and combining them into a simple logic circuit. The NOR gate is one of the universal gates in Boolean logic, meaning that any other logic gate can be built from it alone. The circuit was solved using DNA-based operations, demonstrating the universal nature of this surface DNA computing model.
Pubmed: ABSTRACT Article (Swillens2004) Swillens, S.; Goffard, J.C.; Marechal, Y.; de Kerchove dÉxaerde, A. & El Housni, H. Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve Nucleic Acids Res, 2004, 32, e56 Abstract: Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.
Pubmed: ABSTRACT Article (Sykes1992) Sykes, P.J.; Neoh, S.H.; Brisco, M.J.; Hughes, E.; Condon, J. & Morley, A.A. Quantitation of targets for PCR by use of limiting dilution. Biotechniques, 1992, 13, 444-449 Abstract: We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.
Pubmed: ABSTRACT Article (Syvaenen2001) Syvänen, A.C. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet, 2001, 2, 930-942 Abstract: Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms.
Pubmed: ABSTRACT Article (Syvaenen1999) Syvänen, A.C. From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat, 1999, 13, 1-10 Abstract: In the minisequencing primer extension reaction, a DNA polymerase is used specifically to extend a primer that anneals immediately adjacent to the nucleotide position to be analyzed with a single labeled nucleoside triphospate complementary to the nucleotide at the variant site. The reaction allows highly specific detection of point mutations and single nucleotide polymorphisms (SNPs). Because all SNPs can be analyzed with high specificity at the same reaction conditions, minisequencing is a promising reaction principle for multiplex high-throughput genotyping assays. It is also a useful tool for accurate quantitative PCR-based analysis. This review discusses the different approaches, ranging from traditional gel-based formats to multiplex detection on microarrays that have been developed and applied to minisequencing assays.
Pubmed: ABSTRACT Article (Syvaenen1990) Syvänen, A.C.; Setälä, K.A.; Harju, L.; Kontula, K. & Söderlund, H. A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics, 1990, 8, 684-692 Abstract: We describe a new technique by which single base changes in human genes can be conveniently detected. In this method the DNA fragment of interest is first amplified using the polymerase chain reaction with an oligonucleotide primer biotinylated at its 5'-end. The amplified 5'-biotinylated DNA is immobilized on an avidin matrix and rendered single-stranded. The variable nucleotide in the immobilized DNA is identified by a one-step primer extension reaction directed by a detection step primer, which anneals to the DNA immediately upstream of the site of variation. In this reaction a single labeled nucleoside triphosphate complementary to the nucleotide at the variable site is incorporated. The method is highly sensitive, allowing the use of nucleoside triphosphates labeled with radioisotopes of low specific activity (3H) as well as nonradioactive markers (digoxigenin). The procedure consists of few and simple operations and is thus applicable to the analysis of large numbers of samples. Here we applied it to the analysis of the three-allelic polymorphism of the human apolipoprotein E gene. We were able to correctly identify all possible combinations of the three apo E alleles.
Pubmed: ABSTRACT Article (Syvaenen2005) Syvänen, A. Toward genome-wide SNP genotyping. Nat Genet, 2005, 37 Suppl, S5-10 Abstract: Genome-wide association studies with SNP markers are expected to allow identification of genes that underlie complex disorders. Hundreds of thousands of SNP markers will be required for comprehensive genome-wide association studies. The development of microarray-based methods for SNP genotyping on this scale remains a demanding task, despite many recent advances in technology for the production of high-density microarrays. A key technical obstacle is the PCR amplification step, which is required to reduce the complexity of and gain sufficient sensitivity for genotyping SNPs in large, diploid genomes. The multiplexing level that can be achieved in PCR does not match that of current microarray-based methods, making PCR the limiting step in the assays. Highly multiplexed microarray systems for SNP genotyping have recently been developed by combining well-known reaction principles for DNA amplification and SNP genotyping in clever ways. These new methods offer the potential of genome-wide SNP mapping of genes involved in complex diseases in the foreseeable future, provided that issues related to selection of the optimal SNP markers, sample throughput and the cost of the assays can be addressed.
Pubmed: ABSTRACT Article (Soerlie2006) Sørlie, T.; Rosenvinge, J.H.; Bremnes, R.; Balteskard, L.; Dahl, A.A.; Klepp, O. & Fosså, S.D. Management satisfaction in survivors of testicular cancer. Anticancer Res, 2006, 26, 1463-1469 Abstract: BACKGROUND: The aim of this study was to examine satisfaction with the quality of the doctor-patient relationship (QCD) and the information (INF) made available, as well as the influence of different treatment modalities and psychosocial variables in testicular cancer survivors (TCSs). MATERIALS AND METHODS: Two-hundred and 6 TCSs, seen at 2 Norwegian university hospitals, were studied retrospectively and currently in a cross-sectional follow-up design. Questionnaires were filled in prior to and at the patients' follow-up examination. The mean length of time since their management period had ended was 6.5 years (range 0-17 years). RESULTS: The QCD was rated higher than the INF. The QCD was also the strongest contributor to the variance in the INF. The patients in the surveillance group had the lowest QCD. CONCLUSION: The relatively high QCD scores may reflect the fact that the patients had been cured from a life-threatening disease. The low INF scores may indicate that the TCSs were poorly informed about the potential health problems related to their treatment. Thus, it is suggested that more attention should be focused on the education of the patients.
Pubmed: ABSTRACT Article (Taberlet1996) Taberlet, P.; Griffin, S.; Goossens, B.; Questiau, S.; Manceau, V.; Escaravage, N.; Waits, L.P. & Bouvet, J. Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res, 1996, 24, 3189-3194 Abstract: Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.
Pubmed: ABSTRACT Article (Tang1999) Tang, K.; Fu, D.J.; Julien, D.; Braun, A.; Cantor, C.R. & Koster, H. Chip-based genotyping by mass spectrometry Proc Natl Acad Sci U S A, 1999, 96, 10016-20 Abstract: Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-microl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.
Pubmed: ABSTRACT Article (Tapp2000) Tapp, I.; Malmberg, L.; Rennel, E.; Wik, M. & Syvanen, A.C. Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5nuclease TaqMan assay and Molecular Beacon probes Biotechniques, 2000, 28, 732-8 Abstract: Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan 5nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene. The Molecular Beacon probes distinguished better than the TaqMan probes between homozygous and heterozygous genotypes of a C-to-G transversion in codon 325. The sensitivity of detecting one allele, present as a minority in a mixed sample, varied between the SNPs and was similar for both assays. With the Molecular Beacon assay, the measured signal ratios were proportional to the amount of the minor allele over a wider range than with the TaqMan assay at all three SNPs.
Pubmed: ABSTRACT Article (Tawfik1998) Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat Biotechnol, 1998, 16, 652-656 Abstract: Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10(7)-fold excess of genes encoding another enzyme.
Pubmed: ABSTRACT Article (Taylor1995) Taylor, G.R. & Logan, W.P. The polymerase chain reaction: new variations on an old theme. Curr Opin Biotechnol, 1995, 6, 24-29 Abstract: The polymerase chain reaction (PCR) is firmly established as the method of choice for DNA amplification, though alternative strategies, such as the ligase chain reaction, may also be employed. Despite the continued development of PCR applications for gene mapping and diagnostics, few revolutionary improvements have been made to the technique. The major exception is long-accurate PCR, which has increased the length of amplifiable DNA by an order of magnitude.
Pubmed: ABSTRACT Article (taylor1997) Taylor, T.B.; Deen, E.S.W.; Picozza, E.; Woudenberg, T.M. & Albin, M. Optimization of the performance of the polymerase chain reaction in silicon-based microstructures. Nucleic Acids Res, 1997, 25, 3164-3168 Abstract: We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.
Pubmed: ABSTRACT Article (Tews2003) Tews, B.; Wilhelm, J.; Summerer, D.; Strerath, M.; Marx, A.; Friedhoff, P.; Pingoud, A. & Hahn, M. Application of the C4alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants Biol Chem, 2003, 384, 1533-41 Abstract: This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3terminally modified primers were designed carrying a methyl residue bound to the C4of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.
Pubmed: ABSTRACT Article (Thorisson2003) Thorisson, G.A. & Stein, L.D. The SNP Consortium website: past, present and future Nucleic Acids Res, 2003, 31, 124-7 Abstract: The SNP Consortium website (http://snp.cshl.org) has undergone many changes since its initial conception three years ago. The database back end has been changed from the venerable ACeDB to the more scalable MySQL engine. Users can access the data via gene or single nucleotide polymorphism (SNP) keyword searches and browse or dump SNP data to textfiles. A graphical genome browsing interface shows SNPs mapped onto the genome assembly in the context of externally available gene predictions and other features. SNP allele frequency and genotype data are available via FTP-download and on individual SNP report web pages. SNP linkage maps are available for download and for browsing in a comparative map viewer. All software components of the data coordinating center (DCC) website (http://snp.cshl.org) are open source.
Pubmed: ABSTRACT Article (Thorsen2006) Thorsen, L.; Nystad, W.; Stigum, H.; Hjermstad, M.; Oldervoll, L.; Martinsen, E.W.; Hornslien, K.; Strømme, S.B.; Dahl, A.A. & Fosså, S.D. Cardiorespiratory fitness in relation to self-reported physical function in cancer patients after chemotherapy. J Sports Med Phys Fitness, 2006, 46, 122-127 Abstract: AIM: The aim of this study was to estimate the association between objective cardiorespiratory fitness (CRF) and subjective self-reported physical function, taking into account the influence of mental distress. We hypothesized an association between these parameters, since they might be thought to measure parts of the same phenomenon. METHODS: Approximately 1 month after discontinuation of all primary treatment, 90 cancer patients aged 18-50 years treated with chemotherapy were surveyed. CRF was determined by the Astrand-Ryhming indirect cycle ergometer test, which indicate peak VO(2) in mL.kg(-1).min(-1) (predicted VO(2max)). Self-reported physical function was assessed by The European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30). The relation between VO(2max) and self-reported physical function was estimated by multiple linear regression. Mental distress (assessed by The Hospital Anxiety and Depression scale), age, gender, body mass index (BMI), time from treatment to physical test and diagnoses were included as potential confounders. RESULTS: There was no association between predicted VO(2max) and self-reported physical function. Mental distress was negatively associated with self-reported physical function (P<0.001), but is not associated with predicted VO(2max). CONCLUSIONS: The results suggest that predicted VO(2max) does not reflect self-reported physical function and vice versa in cancer patients after chemotherapy. If information about cardiac and/or pulmonary status is required, direct or indirect measures of VO(2max) should be used.
Pubmed: ABSTRACT Article (Tichopad2003) Tichopad, A.; Dilger, M.; Schwarz, G. & Pfaffl, M.W. Standardized determination of real-time PCR efficiency from a single reaction set-up Nucleic Acids Res, 2003, 31, e122 Abstract: We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.
Pubmed: ABSTRACT Article (Till2004) Till, B.J.; Burtner, C.; Comai, L. & Henikoff, S. Mismatch cleavage by single-strand specific nucleases Nucleic Acids Res, 2004, 32, 2632-41 Abstract: We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery.
Pubmed: ABSTRACT Article (Tillib2001) Tillib, S.V.; Strizhkov, B.N. & Mirzabekov, A.D. Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip Anal Biochem, 2001, 292, 155-60 Abstract: We have developed a method for parallel independent on-chip amplification and the following sequence variation analysis of multiple DNA regions directly using microchip with an array of nanoliter gel pads containing specific sets of tethered primers. The method has three key features. First, DNA to be amplified is enriched at gel pads by its hybridization with immobilized primers. Second, different sets of specific primers are immobilized within various gel pads, and primers are detached within gel pads just before polymerase chain reaction to enhance the amplification. A gel pad may contain an additional permanently immobilized dormant primer that is activated to carry out the allele-specific primer extension reaction to detect mutations. Third, multiple polymerase chain reactions are confined within nanoliter gel pads covered and separated from each other with mineral oil. The method was applied to simultaneously identify several abundant drug-resistant mutations in three genes of Mycobacterium tuberculosis.
Pubmed: ABSTRACT Article (Toepke2006) Toepke, M.W. & Beebe, D.J. PDMS absorption of small molecules and consequences in microfluidic applications. Lab Chip, 2006, 6, 1484-1486 Abstract: Microfluidic devices made out of polydimethylsiloxane (PDMS) have many physical properties that are useful for cell culture applications, such as transparency and gas permeability. Another distinct characteristic of PDMS is its ability to absorb hydrophobic small molecules. Partitioning of molecules into PDMS can significantly change solution concentrations and could potentially alter experimental outcomes. Herein we discuss PDMS absorption and its potential impact on microfluidic experiments.
Pubmed: ABSTRACT Article (Toepke2007) Toepke, M.W.; Brewer, S.H.; Vu, D.M.; Rector, K.D.; Morgan, J.E.; Gennis, R.B.; Kenis, P.J.A. & Dyer, R.B. Microfluidic flow-flash: method for investigating protein dynamics. Anal Chem, 2007, 79, 122-128 Abstract: We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or O2 binding to myoglobin after the laser-induced photolysis of CO from myoglobin by UV/visible absorbance spectral imaging.
Pubmed: ABSTRACT Article (Tost2002) Tost, J.; Brandt, O.; Boussicault, F.; Derbala, D.; Caloustian, C.; Lechner, D. & Gut, I.G. Molecular haplotyping at high throughput Nucleic Acids Res, 2002, 30, e96 Abstract: Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described beta(2)-adrenergic receptor gene.
Pubmed: ABSTRACT Article (Tost2003) Tost, J.; Dunker, J. & Gut, I.G. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing Biotechniques, 2003, 35, 152-6
Pubmed: ABSTRACT Article (Tost2003a) Tost, J.; Schatz, P.; Schuster, M.; Berlin, K. & Gut, I.G. Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry Nucleic Acids Res, 2003, 31, e50 Abstract: As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.
Pubmed: ABSTRACT Article (Tresset2005) Tresset, G. & Takeuchi, S. Utilization of cell-sized lipid containers for nanostructure and macromolecule handling in microfabricated devices. Anal Chem, 2005, 77, 2795-2801 Abstract: We propose an original approach to handle submicrometer-sized biological or inorganic materials in microfabricated devices for micro total analysis applications. Cell-sized liposomes were utilized as containers for nanoparticles, green fluorescent proteins, or DNA and handled within a microfluidic chip. Due to the micrometer size of these liposomes, their detection could be achieved by conventional optical systems. Moreover, liposomes are hardly sensitive to Brownian motion; their trapping or transportation is thereby made easy with electrostatic-based techniques, for instance, developed the past few years for cells and particles. Encapsulated materials were confined for long durations with respect to the diffusive scale time, and the liposome membrane provided excellent protection from the outside environment, inhibiting undesirable interactions. A microfluidic device consisting of a flow cell covering an array of asymmetric electrodes allowed us to convey readily liposomes by the AC electroosmosis effect. We also assessed the electrofusion of liposomes between micromachined electrodes, opening up controlled initiation of reaction inside these containers; it was exemplified by fusing differently colored liposomes. We observed that a large fraction of the liposomes fused for electric field intensity around 6 kV/cm. Applications ranging from ultrasmall biomimetic reactors to large-scale drug delivery or cell labeling can be envisaged.
Pubmed: ABSTRACT Article (Tricarico2002) Tricarico, C.; Pinzani, P.; Bianchi, S.; Paglierani, M.; Distante, V.; Pazzagli, M.; Bustin, S.A. & Orlando, C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies Anal Biochem, 2002, 309, 293-300 Abstract: Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.
Pubmed: ABSTRACT Article (Tsui2003) Tsui, C.; Coleman, L.E.; Griffith, J.L.; Bennett, E.A.; Goodson, S.G.; Scott, J.D.; Pittard, W.S. & Devine, S.E. Single nucleotide polymorphisms (SNPs) that map to gaps in the human SNP map Nucleic Acids Res, 2003, 31, 4910-6 Abstract: An international effort is underway to generate a comprehensive haplotype map (HapMap) of the human genome represented by an estimated 300,000 to 1 million agsingle nucleotide polymorphisms (SNPs). Our analysis indicates that the current human SNP map is not sufficiently dense to support the HapMap project. For example, 24.6% of the genome currently lacks SNPs at the minimal density and spacing that would be required to construct even a conservative tag SNP map containing 300,000 SNPs. In an effort to improve the human SNP map, we identified 140,696 additional SNP candidates using a new bioinformatics pipeline. Over 51,000 of these SNPs mapped to the largest gaps in the human SNP map, leading to significant improvements in these regions. Our SNPs will be immediately useful for the HapMap project, and will allow for the inclusion of many additional genomic intervals in the final HapMap. Nevertheless, our results also indicate that additional SNP discovery projects will be required both to define the haplotype architecture of the human genome and to construct comprehensive tag SNP maps that will be useful for genetic linkage studies in humans.
Pubmed: ABSTRACT Article (Turner2006) Turner, D.J.; Shendure, J.; Porreca, G.; Church, G.; Green, P.; Smith, C.T. & Hurles, M.E. Assaying chromosomal inversions by single-molecule haplotyping. Nat Methods, 2006, 3, 439-445 Abstract: Inversions are an important form of structural variation, but they are difficult to characterize, as their breakpoints often fall within inverted repeats. We have developed a method called 'haplotype fusion' in which an inversion breakpoint is genotyped by performing fusion PCR on single molecules of human genomic DNA. Fusing single-copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. Furthermore, we demonstrated that inversion events with breakpoints embedded within long (>100 kb) inverted repeats can be genotyped by haplotype-fusion PCR followed by bead-based single-molecule haplotyping on repeat-specific markers bracketing the inversion breakpoint. We illustrate this method by genotyping a Yp paracentric inversion sponsored by >300-kb-long inverted repeats. The generality of our methods to survey for, and genotype chromosomal inversions should help our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer.
Pubmed: ABSTRACT Article (Twist2004) Twist, C.R.; Winson, M.K.; Rowland, J.J. & Kell, D.B. Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence Anal Biochem, 2004, 327, 35-44 Abstract: We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are approximately 1fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.
Pubmed: ABSTRACT Article (Twyman2003) Twyman, R.M. & Primrose, S.B. Techniques patents for SNP genotyping Pharmacogenomics, 2003, 4, 67-79 Abstract: Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome, accounting for more than 90% of all differences between individuals. Many complex phenotypes in humans have a significant genetic component and most of the variability is therefore likely to stem from differences in patterns of SNPs. Association studies involving the large-scale analysis of SNPs can help to identify genes affecting many human phenotype variations, including complex diseases and drug responses. SNPs therefore play a major role in all stages of the drug development process, from target identification through to clinical trials. SNPs are also the basis of pharmacogenomics, the tailoring of medicines to suit an individual genome. Given the potential impact of SNPs on healthcare, the biotechnology industry has focussed urgently on the development of high-throughput methods for SNP genotyping. All genotyping methods are a mix and match of different allele discrimination and signal detection technologies and as such may represent the intellectual property of several individuals or organizations. In this review, we explore the patent issues surrounding SNP genotyping and how this is influencing large scale, commercially valuable projects involving SNPs.
Pubmed: ABSTRACT Article (Tyagi1996) Tyagi, S. & Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol, 1996, 14, 303-308 Abstract: We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.
Pubmed: ABSTRACT Article (Uhlmann2002) Uhlmann, K.; Brinckmann, A.; Toliat, M.R.; Ritter, H. & Nurnberg, P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis Electrophoresis, 2002, 23, 4072-9 Abstract: Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot trade mark and Pyrosequencing trade mark to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput.
Pubmed: ABSTRACT Article (Useche2001) Useche, F.J.; Gao, G.; Harafey, M. & Rafalski, A. High-throughput identification, database storage and analysis of SNPs in EST sequences Genome Inform Ser Workshop Genome Inform, 2001, 12, 194-203 Abstract: Single nucleotide polymorphisms (SNPs) are the most frequent form of DNA variation and disease-causing mutations in many genes. Due to their abundance and slow mutation rate within generations, they are thought to be the next generation of genetic markers that can be used in a myriad of important biological, genetic, pharmacological, and medical applications. There are several strategies both experimental, and in-silico for SNP discovery and mapping. Experimental SNP discovery consists of a number of labourious steps that make this process complex and expensive. In-silico discovery has been proposed as an alternative discovery method that makes use and takes advantage of large data sets with potential SNP information that have been generated with other purposes and have not been used as a SNP information source yet. However, in order to successfully apply the in-silico method to large data sets, the following challenges need to be addressed: First it is necessary to build an integrated SNP pipeline that handles data processing steps smoothly from the beginning (collecting sequence information) to end (SNPs in the database). Also, SNP detection tool parameters have to be optimized to satisfy specific goals of the project. Finally, SNP data could not be fully used until the in-silico method is validated experimentally. In this paper we present a design and implementation of an in-silico SNP detection software pipeline that exploits the existence of large EST (expressed sequence tag) data sets and effectively addresses the above challenges. First, the pipeline allows for smooth data transition between its different components by implementing data interfaces that translate the data formats of the different tools in the different stages. Second, we optimized PolyBayes parameters for SNP detection in maize EST. Finally, we implemented a user interface that along with the database structure created allows the scientist to perform preliminary analysis of the data and to perform basic statistics on the SNP data prior to experimental validation. The pipeline works with two different types of sequence assemblers (PHRAP (http://www.phrap.org/) and CAT from DoubleTwist (http://www.doubletwist.com/). It uses a Bayesian engine for SNP detection (PolyBayes), selects relevant polymorphism information which is then uploaded into a database. We detected 2439 SNPs and 822 insertion deletions (INDELs) with a PolyBayes probability higher than 0.99 on the public set of 68,000 maize ESTs. The user interface allowed us analyzing the polymorphism information right after discovery in several ways that allowed us to gain insight into the distribution and significance of the newly acquired data.
Pubmed: ABSTRACT Article (Vaerman2004) Vaerman, J.L.; Saussoy, P. & Ingargiola, I. Evaluation of real-time PCR data. J Biol Regul Homeost Agents, 2004, 18, 212-214 Abstract: If real-time PCR is to be of much worth to its user, some idea regarding the reliability of its data is essential. We discuss here some of the problems associated with interpreting numerical real-time PCR data that lend themselves to analytical evaluation. We translate into the language of molecular biology some of the criteria which are used to evaluate the performance of any new method (linearity, precision, specificity, limit of detection and quantification).
Pubmed: ABSTRACT Article (Van2001) Van Heel, D.A.; McGovern, D.P. & Jewell, D.P. Crohn disease: genetic susceptibility, bacteria, and innate immunity Lancet, 2001, 357, 1902-4
Pubmed: ABSTRACT Article (Van2003) Van Ness, J.; Van Ness, L.K. & Galas, D.J. Isothermal reactions for the amplification of oligonucleotides Proc Natl Acad Sci U S A, 2003, 100, 4504-9 Abstract: We have devised a class of isothermal reactions for amplifying DNA. These homogeneous reactions rapidly synthesize short oligonucleotides (8-16 bases) specified by the sequence of an amplification template. Versions of the reactions can proceed in either a linear or an exponential amplification mode. Both of these reactions require simple, constant conditions, and the rate of amplification depends entirely on the molecular parameters governing the interactions of the molecules in the reaction. The exponential version of the reaction is a molecular chain reaction that uses the oligonucleotide products of each linear reaction to create producers of more of the same oligonucleotide. It is a highly sensitive chain reaction that can be specifically triggered by given DNA sequences and can achieve amplifications of >10(6)-fold. Several similar reactions in this class are described here. The robustness, speed, and sensitivity of the exponential reaction suggest it will be useful in rapidly detecting the presence of small amounts of a specific DNA sequence in a sample, and a range of other applications, including many currently making use of the PCR.
Pubmed: ABSTRACT Article (Vandesompele2002) Vandesompele, J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A. & Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3, 34 Abstract: BACKGROUND: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. RESULTS: We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. CONCLUSIONS: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
Pubmed: ABSTRACT Article (Vega2005) la Vega, F.M.D.; Lazaruk, K.D.; Rhodes, M.D. & Wenz, M.H. Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SNP Genotyping Assays and the SNPlex Genotyping System. Mutat Res, 2005, 573, 111-135 Abstract: In this review we describe the principles, protocols, and applications of two commercially available SNP genotyping platforms, the TaqMan SNP Genotyping Assays and the SNPlex Genotyping System. Combined, these two technologies meet the requirements of multiple SNP applications in genetics research and pharmacogenetics. We also describe a set of SNP selection tools and validated assay resources which we developed to accelerate the cycle of experimentation on these platforms. Criteria for selecting the more appropriate of these two genotyping technologies are presented: the genetic architecture of the trait of interest, the throughput required, and the number of SNPs and samples needed for a successful study. Overall, the TaqMan assay format is suitable for low- to mid-throughput applications in which a high assay conversion rate, simple assay workflow, and low cost of automation are desirable. The SNPlex Genotyping System, on the other hand, is well suited for SNP applications in which throughput and cost-efficiency are essential, e.g., applications requiring either the testing of large numbers of SNPs and samples, or the flexibility to select various SNP subsets.
Pubmed: ABSTRACT Article (Venter2001) Venter, J.C.; Adams, M.D.; Myers, E.W.; Li, P.W.; Mural, R.J.; Sutton, G.G.; Smith, H.O.; Yandell, M.; Evans, C.A.; Holt, R.A.; Gocayne, J.D.; Amanatides, P.; Ballew, R.M.; Huson, D.H.; Wortman, J.R.; Zhang, Q.; Kodira, C.D.; Zheng, X.H.; Chen, L.; Skupski, M.; Subramanian, G.; Thomas, P.D.; Zhang, J.; Gabor Miklos, G.L.; Nelson, C.; Broder, S.; Clark, A.G.; Nadeau, J.; McKusick, V.A.; Zinder, N.; Levine, A.J.; Roberts, R.J.; Simon, M.; Slayman, C.; Hunkapiller, M.; Bolanos, R.; Delcher, A.; Dew, I.; Fasulo, D.; Flanigan, M.; Florea, L.; Halpern, A.; Hannenhalli, S.; Kravitz, S.; Levy, S.; Mobarry, C.; Reinert, K.; Remington, K.; Abu-Threideh, J.; Beasley, E.; Biddick, K.; Bonazzi, V.; Brandon, R.; Cargill, M.; Chandramouliswaran, I.; Charlab, R.; Chaturvedi, K.; Deng, Z.; Di Francesco, V.; Dunn, P.; Eilbeck, K.; Evangelista, C.; Gabrielian, A.E.; Gan, W.; Ge, W.; Gong, F.; Gu, Z.; Guan, P.; Heiman, T.J.; Higgins, M.E.; Ji, R.R.; Ke, Z.; Ketchum, K.A.; Lai, Z.; Lei, Y.; Li, Z.; Li, J.; Liang, Y.; Lin, X.; Lu, F.; Merkulov, G.V.; Milshina, N.; Moore, H.M.; Naik, A.K.; Narayan, V.A.; Neelam, B.; Nusskern, D.; Rusch, D.B.; Salzberg, S.; Shao, W.; Shue, B.; Sun, J.; Wang, Z.; Wang, A.; Wang, X.; Wang, J.; Wei, M.; Wides, R.; Xiao, C.; Yan, C. & others The sequence of the human genome Science, 2001, 291, 1304-51 Abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
Pubmed: ABSTRACT Article (Venter2004) Venter, J.C.; Remington, K.; Heidelberg, J.F.; Halpern, A.L.; Rusch, D.; Eisen, J.A.; Wu, D.; Paulsen, I.; Nelson, K.E.; Nelson, W.; Fouts, D.E.; Levy, S.; Knap, A.H.; Lomas, M.W.; Nealson, K.; White, O.; Peterson, J.; Hoffman, J.; Parsons, R.; Baden-Tillson, H.; Pfannkoch, C.; Rogers, Y.H. & Smith, H.O. Environmental genome shotgun sequencing of the Sargasso Sea Science, 2004, 304, 66-74 Abstract: We have applied hole-genome shotgun sequencingto microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.
Pubmed: ABSTRACT Article (Verma1998) Verma, S. & Eckstein, F. Modified oligonucleotides: synthesis and strategy for users Annu Rev Biochem, 1998, 67, 99-134 Abstract: Synthetic oligonucleotide analogs have greatly aided our understanding of several biochemical processes. Efficient solid-phase and enzyme-assisted synthetic methods and the availability of modified base analogs have added to the utility of such oligonucleotides. In this review, we discuss the applications of synthetic oligonucleotides that contain backbone, base, and sugar modifications to investigate the mechanism and stereochemical aspects of biochemical reactions. We also discuss interference mapping of nucleic acid-protein interactions; spectroscopic analysis of biochemical reactions and nucleic acid structures; and nucleic acid cross-linking studies. The automation of oligonucleotide synthesis, the development of versatile phosphoramidite reagents, and efficient scale-up have expanded the application of modified oligonucleotides to diverse areas of fundamental and applied biological research. Numerous reports have covered oligonucleotides for which modifications have been made of the phosphodiester backbone, of the purine and pyrimidine heterocyclic bases, and of the sugar moiety; these modifications serve as structural and mechanistic probes. In this chapter, we review the range, scope, and practical utility of such chemically modified oligonucleotides. Because of space limitations, we discuss only those oligonucleotides that contain phosphate and phosphate analogs as internucleotidic linkages.
Pubmed: ABSTRACT Article (Vickers2006) Vickers, J.; Caulum, M. & Henry, C. Generation of Hydrophilic Poly(dimethylsiloxane) for High-Performance Microchip Electrophoresis Analytical Chemistry, 2006 Abstract: Poly(dimethylsiloxane) (PDMS) has become one of the most widely used materials for microchip capillary electrophoresis and microfluidics. The popularity of this material is the result of its low cost, simple fabrication, and rugged elastomeric properties. The hydrophobic nature of PDMS, however, limits its applicability for microchip CE, microfluidic patterning, and other nonelectrophoresis applications. The surface of PDMS can be made hydrophilic using a simple air plasma treatment; however, this property is quickly lost through hydrophobic recovery caused by diffusion of unreacted oligomer to the surface. Here, a simple approach for the generation of hydrophilic PDMS with long-term stability in air is presented. PDMS is rendered hydrophilic through a simple two-step extraction/oxidation process. First, PDMS is extracted in a series of solvents designed to remove unreacted oligomers from the bulk phase. Second, the oligomer-free PDMS is oxidized in a simple air plasma, generating a stable layer of hydrophilic SiO2. The conversion of surface-bound siloxane to SiO2 was followed with X-ray photoelectron spectroscopy. SiO2 on extracted-oxidized PDMS was stable for 7 days in air as compared to less than 3 h for native PDMS. Furthermore, the contact angle for modified PDMS was reduced to <40 and remained low throughout the experiments. As a result of the decreased contact angle, capillary channels self-wet through capillary action, making the microchannels much easier to fill. Finally, the modification significantly improved the performance of the devices for microchip electrophoresis. The electroosmotic flow increased from 4.1 × 10-4 to 6.8 × 10-4 cm2/V·s for native compared to oxidized PDMS. Separation efficiencies for electrochemical detection also increased from 50 000 to 400 000 N/m for a 1.1-nL injection volume. The result of this modification is a significant improvement in the performance of PDMS for microchip electrophoresis and microfluidic applications.
PDF: Vickers2006.pdf Article (Vieux2002) Vieux, E.F.; Kwok, P.Y. & Miller, R.D. Primer design for PCR and sequencing in high-throughput analysis of SNPs Biotechniques, 2002, Suppl, 28-30, 32 Abstract: To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.
Pubmed: ABSTRACT Article (Vladkov2006) Vladkov, M. & Barrat, J. Modeling Transient Absorption and Thermal Conductivity in a Simple Nanofluid Nano Letters, 2006
PDF: Vladkov2006.pdf Article (Vlahovicek2003) Vlahovicek, K.; Kajan, L. & Pongor, S. DNA analysis servers: plot.it, bend.it, model.it and IS Nucleic Acids Res, 2003, 31, 3686-7 Abstract: The WWW servers at http://www.icgeb.trieste.it/dna/ are dedicated to the analysis of user-submitted DNA sequences; plot.it creates parametric plots of 45 physicochemical, as well as statistical, parameters; bend.it calculates DNA curvature according to various methods. Both programs provide 1D as well as 2D plots that allow localisation of peculiar segments within the query. The server model.it creates 3D models of canonical or bent DNA starting from sequence data and presents the results in the form of a standard PDB file, directly viewable on the user PC using any molecule manipulation program. The recently established introns server allows statistical evaluation of introns in various taxonomic groups and the comparison of taxonomic groups in terms of length, base composition, intron type etc. The options include the analysis of splice sites and a probability test for exon-shuffling.
Pubmed: ABSTRACT Article (Vo-Dinh2000) Vo-Dinh, T. & Cullum, B. Biosensors and biochips: advances in biological and medical diagnostics Fresenius J Anal Chem, 2000, 366, 540-51 Abstract: In the past two decades, the biological and medical fields have seen great advances in the development of biosensors and biochips capable of characterizing and quantifying biomolecules. This review is meant to provide an overview of the various types of biosensors and biochips that have been developed for biological and medical applications, along with significant advances over the last several years in these technologies. It also attempts to describe various classification schemes that can be used for categorizing the different biosensors and provide relevant examples of these classification schemes from recent literature.
Pubmed: ABSTRACT Article (Vos1995) Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; van de Lee, T.; Hornes, M.; Frijters, A.; Pot, J.; Peleman, J.; Kuiper, M. & e. al. AFLP: a new technique for DNA fingerprinting Nucleic Acids Res, 1995, 23, 4407-14 Abstract: A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
Pubmed: ABSTRACT Article (Vu2000) Vu, H.L.; Troubetzkoy, S.; Nguyen, H.H.; Russell, M.W. & Mestecky, J. A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis Nucleic Acids Res, 2000, 28, E18 Abstract: Accurate quantification of nucleic acids by competitive (RT)-PCR requires a valid internal standard, a reference for data normalization and an adequate mathematical model for data analysis. We report here an effective procedure for the generation of homologous RNA internal standards and a strategy for synthesizing and using a reference target RNA in quantification of absolute amounts of nucleic acids. Further, a new mathematical model describing the general kinetic features of competitive PCR was developed. The model extends the validity of quantitative competitive (RT)-PCR beyond the exponential phase. The new method eliminates the errors arising from different amplification efficiencies of the co-amplified sequences and from heteroduplex formation in the system. The high accuracy (relative error <2%) is comparable to the recently developed real time detection 5nuclease PCR. Also, corresponding computer software has been devised for practical data analysis.
Pubmed: ABSTRACT Article (Walker1992) Walker, G.T.; Fraiser, M.S.; Schram, J.L.; Little, M.C.; Nadeau, J.G. & Malinowski, D.P. Strand displacement amplification--an isothermal, in vitro DNA amplification technique Nucleic Acids Res, 1992, 20, 1691-6 Abstract: Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5 and 3ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5 and 3ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5 and 3sequences modified as desired.
Pubmed: ABSTRACT Article (Wall2003) Wall, J.D. & Pritchard, J.K. Assessing the performance of the haplotype block model of linkage disequilibrium Am J Hum Genet, 2003, 73, 502-15 Abstract: Several recent studies have suggested that linkage disequilibrium (LD) in the human genome has a fundamentally locklikestructure. However, thus far there has been little formal assessment of how well the haplotype block model captures the underlying structure of LD. Here we propose quantitative criteria for assessing how blocklike LD is and apply these criteria to both real and simulated data. Analyses of several large data sets indicate that real data show a partial fit to the haplotype block model; some regions conform quite well, whereas others do not. Some improvement could be obtained by genotyping higher marker densities but not by increasing the number of samples. Nonetheless, although the real data are only moderately blocklike, our simulations indicate that, under a model of uniform recombination, the structure of LD would actually fit the block model much less well. Simulations of a model in which much of the recombination occurs in narrow hotspots provide a much better fit to the observed patterns of LD, suggesting that there is extensive fine-scale variation in recombination rates across the human genome.
Pubmed: ABSTRACT Article (Wallace1979) Wallace, R.B.; Shaffer, J.; Murphy, R.F.; Bonner, J.; Hirose, T. & Itakura, K. Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res, 1979, 6, 3543-3557 Abstract: Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.
Pubmed: ABSTRACT Article (Wang2003) Wang, D.; Gao, H.; Zhang, R.; Ma, X.; Zhou, Y. & Cheng, J. Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays Biotechniques, 2003, 35, 300-2, 304, 306 passim Abstract: Efficiencies of mismatch discrimination using size-varied capture probes were examined at various hybridization temperatures. The probes were 17, 15, 13, 11, 9, and 7 nucleotides long and contained single-base mismatches at their 3ends. The optimal signal intensity and efficiency of base stacking hybridization on mismatch discrimination were observed for capture probes with a melting temperature (Tm) value of 36 degrees C, in the detection of DNA sequence variations at 40 degrees C. We employed asymmetric PCR to prepare single-stranded target DNA labeled with a fluorescent dye, and the PCR product was hybridized on the DNA microarray with no further purification. Our efforts have enhanced the sensitivity and simplified the procedures of base stacking hybridization on mismatch discrimination. As a model experiment, this improved technology was used to identify plasmid templates of human leukocyte antigen (HLA)-A alleles 2601, 2902, and 0206 on oligonucleotide microarrays. It is now possible to apply this simple, rapid, sensitive, and reliable base stacking hybridization technology to detect DNA sequence variations on microarrays in clinical diagnosis and other applications.
Pubmed: ABSTRACT Article (Wang1998) Wang, D.G.; Fan, J.B.; Siao, C.J.; Berno, A.; Young, P.; Sapolsky, R.; Ghandour, G.; Perkins, N.; Winchester, E.; Spencer, J.; Kruglyak, L.; Stein, L.; Hsie, L.; Topaloglou, T.; Hubbell, E.; Robinson, E.; Mittmann, M.; Morris, M.S.; Shen, N.; Kilburn, D.; Rioux, J.; Nusbaum, C.; Rozen, S.; Hudson, T.J.; Lander, E.S. & e. al. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome Science, 1998, 280, 1077-82 Abstract: Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.
Pubmed: ABSTRACT Article (Wang2006a) Wang, J.; Bunimovich, Y.L.; Sui, G.; Savvas, S.; Wang, J.; Guo, Y.; Heath, J.R. & Tseng, H. Electrochemical fabrication of conducting polymer nanowires in an integrated microfluidic system. Chem Commun (Camb), 2006, 3075-3077 Abstract: In this paper, we introduce a new approach for the in situ electrochemical fabrication of an individually addressable array of conducting polymer nanowires (CPNWs) positioned within an integrated microfluidic device and also demonstrate that such an integrated device can be used as a chemical sensor immediately after its construction.
Pubmed: ABSTRACT Article (Wang2005a) Wang, K. & Jones, T.B. Electrowetting dynamics of microfluidic actuation. Langmuir, 2005, 21, 4211-4217 Abstract: When voltage is suddenly applied to vertical, parallel dielectric-coated electrodes dipped into a liquid with finite conductivity, the liquid responds by rising up to reach a new hydrostatic equilibrium height. On the microfluidic scale, the dominating mechanism impeding this electromechanically induced actuation appears to be a dynamic friction force that is directly proportional to the velocity of the contact line moving along the solid surface. This mechanism has its origin in the molecular dynamics of the liquid coming into contact with the solid surface. A simple reduced-order model for the rising column of liquid is used to quantify the magnitude of this frictional effect by providing estimates for the contact line friction coefficient. Above some critical threshold of voltage, the electromechanical force is clamped, presumably by the same mechanism responsible for contact angle saturation and previously reported static height-of-rise limits. The important distinction for the dynamic case is that the onset of the saturation effect is delayed in time until the column has risen more than about halfway to its static equilibrium height.
Pubmed: ABSTRACT Article (Wang2002) Wang, N.; Akey, J.M.; Zhang, K.; Chakraborty, R. & Jin, L. Distribution of recombination crossovers and the origin of haplotype blocks: the interplay of population history, recombination, and mutation Am J Hum Genet, 2002, 71, 1227-34 Abstract: Recent studies suggest that haplotypes are arranged into discrete blocklike structures throughout the human genome. Here, we present an alternative haplotype block definition that assumes no recombination within each block but allows for recombination between blocks, and we use it to study the combined effects of demographic history and various population genetic parameters on haplotype block characteristics. Through extensive coalescent simulations and analysis of published haplotype data on chromosome 21, we find that (1) the combined effects of population demographic history, recombination, and mutation dictate haplotype block characteristics and (2) haplotype blocks can arise in the absence of recombination hot spots. Finally, we provide practical guidelines for designing and interpreting studies investigating haplotype block structure.
Pubmed: ABSTRACT Article (Wang2006) Wang, P.; Dai, M.; Xuan, W.; McEachin, R.C.; Jackson, A.U.; Scott, L.J.; Athey, B.; Watson, S.J. & Meng, F. SNP Function Portal: a web database for exploring the function implication of SNP alleles. Bioinformatics, 2006, 22, e523-e529 Abstract: MOTIVATION: Finding the potential functional significance of SNPs is a major bottleneck in understanding genome-wide SNP scanning results, as the related functional data are distributed across many different databases. The SNP Function Portal is designed to be a clearing house for all public domain SNP functional annotation data, as well as in-house functional annotations derived from different data sources. It currently contains SNP functional annotations in six major categories including genomic elements, transcription regulation, protein function, pathway, disease and population genetics. Besides extensive SNP functional annotations, the SNP Function Portal includes a powerful search engine that accepts different types of genetic markers as input and identifies all genetically related SNPs based on the HapMap Phase II data as well as the relationship of different markers to known genes. As a result, our system allows users to identify the potential biological impact of genetic markers and complex relationships among genetic markers and genes, and it greatly facilitates knowledge discovery in genome-wide SNP scanning experiments. AVAILABILITY: http://brainarray.mbni.med.umich.edu/Brainarray/Database/SearchSNP/snpfunc.aspx.
Pubmed: ABSTRACT Article (Wang2003a) Wang, X. & Seed, B. A PCR primer bank for quantitative gene expression analysis Nucleic Acids Res, 2003, 31, e154 Abstract: Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in approximately 40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.
Pubmed: ABSTRACT Article (Wang2005) Wang, Y.; Moorhead, M.; Neumann, G.K.; Falkowski, M.; Chen, C.; Siddiqui, F.; Davis, R.W.; Willis, T.D. & Faham, M. Allele quantification using molecular inversion probes (MIP). Nucleic Acids Res, 2005, 33, e183 Abstract: Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples.
Pubmed: ABSTRACT Article (Wang2006b) Wang, Z.; Sekulovic, A.; Kutter, J.P.; Bang, D.D. & Wolff, A. Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR. Electrophoresis, 2006, 27, 5051-5058 Abstract: A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.
Pubmed: ABSTRACT Article (Waters1998) Waters, L.C.; Jacobson, S.C.; Kroutchinina, N.; Khandurina, J.; Foote, R.S. & Ramsey, J.M. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing Anal Chem, 1998, 70, 158-62 Abstract: The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device. The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection. Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage lambda DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E. coli genomic and plasmid DNAs are amplified. The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels. Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder.
Pubmed: ABSTRACT Article (Wei2006) Wei Wang, H.W. Silicon inhibition effects on the polymerase chain reaction: A real-time detection approach Journal of Biomedical Materials Research Part A, 2006, 77A, 28-34 Abstract: In the miniaturization of biochemical analysis systems, biocompatibility of the microfabricated material is a key feature to be considered. A clear insight into interactions between biological reagents and microchip materials will help to build more robust functional bio-microelectromechanical systems (BioMEMS). In the present work, a real-time polymerase chain reaction (PCR) assay was used to study the inhibition effects of silicon and native silicon oxide particles on Hepatitis B Virus (HBV) DNA PCR amplification. Silicon nanoparticles with different surface oxides were added into the PCR mixture to activate possible interactions between the silicon-related materials and the PCR reagents. Ratios of silicon nanoparticle surface area to PCR mixture volume (surface to volume ratio) varied from 4.7 to 235.5 mm2/?L. Using high speed centrifugation, the nanoparticles were pelleted to tube inner surfaces. Supernatant extracts were then used in subsequent PCR experiments. To test whether silicon materials participated in amplifications directly, in some cases, entire PCR mixture containing silicon nanoparticles were used in amplification. Fluorescence histories of PCR amplifications indicated that with the increase in surface to volume ratio, amplification efficiency decreased considerably, and within the studied ranges, the higher the particle surface oxidation, the stronger the silicon inhibition effects on PCR. Adsorption of Taq polymerase (not nucleic acid) on the silicon-related material surface was the primary cause of the inhibition phenomena and silicon did not participate in the amplification process directly. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
DOI: http://dx.doi.org/10.1002/jbm.a.30627 Article (Wei2005) Wei, C.; Cheng, J.; Huang, C.; Yen, M. & Young, T. Using a microfluidic device for 1 microl DNA microarray hybridization in 500 s. Nucleic Acids Res, 2005, 33, e78 Abstract: This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 microl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA-DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 mul target was used to hybridize with an array that can hold 5000 probes.
Pubmed: ABSTRACT Article (Weiner2002) Weiner, M.P. & Hudson, T.J. Introduction to SNPs: discovery of markers for disease Biotechniques, 2002, Suppl, 4-7, 10, 12-3
Pubmed: ABSTRACT Article (Wen2007) Wen, J.; Yang, X.; Wang, K.; Tan, W.; Zhou, L.; Zuo, X.; Zhang, H. & Chen, Y. One-dimensional microfluidic beads array for multiple mRNAs expression detection. Biosens Bioelectron, 2007 Abstract: A one-dimensional microfluidic beads array for in vitro rapid measurement of multiple mRNAs expression is presented in this paper. Gene specific capture DNA-functional beads were deposited along a microchannel to form an addressable beads array. We demonstrated that the one-dimensional beads array could perform simultaneous multiple nucleic acid targets detection and a DNA detection limit of 0.02nM was obtained. Using this array, transcripts expression of three tumor-associated genes, including p53, H-ras, and NME1, both in CNE2 nasopharyngeal carcinoma cell lines and in normal human nasopharyngeal epithelial cells were evaluated. The responses of these three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Validation of these results was performed using reverse transcriptase-PCR. The presented methodology combines high throughput of microarrays and low sample consumption, convenient liquid handling of microfluidics. It enables rapid and facile determination of multi-gene expression and holds great potential in early cancer diagnostics and molecular biology.
Pubmed: ABSTRACT Article (Wenzel2003) Wenzel, T.; Elssner, T.; Fahr, K.; Bimmler, J.; Richter, S.; Thomas, I. & Kostrzewa, M. Genosnip: SNP genotyping by MALDI-TOF MS using photocleavable oligonucleotides Nucleosides Nucleotides Nucleic Acids, 2003, 22, 1579-81 Abstract: A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthesised and incorporated within oligonucleotides. After allele-specific primer extension, desalting was performed using genostrep purification plates. Release of the SNP information containing part through photocleavage created shortened molecules that are easily accessible for MALDI-TOF analysis. Additionally, incorporation of mass modified nucleosides enables flexible design of multiplex genotyping.
Pubmed: ABSTRACT Article (West2002) West, J.; Karamata, B.; Lillis, B.; Gleeson, J.P.; Alderman, J.; Collins, J.K.; Lane, W.; Mathewson, A. & Berney, H. Application of magnetohydrodynamic actuation to continuous flow chemistry. Lab Chip, 2002, 2, 224-230 Abstract: Continuous flow microreactors with an annular microchannel for cyclical chemical reactions were fabricated by either bulk micromachining in silicon or by rapid prototyping using EPON SU-8. Fluid propulsion in these unusual microchannels was achieved using AC magnetohydrodynamic (MHD) actuation. This integrated micropumping mechanism obviates the use of moving parts by acting locally on the electrolyte, exploiting its inherent conductive nature. Both silicon and SU-8 microreactors were capable of MHD actuation, attaining fluid velocities of the order of 300 microm s(-1) when using a 500 mM KCl electrolyte. The polymerase chain reaction (PCR), a thermocycling process, was chosen as an illustrative example of a cyclical chemistry. Accordingly, temperature zones were provided to enable a thermal cycle during each revolution. With this approach, fluid velocity determines cycle duration. Here, we report device fabrication and performance, a model to accurately describe fluid circulation by MHD actuation, and compatibility issues relating to this approach to chemistry.
Pubmed: ABSTRACT Article (Wetmur1991) Wetmur, J.G. DNA probes: applications of the principles of nucleic acid hybridization Crit Rev Biochem Mol Biol, 1991, 26, 227-59 Abstract: Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.
Pubmed: ABSTRACT Article (Weusten2002) Weusten, J.J.; Carpay, W.M.; Oosterlaken, T.A.; van Zuijlen, M.C. & van de Wiel, P.A. Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons Nucleic Acids Res, 2002, 30, e26 Abstract: For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.
Pubmed: ABSTRACT Article (Wheeler2006) Wheeler, D.L.; Barrett, T.; Benson, D.A.; Bryant, S.H.; Canese, K.; Chetvernin, V.; Church, D.M.; DiCuccio, M.; Edgar, R.; Federhen, S.; Geer, L.Y.; Helmberg, W.; Kapustin, Y.; Kenton, D.L.; Khovayko, O.; Lipman, D.J.; Madden, T.L.; Maglott, D.R.; Ostell, J.; Pruitt, K.D.; Schuler, G.D.; Schriml, L.M.; Sequeira, E.; Sherry, S.T.; Sirotkin, K.; Souvorov, A.; Starchenko, G.; Suzek, T.O.; Tatusov, R.; Tatusova, T.A.; Wagner, L. & Yaschenko, E. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res, 2006, 34, D173-D180 Abstract: In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Retroviral Genotyping Tools, HIV-1, Human Protein Interaction Database, SAGEmap, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.
Pubmed: ABSTRACT Article (Wheeler2003) Wheeler, D.L.; Church, D.M.; Federhen, S.; Lash, A.E.; Madden, T.L.; Pontius, J.U.; Schuler, G.D.; Schriml, L.M.; Sequeira, E.; Tatusova, T.A. & Wagner, L. Database resources of the National Center for Biotechnology Nucleic Acids Res, 2003, 31, 28-33 Abstract: In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, PubMed, PubMed Central (PMC), LocusLink, the NCBITaxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR (e-PCR), Open Reading Frame (ORF) Finder, References Sequence (RefSeq), UniGene, HomoloGene, ProtEST, Database of Single Nucleotide Polymorphisms (dbSNP), Human/Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker (MM), Evidence Viewer (EV), Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.
Pubmed: ABSTRACT Article (Wheeler2004) Wheeler, E.K.; Benett, W.; Stratton, P.; Richards, J.; Chen, A.; Christian, A.; Ness, K.D.; Ortega, J.; Li, L.G.; Weisgraber, T.H.; Goodson, K. & Milanovich, F. Convectively driven polymerase chain reaction thermal cycler. Anal Chem, 2004, 76, 4011-4016 Abstract: We have fabricated a low-cost disposable polymerase chain reaction thermal chamber that uses buoyancy forces to move the sample solution between the different temperatures necessary for amplification. Three-dimensional, unsteady finite element modeling and a simpler 1-D steady-state model are used together with digital particle image velocimetry data to characterize the flow within the device. Biological samples have been amplified using this novel thermal chamber. Time for amplification is less than 30 min. More importantly, an analysis of the energy consumption shows significant improvements over current technology.
Pubmed: ABSTRACT Article (Whelan2003) Whelan, J.A.; Russell, N.B. & Whelan, M.A. A method for the absolute quantification of cDNA using real-time PCR J Immunol Methods, 2003, 278, 261-9 Abstract: Real-time PCR is an extremely powerful technique, however, often its results are open to interpretation since there is no convention for data presentation. This anomaly has arisen because many applications rely on non-standard calibration genes, which themselves often change in value during experimental manipulation.We present a novel method for absolute quantification of cDNA species using a combination of extremely accurate double-stranded DNA quantification and a plasmid reference curve. PicoGreen and reference standards are used to measure the amount of cDNA present in a sample using fluorescence. Real-time PCR products are cloned into plasmids and then used to calibrate unknown samples. This cloning is achieved using the same primers necessary for real-time PCR and thus does not involve a second design stage. Results are expressed as copy number per microgram of oligo-dT primed cDNA and consequently may be compared between both inter and intra-experimentally. We show results from a sample human system in which absolute levels of interferon-gamma, TNF-alpha, interleukin-2 and interleukin-10 are measured. We further compare the copy numbers of these genes with levels of released protein and find remarkable correlation. Although our interest has been cytokine quantification, we believe that this technique is widely applicable to the majority of real-time PCR applications.
Pubmed: ABSTRACT Article (Whitcombe1998) Whitcombe, D.; Brownie, J.; Gillard, H.L.; McKechnie, D.; Theaker, J.; Newton, C.R. & Little, S. A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping Clin Chem, 1998, 44, 918-23 Abstract: We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.
Pubmed: ABSTRACT Article (Whitcombe1999) Whitcombe, D.; Theaker, J.; Guy, S.P.; Brown, T. & Little, S. Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol, 1999, 17, 804-807 Abstract: Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.
Pubmed: ABSTRACT Article (Whitesides2006) Whitesides, G.M. The origins and the future of microfluidics. Nature, 2006, 442, 368-373 Abstract: The manipulation of fluids in channels with dimensions of tens of micrometres--microfluidics--has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. But the field is still at an early stage of development. Even as the basic science and technological demonstrations develop, other problems must be addressed: choosing and focusing on initial applications, and developing strategies to complete the cycle of development, including commercialization. The solutions to these problems will require imagination and ingenuity.
Pubmed: ABSTRACT Article (Wilding1999) Wilding, P. & Kricka, L.J. Micro-microchips: just how small can we go? Trends Biotechnol, 1999, 17, 465-8
Pubmed: ABSTRACT Article (Wilding1998) Wilding, P.; Kricka, L.J.; Cheng, J.; Hvichia, G.; Shoffner, M.A. & Fortina, P. Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chambers Anal Biochem, 1998, 257, 95-100 Abstract: White blood cells are isolated from whole blood in silicon-glass 4.5-microliter microchips containing a series of 3.5-micron feature-sized 'weir-type' filters, formed by an etched silicon dam spanning the flow chamber. Genomic DNA targets, e.g., dystrophin gene, can be directly amplified using the polymerase chain reaction (PCR) from the white cells isolated on the filters. This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.
Pubmed: ABSTRACT Article (Wilding1994) Wilding, P.; Shoffner, M.A. & Kricka, L.J. PCR in a silicon microstructure Clin Chem, 1994, 40, 1815-8 Abstract: Devices for performing polymerase chain reactions (PCR) have been developed for use with photolithographed silicon. Microchambers capable of holding between 5.0 and 10 microL of PCR reagents were constructed by etching specific areas of rectangular silicon chips (17 x 15 mm), which were then capped with Pyrex glass. These silicon devices (PCRChips), which were etched to depths of 40-80 microns, permitted free flow of fluids in the microchannels and microchambers. Access to the microchambers was through holes in the silicon. Thermal cycling of the PCR reagents was achieved by placing the disposable PCRChip in a small holder containing a computer-controlled Peltier heater-cooler. Successful amplification was demonstrated by electrophoresis of products in agarose gel containing ethidium bromide, and the migration of the product was compared with that obtained in a commercially available thermal cycler. The thermal characteristics of the silicon, coupled with the high surface area to volume ratio in the new devices, are particularly advantageous features for amplification by PCR.
Pubmed: ABSTRACT Article (Wilkinson2002) Wilkinson, M.D. & Links, M. BioMOBY: an open source biological web services proposal Brief Bioinform, 2002, 3, 331-41 Abstract: BioMOBY is an Open Source research project which aims to generate an architecture for the discovery and distribution of biological data through web services; data and services are decentralised, but the availability of these resources, and the instructions for interacting with them, are registered in a central location called MOBY Central. BioMOBY adds to the web services paradigm, as exemplified by Universal Data Discovery and Integration (UDDI), by having an object-driven registry query system with object and service ontologies. This allows users to traverse expansive and disparate data sets where each possible next step is presented based on the data object currently in-hand. Moreover, a path from the current data object to a desired final data object could be automatically discovered using the registry. Native BioMOBY objects are lightweight XML, and make up both the query and the response of a simple object access protocol (SOAP) transaction.
Pubmed: ABSTRACT Article (Wilson1997) Wilson, I.G. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol, 1997, 63, 3741-3751
Pubmed: ABSTRACT Article (Wittwer1990) Wittwer, C.T.; Fillmore, G.C. & Garling, D.J. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal Biochem, 1990, 186, 328-331 Abstract: Hot-air temperature cycling of 1- to 10-microliters samples in glass capillary tubes can amplify DNA by the polymerase chain reaction in 15 min or less. A rapid temperature cycler of low thermal mass was constructed to change sample temperatures among denaturation, annealing, and elongation segments in a few seconds. After 30 cycles of 30 s each, a 536-bp beta-globin fragment of human genomic DNA was easily visualized with ethidium bromide on agarose gels. With rapid cycling, amplification yield depended on polymerase concentration. The time required for DNA amplification can be markedly reduced from prevailing protocols if appropriate equipment and sample containers are used for rapid heat transfer to the sample.
Pubmed: ABSTRACT Article (Wittwer1997) Wittwer, C.T.; Herrmann, M.G.; Moss, A.A. & Rasmussen, R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification Biotechniques, 1997, 22, 130-1, 134-8 Abstract: Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5 by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The sensitivity of SYBR Green I detection is limited by nonspecific product formation. Use of a single exonuclease hydrolysis probe or two adjacent hybridization probes offers increasing levels of specificity. In contrast to fluorescence measurement once per cycle, continuous monitoring throughout each cycle monitors the temperature dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization probes. By using SYBR Green I, product denaturation, annealing and extension can be followed within each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a major cause of the plateau effect. Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.
Pubmed: ABSTRACT Article (Wittwer1997a) Wittwer, C.T.; Ririe, K.M.; Andrew, R.V.; David, D.A.; Gundry, R.A. & Balis, U.J. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control Biotechniques, 1997, 22, 176-81 Abstract: Experimental and commercial microvolume fluorimeters with rapid temperature control are described. Fluorescence optics adopted from flow cytometry were used to interrogate 1-10-microL samples in glass capillaries. Homogeneous temperature control and rapid change of sample temperatures (10 degrees C/s) were obtained by a circulating air vortex. A prototype 2-color, 32-sample version was constructed with a xenon arc for excitation, separate excitation and emission paths, and photomultiplier tubes for detection. The commercial LightCycler, a 3-color, 24-sample instrument, uses a blue light-emitting diode for excitation, paraxial epi-illumination through the capillary tip and photodiodes for detection. Applications include analyte quantification and nucleic acid melting curves with fluorescent dyes, enzyme assays with fluorescent substrates and techniques that use fluorescence resonance energy transfer. Microvolume capability allows analysis of very small or expensive samples. As an example of one application, rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques, Which included using the double-stranded DNA dye SYBR Green I, a dual-labeled 5exonuclease hydrolysis probe, and adjacent fluorescein and Cy5z-labeled hybridization probes. Complete amplification and analysis requires only 10-15 min.
Pubmed: ABSTRACT Article (Wong2004) Wong, K.K.; Tsang, Y.T.; Shen, J.; Cheng, R.S.; Chang, Y.M.; Man, T.K. & Lau, C.C. Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA Nucleic Acids Res, 2004, 32, e69 Abstract: Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single-nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosarcoma tissues and patient-matched blood. SNP calls were then generated by Affymetrix GeneChip DNA Analysis Software. In two osteosarcoma cases, using unamplified DNA, we identified 793 and 1070 SNP loci with allelic imbalance, respectively. In a parallel experiment with amplified DNA, 78% and 83% of these SNP loci with allelic imbalance was detected. The average false-positive rate is 13.8%. Furthermore, using the Affymetrix GeneChip Chromosome Copy Number Tool to analyze the SNP array data, we were able to detect identical chromosomal regions with gain or loss in both amplified and unamplified DNA at cytoband resolution.
Pubmed: ABSTRACT Article (Wong2002) Wong, L. Technologies for integrating biological data Brief Bioinform, 2002, 3, 389-404 Abstract: The process of building a new database relevant to some field of study in biomedicine involves transforming, integrating and cleansing multiple data sources, as well as adding new material and annotations. This paper reviews some of the requirements of a general solution to this data integration problem. Several representative technologies and approaches to data integration in biomedicine are surveyed. Then some interesting features that separate the more general data integration technologies from the more specialised ones are highlighted.
Pubmed: ABSTRACT Article (Wong2005) Wong, M.L. & Medrano, J.F. Real-time PCR for mRNA quantitation. Biotechniques, 2005, 39, 75-85 Abstract: Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.
Pubmed: ABSTRACT Article (Woolley2000) Woolley, A.T.; Guillemette, C.; Li Cheung, C.; Housman, D.E. & Lieber, C.M. Direct haplotyping of kilobase-size DNA using carbon nanotube probes Nat Biotechnol, 2000, 18, 760-3 Abstract: We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.
Pubmed: ABSTRACT Article (Woolley1996) Woolley, A.T.; Hadley, D.; Landre, P.; deMello, A.J.; Mathies, R.A. & Northrup, M.A. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device Anal Chem, 1996, 68, 4081-6 Abstract: Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an ëlectrophoretic valveto couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.
Pubmed: ABSTRACT Article (Wu1991) Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Qian, J. & Wallace, R.B. The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction DNA Cell Biol, 1991, 10, 233-8 Abstract: The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.
Pubmed: ABSTRACT Article (Woelcke2001) Wölcke, J. & Ullmann, D. Miniaturized HTS technologies - uHTS. Drug Discov Today, 2001, 6, 637-646 Abstract: The transition from slow, manual, low-throughput screening to industrialized robotic ultra-high throughput screening (uHTS) in the past few years has made it possible to screen hundreds of thousands of chemical entities against a biological target in a short time-frame. The need to minimize the cost of screening has been addressed primarily by reducing the volume of sample to be screened. This, in turn, has resulted in the miniaturization of HTS technology as a whole. Miniaturization requires new technologies and strategies for compound handling, assay development, assay adaptation, liquid handling and automation in addition to refinement of the technologies used for detection systems and data management. This review summarizes current trends in the field of uHTS and illustrates the technological developments that are necessary to enable the routine application of miniaturized uHTS systems within an industrial environment.
Pubmed: ABSTRACT Article (Xiang2005) Xiang, Q.; Xu, B.; Fu, R. & Li, D. Real time PCR on disposable PDMS chip with a miniaturized thermal cycler. Biomed Microdevices, 2005, 7, 273-279 Abstract: This paper presents the design and implementation of a low-cost miniature PCR device consisting of a disposable reactor chip and a miniature thermal cycler. The simple fabrication of the PCR chip by PDMS (Polydimethylsiloxane) does not need micro-machining or photolithography processes. The thermal cycler was built with a thin film heater for heating and a fan for rapid cooling. This device can perform PCR tests in a single well chip or a multiple-well chip. It can run PCR reactions of different volumes to meet specific application requirements. The smallest reaction volume tested in this work is 0.9 microL. In addition, this device fits any standard fluorescence microscope for real time detection, which makes real time PCR affordable for most research labs and clinics with a fluorescence microscope. Real-time PCR of E. coli stx1 has been demonstrated with the device described.
Pubmed: ABSTRACT Article (Xiao1998) Xiao, C.; Xia, Q.; Wu, H. & Zhang, S. Uniform terminus PCR: amplification of minute unknown DNA fragments Biotechniques, 1998, 25, 780-1, 784
Pubmed: ABSTRACT Article (Xu2003) Xu, H.; Sha, M.Y.; Wong, E.Y.; Uphoff, J.; Xu, Y.; Treadway, J.A.; Truong, A.; E, O.'.; Asquith, S.; Stubbins, M.; Spurr, N.K.; Lai, E.H. & Mahoney, W. Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay Nucleic Acids Res, 2003, 31, e43 Abstract: We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral arcodesare created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.
Pubmed: ABSTRACT Article (Xu1997) Xu, X.H. & Yeung, E.S. Direct measurement of single-molecule diffusion and photodecomposition in free solution Science, 1997, 275, 1106-9 Abstract: Continuous monitoring of submillisecond free-solution dynamics of individual rhodamine-6G molecules and 30-base single-stranded DNA tagged with rhodamine was achieved. Fluorescence images were recorded from the same set of isolated molecules excited either through the evanescent field at the quartz-liquid interface or as a thin layer of solution defined by micron-sized wires, giving diffraction-limited resolution of inter-connected attoliter volume elements. The single-molecule diffusion coefficients were smaller and the unimolecular photodecomposition lifetimes were longer for the dye-DNA covalent complex as compared with those of the dye molecule itself. Unlike bulk studies, stochastic behavior was found for individual molecules of each type, and smaller diffusion coefficients were observed.
Pubmed: ABSTRACT Article (Yager2006) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M.R. & Weigl, B.H. Microfluidic diagnostic technologies for global public health. Nature, 2006, 442, 412-418 Abstract: The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.
Pubmed: ABSTRACT Article (Yakovchuk2006) Yakovchuk, P.; Protozanova, E. & Kamenetskii, M.D.F. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res, 2006, 34, 564-574 Abstract: Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A*T- and G*C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A*T pairing is always destabilizing and G*C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
Pubmed: ABSTRACT Article (Yamada2006) Yamada, M.; Hirano, T.; Yasuda, M. & Seki, M. A microfluidic flow distributor generating stepwise concentrations for high-throughput biochemical processing. Lab Chip, 2006, 6, 179-184 Abstract: In this paper, we describe a microfluidic device in which solutions with stepwise concentrations can be accurately generated by continuously introducing two kinds of miscible liquids from each inlet, and biochemical processing can be conducted at the various conditions. Introduced liquid flows are geometrically divided into a number of downstream flows through multiple distribution channels, and each divided flow is then mixed with the divided flow of another liquid at a confluent point. The lengths of the precisely designed distribution channels determine the mixing ratio of the two liquids, without the influence of flow rate. In this study, a PDMS microfluidic device able to generate nine different concentrations was fabricated, and the performance of this device was estimated via colorimetric assay. As a biological application of this device, cell cultivation was performed under different concentration conditions. Due to its simplicity of operation, this microfluidic flow distributor will be applied to various kinds of biological analysis and screening systems.
Pubmed: ABSTRACT Article (Yang1993) Yang, B.; Yolken, R. & Viscidi, R. Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase Anal Biochem, 1993, 208, 110-6 Abstract: Nucleic acid amplification by polymerase chain reaction (PCR) is a very powerful technique in terms of sensitivity but is limited in terms of ability to perform accurate quantitation. While there is a theoretical correlation between copies of input target sequence and those of PCR product, the quantitative nature of this relationship is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances which might be present in sample preparations, especially those derived from biological fluids. To reliably estimate copies of input DNA target from PCR product, we designed a combination of internal and external control systems coupled to DNA/RNA hybridization and enzymatic immunodetection techniques. The internal control system served to monitor amplification efficiency and to correct for the effects of inhibitors or stimuli on the efficiency of the DNA amplification. The assay is quantitative, nonisotopic, and can be widely applied to assessment of the quantity of DNA present in a wide range of preparations.
Pubmed: ABSTRACT Article (Yang2002) Yang, J.; Liu, Y.; Rauch, C.B.; Stevens, R.L.; Liu, R.H.; Lenigk, R. & Grodzinski, P. High sensitivity PCR assay in plastic micro reactors. Lab Chip, 2002, 2, 179-187 Abstract: Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.
Pubmed: ABSTRACT Article (Yang2007) Yang, L.; Fung, C.; Cho, E. & Ellington, A. Real-Time Rolling Circle Amplification for Protein Detection. Anal Chem, 2007 Abstract: Real-time nucleic acid amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to protein or other analytes. To facilitate the development of real-time rolling circle amplification (RCA) for protein targets, we have developed a novel type of conformation-switching aptamer that can be circularized upon interaction with its protein target, the platelet-derived growth factor (PDGF). Using the structure-switching aptamer, real-time RCA can be used to specifically quantitate PDGF down to the low-nanomolar range (limit of detection, 0.4 nM), even against a background of cellular lysate. The aptamer can also be adapted to RCA on surfaces, although quantitation proved to be more difficult. One of the great advantages of the method described herein is that it can be immediately adapted to almost any aptamer and does not require two or more affinity reagents as do sandwich or proximity assays.
Pubmed: ABSTRACT Article (Yang2003) Yang, Q.; Lishanski, A.; Yang, W.; Hatcher, S.; Seet, H. & Gregg, J.P. Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring Genome Res, 2003, 13, 1754-64 Abstract: We report here a new mechanism for allelic discrimination--allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.
Pubmed: ABSTRACT Article (Yao2005) Yao, L.; Liu, B.; Chen, T.; Liu, S. & Zuo, T. Micro flow-through PCR in a PMMA chip fabricated by KrF excimer laser. Biomed Microdevices, 2005, 7, 253-257 Abstract: As the third PCR technology, micro flow-through PCR chip can amplify DNA specifically in an exponential fashion in vitro. Nowadays many academies in the world have successfully amplified DNA using their own-made flow-through PCR chip. In this paper, the ablation principle of PMMA at 248 nm excimer laser was studied, then a PMMA based flow-through PCR chip with 20 cycles was fabricated by excimer laser at 19 kv and 18 mm/min. The chip was bonded together with another cover chip at 105( composite function)C, 160 N and 20 minutes. In the end, it was integrated with electrical thermal thin films and Pt 100 temperature sensors. The temperature controllers was built standard PID digital temperature controller, the temperature control precision was +/- 0.2( composite function)C. The temperature grads between the three temperature zones were 16.5 and 22.2( composite function)C respectively, the gaps between the temperature zones could realize heat insulation.
Pubmed: ABSTRACT Article (Yi2006) Yi, C.; Zhang, Q.; Li, C.; Yang, J.; Zhao, J. & Yang, M. Optical and electrochemical detection techniques for cell-based microfluidic systems. Anal Bioanal Chem, 2006, 384, 1259-1268 Abstract: The ability to fabricate microfluidic systems with complex structures and with compatible dimensions between the microfluidics and biological cells have attracted significant attention in the development of microchips for analyzing the biophysical and biochemical functions of cells. Just as cell-based microfluidics have become a versatile tool for biosensing, diagnostics, drug screening and biological research, detector modules for cell-based microfluidics have also undergone major development over the past decade. This review focuses on detection methods commonly used in cell-based microfluidic systems, and provides a general survey and an in-depth look at recent developments in optical and electrochemical detection methods for microfluidic applications for biological systems, particularly cell analysis. Selected examples are used to illustrate applications of these detection systems and their advantages and weaknesses.
Pubmed: ABSTRACT Article (Yi2006a) Yi, J.; Zhang, W. & Zhang, D.Y. Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification. Nucleic Acids Res, 2006, 34, e81 Abstract: Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.
Pubmed: ABSTRACT Article (Yoon2002) Yoon, D.S.; Lee, Y.; Lee, Y.; Cho, H.J.; Sung, S.W.; Oh, K.W.; Cha, J. & Lim, G. Precise temperature control and rapid thermal cycling in a micromachined DNA polymerase chain reaction chip Journal of Micromechanics and Microengineering, 2002, 12, 813-823
PDF: Yoon2002.pdf Article (Yu2007) Yu, L.; Li, C.; Zhou, Q. & Luong, J. Poly(vinyl alcohol) Functionalized Poly(dimethylsiloxane) Solid Surface for Immunoassay. Bioconjug Chem, 2007 Abstract: In this communication, we describe a simple and robust method for the covalent bonding of poly(vinyl alcohol) (PVA) on a silanized poly(dimethylsiloxane) (PDMS) surface. Nonspecific adsorption of proteins via hydrophobic-hydrophobic interactions of the PVA-coated surface is greatly reduced, and biomolecules can be rapidly anchored on the PVA-coated surface with high loading and uniformity. On the basis of a sandwich immunoassay with the anti-rabbit IgG and IgG pair as a model, the detection limit for IgG is down to 1 pg/mL with linearity up to 11 mug/mL, the levels often encountered in biological, forensic, and environmental samples.
Pubmed: ABSTRACT Article (Yu2005) Yu, X.M.; Li, T.; Hao, L. & Zhang, D.C. PCR microchip array based on polymer bonding technique JOURNAL OF ELECTRONIC PACKAGING, 2005, 127, 38-42 Abstract: A polymerase chain reaction (PCR) microchip array with a dimension of 21.3 x 17.5 mm(2) has been fabricated by the silicon micromachining technique. The chip is composed of 192 rectangular reaction chambers with the volume of 50 nL. In order to package the silicon-based PCR chip, a low-temperature bonding technique using polymers as the intermediate layers has been developed. The tested polymers include positive photoresist, polyimide, and epoxy. Using stamping and sandwich techniques, void-free and liquid-proof bondings of silicon to a glass cover were achieved with the three polymers. The experimental results of fluorescence-based PCR demonstrate that the DNA amplification can be performed in the chip arrays with nanoliter volumes and the polymer bonding technique is biocompatible and suitable,for the microchip packaging.
Article (Yuan2006) Yuan, J.S.; Reed, A.; Chen, F. & Stewart, C.N. Statistical analysis of real-time PCR data. BMC Bioinformatics, 2006, 7, 85 Abstract: BACKGROUND: Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data. RESULTS: In the first approach, a multiple regression analysis model was developed to derive DeltaDeltaCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the DeltaDeltaCt can be derived from analysis of effects of variables. The other two models involve calculation DeltaCt followed by a two group t-test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS. CONCLUSION: Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.
Pubmed: ABSTRACT Article (Yun2006) Yun, J.J.; Heisler, L.E.; Hwang, I.I.L.; Wilkins, O.; Lau, S.K.; Hyrcza, M.; Jayabalasingham, B.; Jin, J.; McLaurin, J.; Tsao, M. & Der, S.D. Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Res, 2006, 34, e85 Abstract: Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.
Pubmed: ABSTRACT Article (Yuryev2002) Yuryev, A.; Huang, J.; Pohl, M.; Patch, R.; Watson, F.; Bell, P.; Donaldson, M.; Phillips, M.S. & Boyce-Jacino, M.T. Predicting the success of primer extension genotyping assays using statistical modeling Nucleic Acids Res, 2002, 30, e131 Abstract: Using an empirical panel of more than 20 000 single base primer extension (SNP-IT) assays we have developed a set of statistical scores for evaluating and rank ordering various parameters of the SNP-IT reaction to facilitate high-throughput assay primer design with improved likelihood of success. Each score predicts either signal magnitude from primer extension or signal noise caused by mispriming of primers and structure of the PCR product. All scores have been shown to correlate with the success/failure rate of the SNP-IT reaction, based on analysis of assay results. A logistic regression analysis was applied to combine all scored parameters into one measure predicting the overall success/failure rate of a given SNP marker. Three training sets for different types of SNP-IT reaction, each containing about 22 000 SNP markers, were used to assign weights to each score and optimize the prediction of the combined measure. c-Statistics of 0.69, 0.77 and 0.72 were achieved for three training sets. This new statistical prediction can be used to improve primer design for the SNP-IT reaction and evaluate the probability of genotyping success for a given SNP based on analysis of the surrounding genomic sequence.
Pubmed: ABSTRACT Article (Zdobnov2002) Zdobnov, E.M.; Lopez, R.; Apweiler, R. & Etzold, T. The EBI SRS server--recent developments Bioinformatics, 2002, 18, 368-73 Abstract: MOTIVATION: The current data explosion is intractable without advanced data management systems. The numerous data sets become really useful when they are interconnected under a uniform interface--representing the domain knowledge. The SRS has become an integration system for both data retrieval and applications for data analysis. It provides capabilities to search multiple databases by shared attributes and to query across databases fast and efficiently. RESULTS: Here we present recent developments at the EBI SRS server (http://srs.ebi.ac.uk). The EBI SRS server contains today more than 130 biological databases and integrates more than 10 applications. It is a central resource for molecular biology data as well as a reference server for the latest developments in data integration. One of the latest additions to the EBI SRS server is the InterPro database-Integrated Resource of Protein Domains and Functional Sites. Distributed in XML format it became a turning point in low level XML-SRS integration. We present InterProScan as an example of data analysis applications, describe some advanced features of SRS6, and introduce the SRSQuickSearch JavaScript interfaces to SRS.
Pubmed: ABSTRACT Article (Zhang2001) Zhang, D.Y.; Zhang, W.; Li, X. & Konomi, Y. Detection of rare DNA targets by isothermal ramification amplification Gene, 2001, 274, 209-16 Abstract: We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277). This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target. Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo olling circlereplication of bacteriophage. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification. Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity. However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses. Therefore, we tested several DNA polymerases and found that exo(-) Bst DNA polymerase meets the requirement for high sensitivity. By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens.
Pubmed: ABSTRACT Article (Zhang2001a) Zhang, G.; Zhang, S.; Chen, W.; Qiu, W.; Wu, H.; Wang, J.; Luo, J.; Gu, X. & Cotton, R.G. Go!Poly: A gene-oriented polymorphism database Hum Mutat, 2001, 18, 382-7 Abstract: Human genome polymorphisms play a key role in defining the molecular basis of phenotypic differences between individuals in aspects such as disease susceptibility and drug responses. The database requirements for supporting the study of human genetic variation have been well recognized. In order to meet these needs, several generalized databases have been built. However, it is still hard for users to find gene-related variation data from these huge and sophisticated databases. In its role as a gene-oriented directory of polymorphism data, Go!Poly (Gene Oriented Polymorphism Database; http://61.139.84.5/gopoly/) utilizes two new highly curated and non-redundant resources, LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) and RefSeq (http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html), as the standard for identifying and positioning nucleotide variations. As a generalized polymorphism database, Go!Poly extracts human gene-linked sequence variations of all common types (SNP, insertion-deletion, simple tandem repeat, and complex nucleotides variations) from various public resources including scientific journals and internet resources, such as HGBASE (http://hgbase.cgr.ki.se) and dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). The polymorphism data are then categorized into different gene loci, and the reference sequences given by LocusLink are used as positioning references. Through close integration with LocusLink, Go!Poly also provides facilitated connections among sequence data, gene name, and related biological information. This feature also makes Go!Poly easy to search and navigate. Future automated annotations and internal consistency checking may also benefit from this. Extensive efforts are being taken to make the polymorphism information generated by the Chinese scientific community available from this resource.
Pubmed: ABSTRACT Article (Zhang2002) Zhang, J.; Day, I.N. & Byrne, C.D. A novel medium throughput quantitative competitive PCR technology to simultaneously measure mRNA levels from multiple genes Nucleic Acids Res, 2002, 30, e20 Abstract: There is a great demand for technologies to simultaneously measure mRNA levels from multiple genes. Here we report a new quantitative competitive PCR technology and demonstrate simultaneous quantification of mRNA from multiple genes. First, a sequential 2-fold dilution series containing equal amounts of gene-specific standard DNAs for 10-12 genes is prepared. Second, the serially diluted standard DNAs are individually added to equal amounts of tissue-derived cDNA and amplified with gene-specific primers for 19-26 PCR cycles. Each gene/standard DNA pair is amplified individually. All amplified DNA products (n = 80) are resolved by one microplate array diagonal gel electrophoresis using 5% polyacrylamide. Changes in mRNA levels of approximately 15% can be detected by this technology. The mRNA levels from 10-12 genes were simultaneously quantified. mRNA levels were compared in RNA samples from rat liver, kidney and skeletal muscle. This quick, specific, sensitive, reproducible and yet inexpensive technique is ideal for simultaneously studying co-ordinate changes in mRNA levels from multiple genes.
Pubmed: ABSTRACT Article (Zhang2003) Zhang, J. & Li, K. Single-base discrimination mediated by proofreading 3phosphorothioate-modified primers Mol Biotechnol, 2003, 25, 223-8 Abstract: It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3exonuclease activity and the 3phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.
Pubmed: ABSTRACT Article (Zhang2002a) Zhang, J.; Rowe, W.L.; Struewing, J.P. & Buetow, K.H. HapScope: a software system for automated and visual analysis of functionally annotated haplotypes Nucleic Acids Res, 2002, 30, 5213-21 Abstract: We have developed a software analysis package, HapScope, which includes a comprehensive analysis pipeline and a sophisticated visualization tool for analyzing functionally annotated haplotypes. The HapScope analysis pipeline supports: (i) computational haplotype construction with an expectation-maximization or Bayesian statistical algorithm; (ii) SNP classification by protein coding change, homology to model organisms or putative regulatory regions; and (iii) minimum SNP subset selection by either a Brute Force Algorithm or a Greedy Partition Algorithm. The HapScope viewer displays genomic structure with haplotype information in an integrated environment, providing eight alternative views for assessing genetic and functional correlation. It has a user-friendly interface for: (i) haplotype block visualization; (ii) SNP subset selection; (iii) haplotype consolidation with subset SNP markers; (iv) incorporation of both experimentally determined haplotypes and computational results; and (v) data export for additional analysis. Comparison of haplotypes constructed by the statistical algorithms with those determined experimentally shows variation in haplotype prediction accuracies in genomic regions with different levels of nucleotide diversity. We have applied HapScope in analyzing haplotypes for candidate genes and genomic regions with extensive SNP and genotype data. We envision that the systematic approach of integrating functional genomic analysis with population haplotypes, supported by HapScope, will greatly facilitate current genetic disease research.
Pubmed: ABSTRACT Article (Zhang1999) Zhang, J.H.; Chung, T.D. & Oldenburg, K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J Biomol Screen, 1999, 4, 67-73 Abstract: The ability to identify active compounds ((3)hits(2)) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the (3)suitability(2) of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called (3)Z-factor,(2) is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
Pubmed: ABSTRACT Article (Zhao2006) Zhao, W.; Yao, S. & Hsing, I. A microsystem compatible strategy for viable Escherichia coli detection. Biosens Bioelectron, 2006, 21, 1163-1170 Abstract: This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.
Pubmed: ABSTRACT Article (Zhao1997) Zhao, X.; Xia, Y. & Whitesides, G.M. Soft lithographic methods for nano-fabrication Journal of Materials Chemistry, 1997, 7, 1069-1074
Article (Zhao2003) Zhao, Z.; Fu, Y.; Emmett, D.H. & Boerwinkle, E. Investigating single nucleotide polymorphism (SNP) density in the human genome and its implications for molecular evolution. Gene, 2003, 312, 207-213 Abstract: We investigated the single nucleotide polymorphism (SNP) density across the human genome and in different genic categories using two SNP databases: Celera's CgsSNP, which includes SNPs identified by comparing genomic sequences, and Celera's RefSNP, which includes SNPs from a variety of sources and is biased toward disease-associated genes. Based on CgsSNP, the average numbers of SNPs per 10 kb was 8.33, 8.44, and 8.09 in the human genome, in intergenic regions, and in genic regions, respectively. In genic regions, the SNP density in intronic, exonic and adjoining untranslated regions was 8.21, 5.28, and 7.51 SNPs per 10 kb, respectively. The pattern of SNP density based on RefSNP was different from that based on CgsSNP, emphasizing its utility for genotype-phenotype association studies but not for most population genetic studies. The number of SNPs per chromosome was correlated with chromosome length, but the density of SNPs estimated by CgsSNP was not significantly correlated with the GC content of the chromosome. Based on CgsSNP, the ratio of nonsense to missense mutations (0.027), the ratio of missense to silent mutations (1.15), and the ratio of non-synonymous to synonymous mutations (1.18) was less than half of that expected in a human protein coding sequence under the neutral mutation theory, reflecting a role for natural selection, especially purifying selection.
Pubmed: ABSTRACT Article (Zheng2005) Zheng, B. & Ismagilov, R.F. A microfluidic approach for screening submicroliter volumes against multiple reagents by using preformed arrays of nanoliter plugs in a three-phase liquid/liquid/gas flow. Angew Chem Int Ed Engl, 2005, 44, 2520-2523
Pubmed: ABSTRACT Article (Zhou2003) Zhou, Y. & Liu, J. AVA: visual analysis of gene expression microarray data Bioinformatics, 2003, 19, 293-4 Abstract: Summary: AVA (Array Visual Analyzer) is a Java program that provides a graphical environment for visualization and analysis of gene expression microarray data. Together with its interactive visualization tools and a variety of built-in data analysis and filtration methods, AVA effectively integrates microarray data normalization, quality assessment, and data mining into one application. Availability: The software is freely available for academic users on request from the authors. Contact: yihua-zhou2@monsanto.com
Pubmed: ABSTRACT |
||||||||||||||||||||||||||||||
