FISH and CHIPS

FISH applied to metaphase chromosomes allows us to determine the physical position of DNA sequences in respect to known chromosome bands and known genes or reference loci (Sirugo et al. 1996, Kingsley et al. 1997). Probes are not only mapped in humans but also in important animal models including many primates, mouse, rat, chicken, pufferfish and medaka. In metaphase chromosomes, probes that are 1-3 Mb apart are easily distinguishable, but for higher-resolution demands (in the range from 1 to 1000 kb) and probe ordering, FISH on experimentally stretched chromatin fibers (Haaf and Ward 1994) and combed DNA molecules has been established. In addition to the ordering of probes, fiber FISH technology is used for defining the size of gaps and overlaps between clones, and contig building. It also greatly facilitates positional cloning projects (Nothwang et al. 2000) and the construction of sequence-ready maps for probes which lie within 500-1000 kb of each other (Hattori et al. 2000). Combinatorial probe labeling and spectral karyotyping can uniquely identify all 24 human chromosomes by assigning each a different color (Borck et al. 2001). This allows the characterization of very complex chromosome rearrangements, i.e. in tumor cells, using a single hybridization experiment.

All the different applications of FISH depend on the general availability of DNA probes that are specific for a chromosome or chromosome region of interest. To facilitate the molecular cytogenetic analysis of chromosome rearrangements, we have developed a standard set of cytogenetically and genetically anchored YAC probes, approximately one every 1-3 cM, that are more or less evenly spaced throughout the entire human chromosome complement. This probe set will be integrated with human BAC/PAC resources and can be used with unlimited flexibility for a variety of FISH applications, such as positional cloning of disease-associated balanced chromosome rearrangements (Wirth et al. 1999) (Chromosome rearrangement and disease).

For the detection of subtle genome imbalances, i.e cytogenetically cryptic microdeletions and duplications in patient populations, comparative genomic hybridization (CGH) on topographic DNA microarrays is being developed. Initially, we use BAC arrays containing probes for all chromosomes ends or for an entire chromosome, i.e. the X. Our next generation of arrays will have a DNA resolution of one megabase (or better) across the whole genome. Custom region-specific arrays using overlapping BAC/PAC clones will allow high-resolution mapping of translocation breakpoints and other chromosome rearrangements (DNA microarrays).