SNP detection using oligonucleotide microarrays

We developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3´end are covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing SNPs to be typed is achieved by an asymmetric PCR reaction or exonuclease treatment of PTO-modified PCR products. In the presence of DNA polymerase and all four dNTPs with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers takes place along a stretch of target DNA sequence. The yield of elongated products is increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets or used single targets of up to 4.4 kb mitochondrial DNA sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions thereby providing a framework for typing hundreds of mitochondrial DNA polymorphisms.

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

Erdogan F, Kirchner R, Mann W, Ropers HH, Nuber UA.
Nucleic Acids Res. 2001 Apr 1;29(7):E36