High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers

We have developed a new method for high-throughput genotyping of single nuc leotide polymorphisms (SNPs). The technique involves PCR amplification of g enomic DNA with two tailed allele-specific primers that introduce priming s ites for universal energy-transfer-labeled primers. The output of red and g reen light is conveniently scored using a fluorescence plate reader. The ne w method, which was validated on nine model SNPs, is well suited for high-t hroughput, automated genotyping because it requires only one reaction per S NP, it is performed in a single tube with no post-PCR handling, the same en ergy-transfer-labeled primers are used for ail analyses, and the instrument ation is inexpensive. Possible applications include multiple-candidate gene analysis, genomewide scans, and medical diagnostics.