High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR

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 rela tive amounts of each allele in a sample are quantified. This is performed b y dividing equal aliquots of the pooled DNA between two separate PCR reacti ons, each of which contains a primer pail specific to one or the other alle lic SNP variant. For pools with equal amounts of the two alleles, the two a mplifications should reach a detectable level of fluorescence at the same c ycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions call 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 u sing eight different SNPs altogether. The accuracy of measuring known allel e frequencies is very high, with the strength of correlation between measur ed 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 t o sampling error alone, For population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultan eously, inexpensively, and reproducibly, this method is a powerful strategy For detecting meaningful polymorphic differences in candidate gene associa tion studies and genome-wide linkage disequilibrium scans.