High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

The basis of allele frequency measurement using kinetic PCR. Shown are amplification growth curves of PCR reactions performed for the ApoB71 polymorphism. A sample was constructed from two DNAs each homozygous for the different alleles of the ApoB71 SNP and contains 5% of allele 1. Equal aliquots of the pool (20 ng of DNA each) were put into PCRs containing either of the two allele-specific primer sets. Four replicate reactions were performed with each primer set (eight PCRs total). The relative allele frequency is determined on the basis of the ΔC_t using equation 1 (see text and Fig. 2).