Single-Nucleotide Polymorphism Allele Frequencies Determined by Quantitative Kinetic Assay of Pooled Dna (Technical Briefs‪)‬

The identification of genes for complex traits relies on the detection of coinheritance of chromosomal regions in related individuals or the sharing of functional or marker alleles among phenotypically similar individuals. Single-nucleotide polymorphisms (SNPs) comprise the most abundant and accessible class of sequence polymorphisms in the genome and are valuable for high-throughput genotyping (1). Genotyping individual DNA samples is expensive in time, reagents, labor, and DNA. These limitations can be overcome by applying a sample-pooling approach for allele frequency determination before genotyping (2,3). For quantitative allelotyping using pools, equivalent amounts of DNA from multiple individuals are combined in a single-tube assay. Most sample-pooling approaches involve post-PCR processing (4-7). One pooled allelotyping method uses kinetic allele-specific PCR coupled with SYBR Green I fluorescence detection (8). This method requires two separate PCR reactions for each DNA pool, each of which needs to be performed in parallel. Another pooled allelotyping method uses a bioluminometric assay coupled to a modified primer extension reaction. This method also involves multiple procedures (9). Allelotyping by hybridization to DNA chips has been described (10), but its quantitative accuracy has not been fully evaluated. The 5' nuclease assay (TagMan[R]) uses fluorogenic allele-specific detection probes that allow PCR amplification and allele detection in a single procedure (11). To discriminate alleles, each fluorogenic probe is labeled with a different fluorescent reporter dye. The reaction is kinetically monitored in "real time" to quantify the ratio of the two alleles (12). Taking advantage of the specificity and sensitivity of kinetic sequence detection, we developed a method for estimating SNP allele frequencies in pooled DNA samples. Accuracy was evaluated using constructed pools in which allele frequencies were incrementally varied and by comparing the allele frequencies determined from pooled DNA with allele frequencies determined by individual genotyping. For allele frequency determinations, we found that the pooled DNA sample approach is rapid, accurate, and reproducible and requires no post-PCR processing.