Allele frequency estimation in DNA pools of single nuleotide polymorphisms by primer extension and DHPLC

At present, a number of different methods exist for genotyping SNP's through case control and family based studies. However, none provide sufficient economies of cost and labour to permit testing several hundreds of candidate gene hypotheses. We have developed a method for estimating and comparing allele frequencies of SNPs in DNA pools and tested the possibility of using this method to estimate allele frequencies of SNPs in the 5HT2a receptor, proneurotensin and COMT genes in pooled samples of 160 affected and 157 controls. The method uses pooled DNA samples accurately quantified using the pico green method, primer extension and denaturing high performance liquid chromatography (DHPLC). Our data suggest that this technique will allow accurate measurement of both the absolute allele frequencies in pooled DNA samples and the difference in allele frequency between pooled DNA samples from affected and control groups. We have further evaluated this technique on five more assays from SNPs found in the PRODH, STX and mGLUR7 genes.