Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools

Abstract

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.

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“…The method we adopted for determining gene frequencies in a pool combines the genotyping specificity of allele-specific primer extension assay with the quantitative accuracy of high-performance liquid chromatography (HPLC). Previous validation experiments performed in our lab 30 and by others 31 demonstrated that this method is quantitative and highly reproducible and allows an accurate estimation of allele frequencies in pooled samples of DNA. The reported mean experimental error, ie the difference between the allele frequency calculated by individual genotyping and that estimated in the pool, was 7 0.013 30 and 7 0.014 31 respectively, which is a satisfactory level of accuracy.…”

“…Previous validation experiments performed in our lab 30 and by others 31 demonstrated that this method is quantitative and highly reproducible and allows an accurate estimation of allele frequencies in pooled samples of DNA. The reported mean experimental error, ie the difference between the allele frequency calculated by individual genotyping and that estimated in the pool, was 7 0.013 30 and 7 0.014 31 respectively, which is a satisfactory level of accuracy. Previous quantitative analysis indicated that the lower detection threshold of this method lies between 0.01 and 0.05.…”

“…(Transgenomic) instrument. 31 For each SNP, a primer ending at the nucleotide preceding the variation was annealed to the amplified products previously purified by membrane filtration using the Montage PCR Clean up system (Millipore) to remove unincorporated PCR dNTPs and primers and extended by one or more nucleotides to obtain maximum resolution between the two alleles. Primer extension reactions were carried out in 20 ml containing about 40 ng of the purified fragment, 50 mM of the appropriate ddNTPs and/or dNTPs, 15 pmol primer and 0.5 U ThermoSequenase (Amersham), in the buffer provided by the manufacturer.…”

“…The frequency was estimated from the height of the peak corresponding to each extended primer in the HPLC elution profile as previously reported [30][31][32] and is summarised in Figure 1. For each sequence variation, each pool in duplicate (two PCR reactions) and at least three heterozygotes were analysed in the same experiment (including PCR, primer extension and HPLC analysis).…”

“…19,[26][27][28] The markers covered the 5' flanking and the transcribed region of the gene. Basically, the study was performed by the DNA pool method [29][30][31][32] on an extended panel of patients and controls. Haplotypic associations were studied by individual typing of selected markers.…”