Determination of SNP allele frequencies in pooled DNAs by primer extension genotyping and denaturing high-performance liquid chromatography

By testing DNA pools rather than single samples the number of tests for a case-control association study can be decreased to only two for each marker: one on the patient and one on the control pool. A fundamental requirement is that each pool represents the frequency of the markers in the corresponding population beyond the influence of experimental errors. Consequently the latter must be carefully determined. To this aim, we prepared pools of different size (49-402 individuals) with accurately quantified DNAs, estimated the allelic frequencies in the pools of two SNPs by primer extension genotyping followed by DHPLC analysis and compared them with the real frequencies determined in the single samples. Out data show that (1) the method is highly reproducible: the standard deviation of repeated determinations was +/-0.014: (2) the experimental error (i.e., the discrepancy between the estimated and teal frequencies) was +/-0.013 (95% C.I.: 0.0098-0.0165). The magnitude of this error was not correlated to the pool size ol to the type of SNP. The effect of the observed experimental error on the power of the association test was evaluated. We conclude that this method constitutes an efficient tool for high-throughput association screenings provided that the experimental error is low. We therefore recommend that before a pool is used for extensive association studies, its quality, i.e., the experimental error. is verified by determining the difference between estimated and real frequencies for at least one marker. (C) 2001 Elsevier Science B.V. All rights reserved.