Original Investigation

Human Genetics

, Volume 110, Issue 5, pp 471-478

Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools

  • Nadine NortonAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Nigel M. WilliamsAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Hywel J. WilliamsAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Gillian SpurlockAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , George KirovAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Derek W. MorrisAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Bastiaan HoogendoornAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Michael J. OwenAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
  • , Michael C. O'DonovanAffiliated withDepartment of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK

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Abstract.

Detecting alleles that confer small increments in susceptibility to disease will require large-scale allelic association studies of single-nucleotide polymorphisms (SNPs) in candidate, or positional candidate, genes. However, current genotyping technologies are one to two orders of magnitude too expensive to permit the analysis of thousands of SNPs in large samples. We have developed and thoroughly validated a highly accurate protocol for SNP allele frequency estimation in DNA pools based upon the SNaPshot (Applied Biosystems) chemistry adaptation of primer extension. Using this assay, we were able to estimate the difference in allele frequencies between pooled cases and controls (Δ) with a mean error of 0.01. Moreover, when we genotyped seven different SNPs in a single multiplex reaction, the results were similar, with a mean error for Δ of 0.008. The assay performed well for alleles of low frequency alleles (f~0.05) and was accurate even with relatively poor quality DNA template extracted from mouthwashes. Our assay conditions are generalisable, universal, robust and, therefore, for the first time, permit high-throughput association analysis at a realistic cost.