title: Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools.
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 (Delta) 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 Delta of 0.008. The assay performed well for alleles of low frequency alleles (f approximately 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.
authors.affiliation: Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
authors.forename: Nadine
authors.initials: N
authors.lastname: Norton
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Williams
authors.forename: Hywel J
authors.initials: HJ
authors.lastname: Williams
authors.forename: Gillian
authors.initials: G
authors.lastname: Spurlock
authors.forename: George
authors.initials: G
authors.lastname: Kirov
authors.forename: Derek W
authors.initials: DW
authors.lastname: Morris
authors.forename: Bastiaan
authors.initials: B
authors.lastname: Hoogendoorn
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Owen
authors.forename: Michael C
authors.initials: MC
authors.lastname: O'Donovan
mesh_terms: D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D005787:Gene Frequency / Q000235:genetics*; D005819:Genetic Markers; D006801:Humans; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D004247:DNA