Ranade K, Chang M S, Ting C T, Pei D, Hsiao C F, Olivier M, Pesich R, Hebert J, Chen Y D, Dzau V J, Curb D, Olshen R, Risch N, Cox D R, Botstein D
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA.
Genome Res. 2001 Jul;11(7):1262-8. doi: 10.1101/gr.157801.
To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.
为了使大规模关联研究成为现实，需要用于单核苷酸多态性（SNP）基因分型的自动化高通量方法。我们描述了允许使用TaqMan或5'核酸酶等位基因鉴别分析对任何SNP的大量个体进行分型的PCR条件，以及能够自动分配基因型的计算方法。为了证明这些方法的实用性，我们对1600多名个体进行了基因分型，其中一个是内皮型一氧化氮合酶基因第298位由谷氨酸到天冬氨酸取代的G到T颠换，另一个是11-β羟化酶基因内含子8中的G/C多态性（在我们实验室新发现）。该基因分型方法准确——我们估计基因型错误率低于两千分之一，快速——使用五台96孔PCR仪、一台荧光读数仪且无需自动移液设备，一个人一天就能产生一千多个基因型，并且灵活——一个新的SNP在不到一周的时间内就能进行关联测试。实际上，使用该方法已完成了13个不同基因中另外23个SNP的大规模基因分型。此外，我们鉴定出三个可能是重复结果的“假SNP”（WIAF1161、WIAF2566和WIAF335）。
