Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools
Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools
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DOI：
10.1007/s00439-002-0706-6
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发表时间：
2002-05-01
期刊：
影响因子：
5.3
通讯作者：
O'Donovan, MC
中科院分区：
文献类型：
--
作者：
Norton, N;Williams, NM;O'Donovan, MC
检测那些使疾病易感性有小幅增加的等位基因，需要对候选基因或定位候选基因中的单核苷酸多态性（SNP）进行大规模的等位基因关联研究。然而，当前的基因分型技术成本过高，比允许在大样本中分析数千个SNP所需的成本高出一到两个数量级。我们已经开发并全面验证了一种基于引物延伸的SNaPshot（应用生物系统公司）化学改良法的、用于DNA池SNP等位基因频率估算的高精度方案。利用这种检测方法，我们能够估算混合病例组和对照组之间等位基因频率的差异（Δ），平均误差为0.01。此外，当我们在一次多重反应中对7个不同的SNP进行基因分型时，结果相似，Δ的平均误差为0.008。该检测方法对低频等位基因（频率约为0.05）表现良好，即使是从漱口水提取的质量相对较差的DNA模板也能准确检测。我们的检测条件具有通用性、普遍性和稳健性，因此，首次使得以合理成本进行高通量关联分析成为可能。
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 (fsimilar to0.05) 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.