A high-throughput SNP typing system for genome-wide association studies
A high-throughput SNP typing system for genome-wide association studies
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DOI：
10.1007/s100380170047
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发表时间：
2001-01-01
影响因子：
3.5
通讯作者：
Nakamura, Y
中科院分区：
文献类型：
--
作者：
Ohnishi, Y;Tanaka, T;Nakamura, Y
全基因组关联研究中最难解决的问题之一是减少基因分型所需的基因组DNA的量。目前可用的技术需要太大量的基因组DNA来用数百或数千个单核苷酸多态性（SNP）进行基因分型。为了克服这个问题，我们结合了入侵者检测与多重聚合酶链反应（PCR），在抗体的Taq聚合酶的存在下进行，以及使用一种新的384孔卡系统，可以减少所需的反应体积。我们在一个试管中扩增了100个基因组DNA片段，每个片段包含一个SNP，并使用Invader检测分析每个SNP。该程序正确地对来自10个个体的PCR扩增样品中检查的100个SNP位点中的98个进行了基因分型;通过直接测序确认了基因型。用另外两组100个SNP证实了该方法的重现性和通用性。因为我们使用40 ng的基因组DNA作为多重PCR的模板，检测一个SNP所需的量仅为0.4ng;因此，理论上，当100 μ g的基因组DNA可用时，可以一次对超过200，000个SNP进行基因分型。我们的研究结果表明，仅使用5- 10 ml血液样本进行全基因组关联研究是可行的。
One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals; the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 mug of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10ml.