Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y
Laboratory for Cardiovascular Diseases, SNP Research Center, The Institute of Physical and Chemical Research (RIKEN), Tokyo, Japan.
J Hum Genet. 2001;46(8):471-7. doi: 10.1007/s100380170047.
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 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 microg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 ml.
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量。目前可用的技术需要大量的基因组DNA才能对数百或数千个单核苷酸多态性（SNP）进行基因分型。为克服这一问题，我们将侵入法与多重聚合酶链反应（PCR）相结合，在存在抗Taq聚合酶抗体的情况下进行，并使用一种新型的384孔板系统，该系统可减少所需的反应体积。我们在单个试管中扩增了100个基因组DNA片段，每个片段包含一个SNP，并使用侵入法分析每个SNP。该程序对来自十个人的PCR扩增样本中检测的100个SNP位点中的98个进行了正确的基因分型：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。由于我们使用40 ng基因组DNA作为多重PCR的模板，分析一个SNP所需的量仅为0.4 ng；因此，理论上，当有100 μg基因组DNA时，一次可以对超过200,000个SNP进行基因分型。我们的结果表明，使用仅5-10 ml的血样进行全基因组关联研究是可行的。
