Bannai Makoto, Higuchi Kaori, Akesaka Tamao, Furukawa Masako, Yamaoka Megumi, Sato Kazuhisa, Tokunaga Katsushi
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Anal Biochem. 2004 Apr 15;327(2):215-21. doi: 10.1016/j.ab.2004.01.012.
Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500-10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.
全基因组扩增（WGA）方法被用于单核苷酸多态性（SNP）分型，以尽量减少在大规模研究中对数千种不同SNP进行分型时所需的基因组DNA量；通过WGA方法（改进的引物延伸预扩增聚合酶链反应（I-PEP-PCR）、简并寡核苷酸引物PCR（DOP-PCR）或多重置换扩增（MDA））对5-10 ng基因组DNA进行扩增。以全基因组扩增产物1/100至1/500的量作为模板，成功进行了后续分析。通过序列特异性引物（SSP）-PCR方法，随后进行荧光相关光谱法（FCS）对SNP进行基因分型。对肿瘤坏死因子受体1和2基因（TNFR1和TNFR2）上的四个不同SNP的分型结果进行了评估。使用WGA产物通过SSP-FCS方法确定的基因型与使用基因组DNA通过核苷酸测序确定的基因型100%一致。我们已经对300多种不同的SNP进行了分型，目前每天使用来自几种常见疾病患者的WGA样本进行7500-10000次分型。WGA与FCS相结合，能够对数千个样本中的数千种不同SNP进行特异性和高通量基因分型。
