Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy
Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy
复制标题
DOI：
10.1016/j.ab.2004.01.012
复制
发表时间：
2004-04-15
影响因子：
2.9
通讯作者：
Tokunaga, K
中科院分区：
文献类型：
--
作者：
Bannai, M;Higuchi, K;Tokunaga, K
采用全基因组扩增（WGA）方法进行单核苷酸多态性（SNP）分型，以最大程度地减少大规模研究中数千种不同 SNP 分型所需的基因组 DNA 量；通过WGA方法（改进的引物延伸预扩增聚合酶链式反应（I-PEP-PCR）、简并寡核苷酸引物-PCR（DOP-PCR）或多重置换扩增（MDA））扩增5-10ng基因组DNA。以1/100至1/500的全基因组扩增产物为模板，成功进行了后续分析。通过序列特异性引物 (SSP)-PCR 方法和荧光相关光谱 (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）上的四种不同 SNP 的分型结果进行了评估。使用 WGA 产品通过 SSP-FCS 方法确定的基因型与使用基因组 DNA 进行核苷酸测序确定的基因型 100% 一致。我们已经对 300 多个不同的 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 7,500-10,000 次分型。 WGA 与 FCS 相结合，可以对数千个样本的数千个不同的 SNP 进行特异性和高通量基因分型。 (C) 2004 Elsevier Inc. 保留所有权利。
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. (C) 2004 Elsevier Inc. All rights reserved.