应用复合诱导突变分离PCR法检测mtDNA的单核苷酸多态性

目的应用复合诱导突变分离PCR(multiplexed mutagenically separated PCR,MS-PCR)技术、银染分型,建立线粒体DNA(mitochondrial DNA,mtDNA)编码区单核苷酸多态(single nucleotide polymorphism,SNP)分型系统,探讨其应用价值。并调查了成都汉族群体mtDNA编码区4个SNP基因座等位基因频率和单倍型分布情况。方法根据SNP基因座(C12705T、A8701G、G8584A、C10400T)设计两条片段相差4个碱基的等位基因特异性引物和一条公共引物,4个SNP基因座复合扩增,PCR产物经聚丙烯酰胺凝胶电泳、银染显带后确定样本的基因型。结果不同SNP基因座为长度不同的单一谱带,其分型结果与直接测序一致。在成都汉族160名无关个体中,4个SNP基因座C12705T、A8701G、G8584A、C10400T等位基因频率分别为0.3813/0.6187、0.4813/0·5187、0.8250/0.1750、0.4938/0.5062;共检出6种单倍型,单倍型的基因多样性为0.7137。结论建立的MMS-PCR银染分型系统是一种简单、快速、准确、有效的SNP分型方法,对建立mtDNA编码区SNP数据库,研究群体遗传学、进化学和进行法医学个人识别和亲子鉴定有重要意义。

Multiplexed mutagenicaliy separated PCR assay for rapid detection of SNP loci in mitochondrial DNA coding region

Objective To develop a multiplexed mutagenically separated PCR (MS-PCR) for single nucleotide polymorphism (SNP) loci typing in mitochondrial DNA coding regions and to study the applications in investigating the allele frequencies and haplotypes of four SNP loci in mitochondrial DNA coding regions in Chinese Chengdu Han population. Methods Four SNP loci C12705T, A8701G, G8584A and C10400T, two allele specific forward primer with 4 bases different in size and a common reverse primer were designed for SNP typing. The primers simultaneously were amplifyed in a single tube. The genotyping of SNPs was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining. Results The different SNP loci comprised a single band with different size respectively. Typing results were completely consistent with those by direct sequencing. The allelic frequencies of C12705T, A8701G, G8584A and C10400T were 0.3813/0.6187, 0.4813/0.5187, 0.8250/0.1750 and 0.4938/0.5062 respectively. A total of 6 different haplotypes was identified and the genetic diversity reached 0.7137. Conclusion Multiplexed MS-PCR is a simple, rapid, accurate and efficient method for SNP typing,which will be very powerful for SNPs in the database establishing of mitochondrial DNA coding regions, the testing of forensic and population genetics research.