基于荧光定量PCR扩增反应的SNP测定法  

A Novel SNP Typing Based on Real-time Polymerase Chain Reaction Amplification

作　　者：单志新[1] 谭虹虹[1] 余细勇[1] 林秋雄[1] 

机构地区：[1]广东省人民医院医学研究中心,广州510080

出　　处：《中国生物化学与分子生物学报》2005年第6期827-830,共4页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30300421)~~

摘　　要：建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.A new effective single nucleotide polymorphism（SNP） typing method was developed by using real-time polymerase chain reaction（PCR）. To determine Arg16Gly locus alleles of human β2-adrenergic receptor （β2-AR） genetic polymorphisms, genotypes were assigned based on PCR growth curves and melt curve analysis with the indicating fluorophore SYBR. Green Ⅰ. Allele-specific primers were developed for both wild-type and mutant alleles, and an additional artificial mismatch base at the third position from the 3＇end of each primer was used to improve amplification specificity and to prevent generation of nonspecific products. The accuracy of SNP typing determined by real-time PCR was validated by comparison with results from direct DNA sequencing. The data revealed that the melt temperature was nearly identical for PCR products formed using either wild-type or mutant specific primers with a peak at 86℃, while the plot of fluorescence versus cycle number can be used to discriminate between A or G allele using the same primers. The results of SNP typing for Arg16Gly locus alleles of β2-AR gene showed that the presented allelic discrimination assay was specific, easy to operate and applicable for high-throughput sample analysis in large population genotyping studies.

关 键 词：单核苷酸多态性 实时聚合酶链反应 等位基因特异性延伸反应 人为错配碱基 

分 类 号：Q503[生物学—生物化学]