四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基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 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .

A Rapid Single-tube SNP Typing Based on Tetra-primer PCR Amplification

A new effective single nucleotide polymorphism (SNP) typing approach was developed in a single tube. With human I823M, four primers were designed for PCR amplification, two of which were used for amplifying DNA fragments containing SNP point, the others were SNP specific primers. The templates were amplified in one tube with four primers by PCR and the type of SNP was determined by the length of products to be extended. An accurate and effective method was developed for the rapid detection of SNP, which was based on di allele specific amplification with artificially modified primers. A mismatched base was introduced on the third position upstream from 3′ terminal of a SNP specific primer to increase the accuracy of SNP typing and to decrease the probability of spurious extension reaction. The results for typing mutation point of I823M in human ABCA1 gene showed that the method was specific, rapid and easy to operate.