高保真DNA聚合酶在SNP检测中的应用

Application of DNA Polymerase With 3′ Exonuclease in SNP Assay

DNA测序技术 ,使人类步入了后基因时代 ;而后基因时代对DNA序列分析的新要求 ,尤其是有关单碱基多态性的检测 ,急需更高效和更特异的分析方法。作者介绍两种可靠性高且适应性广的单碱基多态性检测新方法。基于高保真DNA聚合酶对引物 3′末端错配碱基的校正机制 ,通过标记引物 3′末端 ,配对引物得到带标记信号的产物而不配对的引物则只能得到不含标记信号的产物。最近 ,我们新发现高保真DNA聚合酶具有通过聚合反应非成熟性终止以维持成熟性终产物保真度的能力。利用 3′硫化磷酸修饰的引物 ,可强化由不配对引物所激发的对聚合反应的终止作用。3′硫化磷酸修饰与高保真DNA聚合酶共同形成一种由单碱基多态性调控的分子开关。这一新的分子开关可与电泳等多种现有的技术联合使用 ,在分析单一或多个已知SNP位点时 ,具有极大的应用价值。

Two assays for SNP analysis were developed based on 3′ exonuclease of DNA polymerase. Proofreading or mismatch editing by 3′ exonuclease exhibited a reliable discrimination to single base differences, which is used in terminal labeled allele-specific primer extenstion for SNP assay. In addition to the well-known proofreading mechanism, we recently revealed a novel 'OFF-switch' effect by which the 3′exonuclease is attributed to fidelity maintenance in DNA replication. With phosphorothioate-modified allele-specific primer, polymerase with 3′ exonuclease showed a powerful'ON/OFF switch': matched primer turned on and mismatched primer turned off DNA polymerization. These new methods especially the SNP-operated 'ON/OFF switch' have immediate applications in screening single and multiple SNPs.