One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA

A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3′–5′ exonuclease activity of T4 DNA polymerase were used in the assay.

本研究提出了一种利用MutS蛋白(MutS protein)识别DNA非互补序列能力的新型通用突变检测方法。将待检测DNA片段与对照DNA片段经聚合酶链式反应(PCR)扩增，扩增产物经纯化后混合，通过加热、冷却处理形成异源双链。若存在突变，携带碱基错配的异源双链DNA可被MutS蛋白保护，免受核酸酶降解；而无错配的DNA则会被降解。通过直接添加可特异性结合DNA的高灵敏度荧光染料SYBR-Gold，即可直观展示该保护效果。本检测体系采用了嗜热栖热菌(Thermus thermophilus)重组的带His标签的MutS蛋白，以及T4 DNA聚合酶的3′–5′核酸外切酶活性。