基于缺口酶的链置换等温扩增技术

Nicking enzyme-based isothermal strand displacement amplification

传统的链置换等温扩增技术中需要加入修饰的底物,为了解决这一技术难题,实现常规底物下的链置换等温扩增技术,采用缺口酶Nt.BstNBI代替传统链置换等温扩增技术中的限制性内切酶,通过优化两种酶的比例、反应温度、反应时间、两种原装buffer的比例、buffer中的离子浓度等反应条件和引物浓度、引物序列以及引物前端保护序列的长度,最终建立了一种新型的链置换等温扩增技术.该方法可以检测到2 pmol/L的质粒DNA.扩增反应在15-30 min即可完成,并且产物可通过琼脂糖凝胶电泳进行直接检测.本研究建立的基于缺口酶的链置换等温扩增技术较之传统方法更加简便、快捷、环保且价格低廉,具有非常强的实际应用潜力,特别适用于现场检测.

Modified substrate（d ATPαS） is needed in traditional isothermal strand displacement amplification. In order to realize isothermal strand displacement amplification at conventional substrate, this study replaced the restriction enzyme in traditional isothermal strand displacement amplification with nicking enzyme Nt.Bst NBI. The detection limit of plasmid DNA was as low as 2 pmol/L. The amplification reaction could be completed in 15-30 min, with products directly detectable by agarose gel electrophoresis. The results showed that the nicking enzyme-based isothermal strand displacement amplification is an environment-friendly, cost effective new technology that is faster, more convenient, and practical than the traditional method, especially for on-site testing.