Wu Wanghua, Zhang Tao, Han Da, Fan Hongliang, Zhu Guizhi, Ding Xiong, Wu Cuichen, You Mingxu, Qiu Liping, Li Juan, Zhang Liqin, Lian Xiang, Hu Rong, Mu Ying, Zhou Jianguang, Tan Weihong
Research Center for Analytical Instrumentation , Institute of Cyber-Systems and Control , State Key Laboratory of Industrial Control Technology , Zhejiang University , Hangzhou 310027 , China . Email:
Center for Research at Bio/nano Interface , Department of Chemistry , Department of Physiology and Functional Genomics , Health Cancer Center , UF Genetics Institute and McKnight Brain Institute , University of Florida , Gainesville , Florida 32611-7200 , USA . Email:
Chem Sci. 2018 Feb 28;9(11):3050-3055. doi: 10.1039/c7sc05141g. eCollection 2018 Mar 21.
We herein describe a simple and versatile approach to use conventional nicking endonuclease (NEase) for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC), and its application to DNA isothermal exponential amplification (AMC-based strand displacement amplification, AMC-SDA). AMC uses a hairpin-shaped DNA aligner (DA) that contains a recognition site in its stem and two side arms complementary to target DNA. Thus, it enables the loading of an NEase on DA's stem, localization to a specific locus through hybridization of the side arms with target DNA, and cleavage thereof. By using just one NEase, it is easy to make a break at any specific locus and tune the cleavage site to the single-nucleotide scale. This capability also endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence. Moreover, this manner of initiation facilitates the adoption of 3'-terminated primers, thus making AMC-SDA highly sensitive and highly specific, as well as simple primer design.
我们在此描述了一种简单且通用的方法，即使用传统的切口内切核酸酶（NEase）对DNA进行可编程的序列特异性切割，称为比对介导切割（AMC），及其在DNA等温指数扩增中的应用（基于AMC的链置换扩增，AMC-SDA）。AMC使用一种发夹形DNA比对物（DA），其茎部包含一个识别位点，两个侧臂与靶DNA互补。因此，它能够将NEase加载到DA的茎部，通过侧臂与靶DNA杂交将其定位到特定位点并进行切割。仅使用一种NEase，就很容易在任何特定位点产生断裂，并将切割位点调整到单核苷酸水平。这种能力还赋予了所提出的AMC-SDA出色的通用性，因为靶DNA的切割，随后沿着特定引物进行聚合酶催化的延伸作为启动SDA的关键步骤，不再依赖于任何特殊序列。此外，这种起始方式有利于采用3'端终止引物，从而使AMC-SDA具有高灵敏度、高特异性以及简单的引物设计。
