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.

中文翻译：

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对准剂介导的核酸裂解及其在等温指数扩增中的应用†

我们在此描述了一种简单通用的方法，可将常规的切口内切核酸酶（NEase）用于DNA的可编程序列特异性切割（称为比对子介导的切割（AMC））及其在DNA等温指数扩增（基于AMC的链置换扩增， AMC-SDA）。AMC使用发夹形DNA对齐器（DA），该对齐器在其茎中包含一个识别位点，并与靶DNA互补的两个侧臂。因此，它使得能够将NEase装载在DA的茎上，通过侧臂与靶DNA的杂交并将其切割定位在特定的基因座上。通过仅使用一种NEase，很容易在任何特定位点进行断裂，并将切割位点调节至单核苷酸水平。此功能还使拟议的AMC-SDA具有出色的通用性，由于切割目标DNA，然后沿着特定引物进行聚合酶催化的延伸是启动SDA的关键步骤，因此不再依赖任何特殊序列。而且，这种起始方式促进了3'-末端引物的采用，从而使AMC-SDA具有高灵敏度和高特异性，并且引物设计简单。