Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.
Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.
复制标题
使用自回避分子识别系统对 DNA 和 RNA 进行解旋酶依赖性等温扩增。
DOI：
10.1002/cbic.201500135
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Benner,StevenA
中科院分区：
文献类型：
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作者：
Yang,Zunyi;McLendon,Chris;Hutter,Daniel;Bradley,KevinM;Hoshika,Shuichi;Frye,CaroleB;Benner,StevenA
检测DNA或RNA （xNA）的试验是高度敏感的，因为少量的xNA可以通过PCR扩增。不幸的是，PCR在资源匮乏的环境中是不方便的，并且需要在这些环境中可能无法获得的设备和电源。等温过程，避免热循环，经常混淆引物二聚体，脱靶引物，和其他人为因素。在这里，我们展示了“自我避免分子识别系统”（SAMRS）如何消除这些伪像，并在解旋酶依赖的等温扩增（SAMRS - HDA）中获得干净的扩增子。我们还发现，将SAMRS结合到引物的3 '‐端有助于设计和筛选HDA检测的引物。最后，我们证明SAMRS - HDA可以实现双重复用，这是使用标准引物的HDA难以实现的。因此，SAMRS - HDA是一种比标准HDA更通用的方法，对xNA靶向诊断和研究具有更广泛的适用性。
Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low‐resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off‐target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase‐dependent isothermal amplification (SAMRS‐HDA). We also show that incorporating SAMRS into the 3′‐ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS‐HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS‐HDA is a more versatile approach than standard HDA, with a broader applicability for xNA‐targeted diagnostics and research.