Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.
Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.
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DOI：
10.1021/ac301944v
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发表时间：
2012-10-02
影响因子：
7.4
通讯作者：
Ellington, Andrew D.
中科院分区：
文献类型：
--
作者：
Li, Bingling;Chen, Xi;Ellington, Andrew D.
环介导的DNA等温扩增(LAMP)是一种强大的等温核酸扩增技术，它可以在一到两个小时内从不到10个拷贝的输入模板中积累~109个拷贝。不幸的是，尽管扩增反应非常强大，但LAMP产物的定量检测仍然很难分析。在本文中，为了提高LAMP检测的特异性和使LAMP扩增的直接读出更加简单和可靠，我们开发了一种非酶核酸电路(催化发夹组装，CHA)，它既可以扩增和整合LAMP扩增中存在的特定序列信号。通过发夹受体，从LAMP扩增的四个环产物中的一个被转导到活性催化剂ssDNA，该催化剂反过来可以引发CHA反应。在CHA检测后，即使少于10个分子/μ的L模型模板(M13mp18)也能产生显著的信号，非特异性模板和寄生扩增都不会带来干扰。更重要的是，为了进一步提高特异性，我们设计了一种双CHA电路，只有在存在两个灯回路的情况下才会给出积极的响应。AND门探测器将作为灯产品的同步、特定读数，而不是竞争和寄生放大器的读数。
Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~109 copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, in order to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a non-enzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both non-specific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons.
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无酶DNA电路对多种检测方法的有理模块化适应。
影响因子：
14.9
作者：
Li B;Ellington AD;Chen X
通讯作者：
Chen X
DOI：
10.1073/pnas.0407024101
发表时间：
2004-10-26
影响因子：
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作者：
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通讯作者：
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作者：
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作者：
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通讯作者：
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影响因子：
3.3
作者：
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通讯作者：
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