Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
J Am Chem Soc. 2013 May 22;135(20):7430-3. doi: 10.1021/ja4023978. Epub 2013 May 9.
Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.
催化发夹组装 (CHA) 是一种无需酶的扩增方法，它以前已被证明在扩增和转导核酸扩增反应末端的信号方面非常有用。在这里，我们首次对 CHA 进行了工程改造，使其在 37 至 60°C 之间具有热稳定性，因此将其应用范围推广到了等温扩增反应的实时检测。我们为高温和低温滚环扩增 (RCA) 和链置换扩增 (SDA) 设计和优化了 CHA 回路。由此产生的回路不仅提高了检测的特异性，而且与可比的实时检测方法相比，灵敏度提高了 25 至 10000 倍。这些方法已被浓缩为一组通用规则，用于设计具有高信号和低噪声的热稳定 CHA 回路。
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