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J Am Chem Soc. 2013 May 22;135(20):7430-3. doi: 10.1021/ja4023978. Epub 2013 May 9.

Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly.

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  • 1Institute 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.

Abstract

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.

PMID:
23647466
[PubMed - indexed for MEDLINE]
PMCID:
PMC3724415
Free PMC Article

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