Real-Time Detection of Isothermal Amplification Reactions
with Thermostable Catalytic Hairpin Assembly
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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