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

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 be thermostable from 37 °C to 60 °C and in consequence have generalized its application to 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, they also improved sensitivity by as much as 25- to 10,000-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.

Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform

“…The usage of enzymes will need particular reaction time and conditions to maintain the enzyme's activities. Recently, an enzyme-free amplification (EFA) method has been developed (Yin et al, 2008;Li et al, 2011;Chen et al, 2013;Jiang et al, 2013;Liao et al, 2014). It overcomes the disadvantages of enzymatic amplification.…”

“…However, these methods all used luminophore labeled probes as signal probes. (Yin et al, 2008;Li et al, 2011;Chen et al, 2013;Jiang et al, 2013;Liao et al, 2014).…”

Designed diblock hairpin probes for the nonenzymatic and label-free detection of nucleic acid

“…However, these enzyme-based assays are labor-intensive, require complex operation, and have a relatively high cost. In contrast, catalytic hairpin assembly (CHA) is a simple and enzyme-free amplifying strategy that can effectively amplify and transduce the signal from DNA hybridizing events (Yin et al, 2008;Jiang et al, 2013b;Chen et al, 2015b). The CHA employs the target-initiated circular assembly of metastable hairpin probes to fabricate branched DNA junctions.…”

A Spinach molecular beacon triggered by strand displacement

“…Such a molecular beacon should prove to be generally useful for directly generating target sequence biosensors by transcription both in vitro and in vivo. In particular, strand displacement nucleic acid circuits have proven to be useful tools for the sequence-specific detection of amplicons, especially those generated by isothermal enzymatic amplification reactions such as rolling circle amplification (RCA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP) (Jiang et al 2013). In the current work we demonstrate that sequence-dependent Spinach can be transcribed in parallel with and function as a biosensor for an isothermal amplification reaction, NASBA.…”