Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions

Abstract

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.

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

“…As was previously the case for LAMP, 30–32 the use of CHA to transduce and report the presence of correct isothermal amplification products leads to much greater specificity of detection than would just monitoring the accumulation of DNA (Figures 4A–4D). While false amplicons accumulate during RCA even in the absence of template, these amplicons are not reported by the EHT-CHA circuit.…”

“…26–29 Recently, CHA has been used as a specific end-point transducer 30 for enzyme-based isothermal amplification reactions, such as LAMP, that sometimes produce parasitic amplicons and result in a high false positive rate. 30–32 …”

Design, Synthesis, and Application of Spinach Molecular Beacons Triggered by Strand Displacement

“…We have previously used nucleic acid circuits for transducing signals from the amplification reactions that underlie molecular diagnostics (Jiang, Li, Milligan, Bhadra, & Ellington, 2013; Li, Chen, & Ellington, 2012). In one such amplification reaction, NASBA (Compton, 1991), RNA templates are reverse transcribed into double-stranded DNA using forward and reverse primers, one of which carries the T7 RNA polymerase promoter sequence.…”

Nucleic acid quantification using nicking–displacement, rolling circle amplification and bio-bar-code mediated triple-amplification

“…The most widely used amplification techniques for bioanalysis are based on enzyme cycle enhancement. These techniques include polymerase chain reaction (PCR) [4,5], ligase chain reaction (LCR) [6], rolling circle amplification (RCA) [7,8], Loop-mediated isothermal amplification (LAMP) [9,10], self-sustained sequence replication [11], endonuclease [12], exonuclease [13] and duplex-specific nuclease (DSN) [14,15]-induced amplification. Recently, multiple amplification approaches have developed to further improve the sensitivity [16,17].…”