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 ~10(9) 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, 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 nonenzymatic 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 nonspecific 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.