Method for target nucleic acid detection based on three-way junction structure-induced isothermal amplification reaction using specially designed combinatorial probe

Abstract

Herein, we developed a three-way junction (3WJ) structure-induced isothermal amplification reaction (ThIsAmp) using specially designed combinatorial probe for nucleic acid detection. In this strategy, we employed specially designed ThIsAmp template which can serve as a component of 3WJ structure as well as an EXPAR template. In the presence of target DNA, ThIsAmp template is hybridized with target DNA and ThIsAmp primer which is additionally introduced to form the 3WJ structure. After the 3WJ structure is formed, the trigger strand is generated by DNA polymerase and nicking endonuclease. The produced trigger strand then induces two pathways for double-stranded ThIsAmp template (dsDNA product) amplification. First, the trigger strand binds to the 3’ end region of ThIsAmp template in 3WJ-structure, then polymerase extends the 3’ end of trigger strand, consequently generating the dsDNA product and releasing the target DNA from the 3WJ structure (pathway 1). Second, the trigger strand binds to free ThIsAmp template, which promotes exponential amplification of dsDNA products by following the conventional EXPAR system (pathway 2). As a result of the pathway 1 and 2, a lot of dsDNA products are produced and its fluorescence intensity is monitored in real-time by SYBR Green I staining. By employing newly designed probes, we finally developed the novel isothermal nucleic acid amplification strategy which can overcome the critical disadvantage of conventional EXPAR technology which has difficulty in detecting a 3’-OH-less nucleic acid or a nucleic acid whose length is too long to be used as a primer for EXPAR. Based on this strategy, we successfully detected target nucleic acid with the high specificity and sensitivity in the dynamic range of 1 fM to 1 nM with a low detection limit down to 78.1 aM.