(A) new target DNA detection method based on hairpin probe-assisted isothermal probe amplification

Abstract

We herein developed a novel target DNA detection method called hairpin probe-assisted isothermal probe amplification (HPA IPA) to overcome a disadvantage of exponential amplification reaction (EXPAR) method. In spite of high amplification efficiency, the EXPAR can be used exclusively for the detection of short oligonucleotide, trigger, which confines the use of the EXPAR as a diagnostic method. To solve the problem of the EXPAR method, we employed hairpin probe (HP) which contains an anti-trigger probe (aTP) at its 3’-end and a nicking enzyme recognition sequence followed by additional aTP at its 5’-end. As the target DNA binds to the loop region of the HP, primer can bind to the free stem region of the HP and be extended by DNA polymerase, generating double-stranded (ds) DNA. Subsequently, the trigger probe (TP) can be continuously produced by combination reaction of the DNA polymerase and nicking enzyme and induce additional production of dsDNA by binding to the free aTP at 3’-end of the HP. Consequently, as the HPA IPA reaction takes place, the fluorescent signal from SYBR Green I that fluoresces when it is intercalated into the dsDNA is increased. By employing this amplification principle, the target DNA was sensitively detected down to 0.33 fM with high specificity over other mismatched target DNAs. Finally, the practical utility of this strategy was successfully verified by detecting long target DNAs with high reliability.