Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection

Dual enzyme cleavage-based cascade signal amplification for nucleic acids detection was developed. In this system, a modified DNA aligner(MDA) that contains an abasic site was first cleaved by Tth Endonuclease IV in the presence of target DNA, and then served as an aligner to mediate the cleavage of molecular beacon by nicking endonuclease Nt. BstNBI. These cascade reactions not only overcame the sequence dependence of Nt.BstNBI, but also improved the detection sensitivity. The results showed a good linear correlation between the fluorescence intensity and the logarithm of target DNA concentration (lgc) ranging from 1 pmol/L to 1 nmol/L. Moreover, the proposed method also showed a good capability of identifying single base mutation in target DNA. In addition, this method also features simple probe design and excellent universality. By modifying a small fragment on MDA's loop, it can be used to sense various target DNAs. Experiments with target DNA spiked in human serum showed the potential of applying this method to real samples.