Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch

Abstract

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5′-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.