Expanding the Design Rules for Discriminating Nucleic Acid Mutations via Mismatch‐Exchange

ABSTRACT Complementarity between nucleic acids via Watson–Crick base pairing formulates the basic principle for designing hybridization probes but often suffers low sequence selectivity against single nucleotide mutations. Herein, we report mismatch‐exchange as a new design principle that allows the highly sensitive and robust discrimination of single nucleotide polymorphisms (SNPs) by simply manipulating the number and position of mismatches in both probes and the reaction products. Leveraging mismatches to drive the strand‐exchange and finetuning the specificity, mismatch‐exchange is particularly advantageous for analyzing complex nucleic acid targets containing multiple nearby SNPs. Both selective tolerance to synonymous SNPs and OR‐gate‐based detection of clustered drug‐resistant SNPs were demonstrated. Once deployed to nucleic acid testing in clinical settings, mismatch‐exchange enabled the discrimination of multiple lamivudine‐resistant hepatitis B virus mutants in a clinical cohort containing 65 clinical plasma samples.