A two-layer assay for single-nucleotide variants utilizing strand displacement and selective digestion.

Point mutations have emerged as prominent biomarkers for disease diagnosis, particularly in the case of cancer. Discovering single-nucleotide variants (SNVs) is also of great importance for the identification of single-nucleotide polymorphisms within the population. The competing requirements of thermodynamic stability and specificity in conventional nucleic acid hybridization probes make it challenging to achieve highly precise detection of point mutants. Here, we present a fluorescence-based assay for low-abundance mutation detection based on toehold-mediated strand displacement and nuclease-mediated strand digestion that enables highly precise detection of point mutations. We demonstrate that this combined assay provides 50-1000-fold discrimination (mean value: 255) between all possible single-nucleotide mutations and their corresponding wild-type sequence for a model DNA target. Using experiments and kinetic modeling, we investigate probe properties that obtain additive benefits from both strand displacement and nucleolytic digestion, thus providing guidance for the design of enzyme-mediated nucleic acid assays in the future.