Simple protocol for sequence-specific detection of mixed-base nucleic acids using a smart probe with NABs

A fluorescent smart probe (SP) was used to detect a mixed-base ribonucleic acids sequence. While the SP presents excellent sensitivity for the target, it gives subtle discrimination between the perfect target sequence and several mismatch sequences. Its sequence-specificity for the target was greatly enhanced by using nucleic acid blockers (NABs), which are unlabeled, non-fluorescent hairpin oligonucleotides that are perfectly complementary to those mismatch sequences. This approach is simple, feasible at room temperature, requires no amplification enzymes, and it is suitable for applications requiring routine nucleic acids sequence detection and quantification methods such as genetic testing and biomedical diagnostics. •Poor sequence discrimination due to non-specific hybridization.•Marginal signal difference in the absence of NABs.•Large signal difference in the presence of NABs.•Very low SP/NABs molar ratio and no decrease in signal intensity in the presence of NABs.•SP/NABs-based protocol is simple and elegant, and feasible at ambient temperatures of 20–40 °C.