Enzyme-mediated single-nucleotide variation detection at room temperature with high discrimination factor

We demonstrate a new powerful tool to detect single-nucleotide variation in DNA at room temperature with high selectivity, based on predetermined specific interactions between Lambda exonuclease and a chemically modified DNA substrate structure which comprises two purposefully introduced mismatches and a covalently attached fluorophore. The fluorophore not only acts as a signal reporter in the detection system, but also plays a notable role in the specific molecular recognition between the enzyme and the probe/target hybrid substrate. The method is single-step, rapid, and can be easily adapted to different high-throughput micro-devices without the need for temperature control.