Visual DNA Detection and SNP Genotyping Using Asymmetric PCR and Split DNA Enzymes

Abstract

We describe a method to detect DNA sequences visually through a color change reaction using DNAzymes. We successfully applied the assay for the detection of Salmonella and Mycobacterium DNA, as well as for genotyping single base differences from within human genomic DNA samples. Our approach adopts a split probe targeting system, designed with G-rich sequences, which reassembles in the presence of target DNA, producing G-quadruplexes with catalytic activity. Asymmetric PCR is first performed to amplify the target region into single-stranded copies, with primer ratios tailored for optimum amplification. This is followed by direct addition of the visual probes, substrates, and reagents to produce a color change within 15 min should the desired target sequences be present. This approach hence offers a rapid readout, ease-of-use, and handling convenience, especially at the point-of-care.