title: 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.
authors.affiliation: Department of Chemistry, Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore, Singapore.
authors.forename: Jia Ling
authors.initials: JL
authors.lastname: Neo
authors.forename: Mahesh
authors.initials: M
authors.lastname: Uttamchandani
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D021881:DNA, Catalytic / Q000737:chemistry*; D054856:G-Quadruplexes; D005798:Genes, Bacterial; D060005:Genotyping Techniques; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D019779:Salmonella enterica / Q000235:genetics
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D021881:DNA, Catalytic; D008562:Membrane Glycoproteins; C030340:elastin microfibril interface located protein