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.
Visual DNA detection Genotyping Single-nucleotide polymorphism DNAzymes G-quadruplex Pathogen detection
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This work was supported by the Defence Science and Technology Agency, Grant No. 20080054-R6.
Neo JL, Kamaladasan K, Uttamchandani M (2012) G-quadruplex based probes for visual detection and sensing. Curr Pharm Des 18:2048–2057PubMedCrossRefGoogle Scholar
Kolpashchikov DM (2008) Split DNA enzyme for visual single nucleotide polymorphism typing. J Am Chem Soc 130:2934–2935PubMedCrossRefGoogle Scholar
Darius AK, Ling NJ, Mahesh U (2010) Visual detection of DNA from salmonella and mycobacterium using split DNAzymes. Mol Biosyst 6:792–794PubMedCrossRefGoogle Scholar
Neo JL, Aw KD, Uttamchandani M (2011) Visual SNP genotyping using asymmetric PCR and split DNA enzymes. Analyst 136:1569–1572PubMedCrossRefGoogle Scholar
Zacchigna L, Vecchione C, Notte A, Cordenonsi M, Dupont S, Maretto S et al (2006) Emilin1 links TGF-beta maturation to blood pressure homeostasis. Cell 124:929–942PubMedCrossRefGoogle Scholar
Shimodaira M, Nakayama T, Sato N, Saito K, Morita A, Sato I et al (2010) Association study of the elastin microfibril interfacer 1 (EMILIN1) gene in essential hypertension. Am J Hypertens 23:547–555PubMedCrossRefGoogle Scholar