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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Li Y, Geyer CR, Sen D (1996) Recognition of anionic porphyrins by DNA aptamers. Biochemistry 35:6911–6922
Travascio P, Li Y, Sen D (1998) DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chem Biol 5:505–517
Gellert M, Lipsett MN, Davies DR (1962) Helix formation by guanylic acid. Proc Natl Acad Sci U S A 48:2013–2018
Neo JL, Kamaladasan K, Uttamchandani M (2012) G-quadruplex based probes for visual detection and sensing. Curr Pharm Des 18:2048–2057
Kolpashchikov DM (2008) Split DNA enzyme for visual single nucleotide polymorphism typing. J Am Chem Soc 130:2934–2935
Darius AK, Ling NJ, Mahesh U (2010) Visual detection of DNA from salmonella and mycobacterium using split DNAzymes. Mol Biosyst 6:792–794
Neo JL, Aw KD, Uttamchandani M (2011) Visual SNP genotyping using asymmetric PCR and split DNA enzymes. Analyst 136:1569–1572
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 124:929–942
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 23:547–555
Acknowledgment
This work was supported by the Defence Science and Technology Agency, Grant No. 20080054-R6.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Neo, J.L., Uttamchandani, M. (2013). Visual DNA Detection and SNP Genotyping Using Asymmetric PCR and Split DNA Enzymes. In: Kolpashchikov, D., Gerasimova, Y. (eds) Nucleic Acid Detection. Methods in Molecular Biology, vol 1039. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-535-4_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-535-4_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-534-7
Online ISBN: 978-1-62703-535-4
eBook Packages: Springer Protocols