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

  • Jia Ling Neo
  • Mahesh Uttamchandani
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1039)

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.

Key words

Visual DNA detection Genotyping Single-nucleotide polymorphism DNAzymes G-quadruplex Pathogen detection 

Notes

Acknowledgment

This work was supported by the Defence Science and Technology Agency, Grant No. 20080054-R6.

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Jia Ling Neo
    • 1
  • Mahesh Uttamchandani
    • 1
    • 2
    • 3
  1. 1.Department of Chemistry, Defence Medical and Environmental Research InstituteDSO National LaboratoriesSingaporeSingapore
  2. 2.Department of ChemistryNational University of SingaporeSingaporeSingapore
  3. 3.Department of Biological SciencesNational University of SingaporeSingaporeSingapore

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