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Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Venkatesan, Vasuki and Hoti, Sugeerappa Laxmanappa and Kamaraj, Nagalakshmi and Ghosh, Somnath and Rajaram, Kaushik (2013) Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection. In: MEMORIAS DO INSTITUTO OSWALDO CRUZ, 108 (6). pp. 804-807.

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Official URL: http://dx.doi.org/10.1590/0074-0276108062013020

Abstract

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Item Type: Journal Article
Additional Information: Copyright for this article belongs to Fundação Oswaldo Cruz
Keywords: asymmetric PCR; single-stranded DNA; electrochemical detection; Wuchereria bancrofti
Department/Centre: Division of Chemical Sciences > Solid State & Structural Chemistry Unit
Depositing User: Id for Latest eprints
Date Deposited: 25 Oct 2013 06:46
Last Modified: 25 Oct 2013 06:46
URI: http://eprints.iisc.ac.in/id/eprint/47581

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