title: A Label-Free, Sensitive, Real-Time, Semiquantitative Electrochemical Measurement Method for DNA Polymerase Amplification (ePCR).
abstract: Oligonucleotide hybridization to a complementary sequence that is covalently attached to an electrochemically active conducting polymer (ECP) coating the working electrode of an electrochemical cell causes an increase in reaction impedance for the ferro-ferricyanide redox couple. We demonstrate the use of this effect to measure, in real time, the progress of DNA polymerase chain reaction (PCR) amplification of a minor component of a DNA extract. The forward primer is attached to the ECP. The solution contains other PCR components and the redox couple. Each cycle of amplification gives an easily measurable impedance increase. Target concentration can be estimated by cycle count to reach a threshold impedance. As proof of principle, we demonstrate an electrochemical real-time quantitative PCR (e-PCR) measurement in the total DNA extracted from chicken blood of an 844 base pair region of the mitochondrial Cytochrome c oxidase gene, present at ∼1 ppm of total DNA. We show that the detection and semiquantitation of as few as 2 copies/μL of target can be achieved within less than 10 PCR cycles.
authors.forename: Nihan
authors.initials: N
authors.lastname: Aydemir
authors.forename: Hazel
authors.initials: H
authors.lastname: McArdle
authors.affiliation: ∇School of Biological Sciences, University of Auckland, Auckland 1022, New Zealand.
authors.forename: Selina
authors.initials: S
authors.lastname: Patel
authors.affiliation: ∇School of Biological Sciences, University of Auckland, Auckland 1022, New Zealand.
authors.forename: Whitney
authors.initials: W
authors.lastname: Whitford
authors.affiliation: ∇School of Biological Sciences, University of Auckland, Auckland 1022, New Zealand.
authors.forename: Clive W
authors.initials: CW
authors.lastname: Evans
authors.forename: Jadranka
authors.initials: J
authors.lastname: Travas-Sejdic
authors.forename: David E
authors.initials: DE
authors.lastname: Williams
mesh_terms: D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004563:Electrochemistry; D005292:Ferricyanides / Q000737:chemistry; D010084:Oxidation-Reduction; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: D017931:DNA Primers; D005292:Ferricyanides; C007931:hexacyanoferrate III; D004247:DNA