A Label-Free, Sensitive, Real-Time, Semiquantitative
Electrochemical Measurement Method for DNA Polymerase Amplification
(ePCR)
- Publication date
- Publisher
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