Nucleic hybridisation is core to many biological processes and protocols used in molecular biology such as nucleic acid amplification, e.g. by PCR. This project aims to radically simplify nucleic acid amplification by driving the reaction via means of electrochemistry. To fulfil this aim, specialised expertise in biosensors, physical chemistry, biophysics and microsystems engineering is brought together.
We have recently demonstrated reversible, isothermal control of DNA denaturation and renaturation by means of electrochemistry using the electroactive DNA intercalator daunomycin. The operational principle has been successfully demonstrated, with UV-vis and circular dichroism spectroelectrochemistry, using a model system of complementary synthetic DNA strands.
We propose a radically new way of controlling reversible hybridisation of nucleic acids as a novel tool to control potentially all types of biological reactions involving nucleic acid base pairing. A wide range of biological applications can make use of this new tool and, thus, addresses any of the strategic priorities of BBSRC relying on nucleic acid-based testing. To pump prime these developments and to show the proof-of-principle, we aim to apply this new tool to develop electrochemically controlled PCR (ePCR).
Preliminary investigations confirmed that daunomycin does not inhibit PCR. The proof-of-principle will be demonstrated on screen-printed electrodes via electrolysis. Standard PCR carried out in thermal cyclers will be employed as benchmark for comparison and assessment of ePCR. Gel electrophoresis will be utilised as the main method for amplification analysis.
Furthermore, for optimisation, in-depth biophysical characterisation of the intercalator-DNA complex will be carried out using isothermal calorimetry and NMR. A suitable real-time electrochemical detection method will be indentified.
