Electrochemical analysis of template-free DNA synthesis: Lowering pH and the active component of the impedance spectrum

Аннотация

The process of template-free DNA synthesis was detected in two ways: by measuring the pH of the solution by a semiconductor sensor and by measuring the conductivity in the recording of impedance spectra. Synthesis was carried out without using template DNA, with only two enzymes being involved in the reaction: DNA polymerase and nicking endonuclease (nickase) in the presence of deoxynucleotide triphosphates. Previously, Purushothaman et al., by applying the recording of the results of template-directed synthesis with a pH-sensitive field-effect transistor, showed that protons are released into solution in the incorporation of nucleotides [4]. Regarding this, it was important to establish that the same release of protons into solution occurred in the template-free synthesis as in the template-directed synthesis and to identify the changes by measuring the conductivity of the solution using impedance spectra. It was found that the template-free synthesis was accompanied by the generation of protons (ΔpH is ~1.5 pH at an initial concentration of deoxynucleotide triphosphates of 150 µM) and a decrease in the value of active impedance component by ~25% of the initial value. The effect of a decrease in the active impedance component was explained as being due to an increasing conductivity of medium due to a growth in the concentration of protons.