Label-free voltammetric detection of products of terminal deoxynucleotidyl transferase tailing reaction

A label-free approach that takes advantage of intrinsic electrochemical activity of nucleobases has been applied to study the products of terminal deoxynucleotidyl transferase (TdT) tailing reaction. DNA homooligonucleotides A 30 , C 30 and T 30 were used as primers for the tailing reaction to which a dNTP – or a mixture of dNTPs – and TdT were added to form the tails. Electrochemical detection enabled study of the tailing reaction products created by various combinations of primers and dNTPs, with pyrolytic graphite electrode (PGE) being suitable for remarkably precise analysis of the length of tailing reaction products. Furthermore, the hanging mercury drop electrode (HMDE) was able to reveal formation of various DNA structures, such as DNA hairpins and G-quadruplexes, which influence the behavior of DNA molecules at the negatively charged surface of HMDE. Thus, the described approach proves to be an excellent tool for studying the TdT tailing reactions and for exploring how various DNA structures affect both the tailing reactions and electrochemical behavior of DNA oligonucleotides at electrode surfaces