nanopore sequencing), massive efforts are currently being undertaken to develop working principles for faster, more accurate and consequently more cost-effective assays 1, 2, designed ultimately, to operate at the single molecule level in medical applications. In particular in the field of sequencing-by-synthesis (e.g. The DNA polymerases are the workhorses for replication of genomic information in living cells but more recently also for biotechnology applications such as the polymerase chain reaction (PCR) and DNA sequencing. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities.

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes.

Klenow fragment