Enzymatic Synthesis of Modified DNA by PCR and its Application

Thymidine analogue bearing a methyl ester at the C5 position were synthesized. Thermophilic family B DNA polymerases accepted these analogues as substrate to form the corresponding PCR product. Modified DNA containing this analogue were prepared by PCR with KOD Dash DNA polymerase and were further post-modified to form the corresponding derivatized DNA. These DNA could act as a template for PCR and could accept natural TTP or modified analogue as substrate. The thymidine analogue with an amino-linker was also reacted with fluorescein isothiocyanate, yielding a new analogue which was used as a substrate for PCR to get the corresponding fluorescent labeled DNA. Finally, a novel modified DNA aptamer for sialyllactose that is ubiquitous component of the cell surface responsible for the infections of several viruses was produced by using SELEX method with strong binding ability.