Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Simultaneous incorporation of three different modified nucleotides during PCR.

Modified analogs of 2'-deoxycytidine triphosphates bearing (6-aminohexyl)carbamoylmethyl or 7-amino-2,5-dioxaheptyl linker at a C5 position were designed and synthesized. Both analogs were found to be good substrates for Vent(exo-) DNA polymerase during PCR, resulting in the corresponding full-length modified DNAs, respectively. Moreover, we have demonstrated simultaneous incorporation of three different modified nucleotides into a DNA strand by PCR using triphosphates of 5-(3-aminopropynyl)dUTP, 5-[(6-aminohexyl)carbamoylmethyl]dCTP and 2-amino-dATP (dDTP) or N6-methyl-dATP in place of the natural nucleoside triphosphates TTP, dCTP and dATP.