Capacity of N4-methyl-2′-deoxycytidine 5′-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases

Abstract

The dCTP analog N4-methyl-2′-deoxycytidine 5′-triphosphate (N4medCTP) was evaluated for its performance in the polymerase chain reaction (PCR). Using the HotStart Taq DNA polymerase with a standard thermal protocol, test segments 85 or 200bp long were amplified equally well using dCTP or N4medCTP:dCTP mixtures ranging in molar ratio from 3:1 to 10:1, while complete replacement of dCTP by N4medCTP gave clearly lower amplicon yields and higher Cq values. Comparable yields with N4medCTP or dCTP were achieved only by using a slowdown protocol. Post-PCR melting analyses showed decreasing Tm values for amplicons obtained with increasing N4medCTP:dCTP input ratios; for the 200-bp amplicon, complete replacement of dCTP by N4medCTP in the reaction reduced the Tm by 11°C; for the 85-bp amplicon the Tm reduction was 7°C. In experiments aiming at the 200-bp amplicon, Pfu exo− DNA polymerase did not sustain PCR when dCTP was fully replaced by N4medCTP, even with the slowdown protocol, except at elevated N4medCTP concentrations, and, compared to PCR conducted exclusively with dCTP, the use of N4medCTP:dCTP mixtures gave reduced yields and distinctly higher Cq values, regardless of the thermal program employed. PCR experiments with 9°N DNA polymerase using N4medCTP in the conventional thermal protocol failed to produce the 200-bp amplicon.