Capacity of N~4-methyl-2′-deoxycytidine 5′-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases

摘要

The dCTP analog N~4-methyl-2′-deoxycytidine 5′-triphosphate (N~4medCTP) 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 200 bp long were amplified equally well using dCTP or N~4medCTP:dCTP mixtures ranging in molar ratio from 3:1 to 10:1, while complete replacement of dCTP by N 4medCTP gave clearly lower amplicon yields and higher C_q values. Comparable yields with N~4medCTP or dCTP were achieved only by using a slowdown protocol. Post-PCR melting analyses showed decreasing T_m values for amplicons obtained with increasing N~4medCTP: dCTP input ratios; for the 200-bp amplicon, complete replacement of dCTP by N~4medCTP 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 N~4medCTP, even with the slowdown protocol, except at elevated N~4medCTP concentrations, and, compared to PCR conducted exclusively with dCTP, the use of N~4medCTP:dCTP mixtures gave reduced yields and distinctly higher C_q values, regardless of the thermal program employed. PCR experiments with 9 N DNA polymerase using N~4medCTP in the conventional thermal protocol failed to produce the 200-bp amplicon.