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

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 200 bp 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 N ⁴medCTP gave clearly lower amplicon yields and higher C_q values. Comparable yields with N⁴medCTP or dCTP were achieved only by using a slowdown protocol. Post-PCR melting analyses showed decreasing T _m 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 T_m by 11 C; for the 85-bp amplicon the T_m 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 C_q 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.