124 Evaluation of different DNA polymerases for their effectiveness in using a cytosine analogue during real-time PCR amplification

Abstract

GC-rich regions of the DNA are of special interest, as they generally occur within transcribed or control regions of the genome. However, the high GC-content makes these sequences prone to the formation of hairpin structures, which may persist even at the relatively high temperature of the extension step in PCR protocols, making their amplification difficult in many instances. Conversion to a different nucleotidic alphabet, resulting in thermodynamically weaker versions of the G:C base pair, may provide a resolution of this problem. The 7-deaza analogue of the guanine base has been tried in this context; however, it appears that the c7G:C base pair, while less stable than the canonical G:C pair, is still too strong, so that problems due to hairpin formation persist. N4-alkylated cytosines form specific, but much weaker base pairs with guanines, and may represent a more effective solution to the problem, provided not only that they are stably inserted into the newly formed DNA during the extension step, but also correctly read in subsequent extensions. We have examined the dCTP analogue, N4methyl-dCTP, for its ability to sustain a PCR, both with HotStart Taq DNA polymerase and with Pfu exo− DNA polymerase, amplifying a 200-bp amplicon within the pUC18 sequence. The Taq enzyme produced the expected product with the nucleotide complement dATP/dGTP/dTTP/N4methyl-dCTP, albeit in reduced yield, compared to the yield obtained with the use of dCTP or of dCTP/N4methyl-dCTP mixtures. A slowdown thermal protocol (Frey et al., 2008, Nature Protocols, 3, 1312–1318), using successively lower hybridization temperatures and slow temperature ramps, proved beneficial in giving a high yield of the desired amplicon, with the expected size and the correct nucleotide sequence. The all-N4-methylC amplicon showed a T m reduced by 11 °C, compared to the amplicon obtained with the canonical set of nucleotides, while amplifications performed with mixtures of dCTP and N4methyl-dCTP gave products of the expected size, showing intermediate melting temperatures (see Figure), all of which attests to the lower thermal stability of the N4-methylC:G pair, compared to the G:C base pair. In contrast, the amplicon synthesized with the dATP/c7dGTP/dTTP/dCTP nucleotide set showed a Tm reduction of only 5°C. The Pfu exo−enzyme was less capable of sustaining PCR with the N4-methyl nucleotide, providing a small yield of 200-bp amplicon only in the presence of higher concentrations of N4-methyl-dCTP.