DNA polymerases need to elongate rapidly and accurately to function effectively in vivo and in vitro, yet certain DNA regions appear to interfere with their progress. One common problem is pause sites, at which DNA polymerase molecules cease elongation for varying lengths of time. Many strong DNA polymerase pauses are at the beginnings of regions of strong secondary structure such as template hairpins. Taq polymerase use in PCR suffers the same fate and GC-rich DNA sequences often require laborious work to optimize the amplification assay. The GC-rich sequences possess high thermal and structural stability, presumably because the high duplex melting temperature that permits stable secondary structures to form, thus preventing completion of a faithful replication.
Nucleotide analog 7-deaza dGTP is effective in reducing the secondary structure associated with GC rich region by reducing the duplex stability. Betaine, DMSO and formamide reduces the Tm and the complex secondary structure thus the duplex stability. Tetramethyl ammonium chloride (TMAC) actually increases the specificity of hybridization and increases the Tm. The use of TMAC is recommended in PCR conditions using degenerate primers.
These PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions. These additives may have beneficial effects on some amplification and it is impossible to predict which agents will be useful in a particular context and therefore they must be empirically tested for each combination of template and primers.