Priming efficiency in PCR

Taq and Pfu DNA polymerases were tested for their propensity to prime from mismatched primers. Two series of bacteriophage lambda primers were designed with progressively longer mismatched 5' termini. Effects of the primer concentration, annealing temperature, salt and solvent concentrations on PCR yield were tested. At the standard PCR conditions, priming was detectable when the 3'-terminal portion of the partially mismatched primer formed a continuous duplex more stable than -11 kcal/mol with the target DNA. In the presence of low magnesium ion concentrations, priming was significantly reduced, but glycerol (5%) and formamide (2.5%) had only a slight effect (Taq DNA polymerase). Although priming specificities of Taq and Pfu DNA polymerases were similar, the solvents had no effect on Pfu DNA polymerase-directed PCR. Oligonucleotides that are GC rich at their 3' ends exhibit high priming efficiency but are also prone to false priming, since the shorter fragments of their 3' ends are stable enough to serve as primers.