Maximizing sensitivity and specificity of PCR by pre-amplification heating

We have found that assembling the reaction mixture at a temperature greater than the annealing temperature improved both product yield and specificity of PCR. When reactions were maintained at 70°C in a dry heating block during addition of denatured samples to aliquotted reagent master mix, a reproducible increase in product yield was observed compared to duplicates maintained at room temperature (Table 1). Greater specificity was also seen with heating in a gel electrophoretic analysis (not shown). In addition, improved sensitivity and specificity was seen with pre-amplification heating using templates of double stranded plasmid DNA that had not been previously denatured (Figure 1), both with (Figure 1) and without (not shown) the addition of tetramethylammonium chloride (TMAC) (Fisher Scientific) (3) to the reaction master mix. This effect was observed with 3 different primer-template pairs (Table I and Figure 1). It is of note that pre-amplification heating did not improve product yield with primers of the same sequence as those used in the experiment in Table I that had been documented to lack failure sequences by HPLC. We speculate that preamplification heating may promote stringent primer annealing and subsequent extension, thereby increasing effective primer length. Minimization of 'primer-dimer'or primer self-annealing may also contribute. Preamplification heating has allowed the use of primer pairs which were inadequate for amplification of low copy number templates when reactions were assembled at room temperature.