Biochimica et biophysica acta
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Biochim. Biophys. Acta · Nov 2000
The PCR plateau phase - towards an understanding of its limitations.
The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. ⋯ PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.