[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

Objective To determine whether 3′phosphorothioate-modified -2 terminal mismatched primers can turn off DNA polymerization mediated by Exo+ polymerase. Methods Two-directional primer extension was performed using polymerase with and without 3′ exonuclease activity. The effects of unmodified primers and 3′ phosphorothioate-modified primers on primer extension were evaluated. Results Exo- polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3′ phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported “off-switch” of Exo+ polymerase. Conclusion These data suggested that the “off-switch” can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.