Nucleic Acids Research, 1992, Vol. 20, No. 14 3551-3554
© 1992
METHODS |
Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity
Max-Planck-Institute für Biophysik Heinrich-Hoffmann-Straße 7, W–6000 Frankfurt/Main 71, Germany
Received June 15, 1992. Accepted June 25, 1992.
Two thermostable DNA polymerases with proofreading activityVent DNA polymerase and Pfu DNA polymerase–have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the ollgodeoxynucleotldes serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the Introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.
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