Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.
Two thermostable DNA polymerases with proofreading activity--Vent 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 oligodeoxynucleotides 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.
Skerra
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Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D013873:Thionucleotides / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase