The 3'-~S' exonuclease activity of Vent, a thermostable polymerase from Thermococcus litoralis, enhances DNA replication fidelity but also diverts PCR primers (amplimers) from targeted amplification by degrading their 3' termini. We demonstrate that amplimers with a 3-base 3'-terminal mismatch can be efficiently truncated by Vent to prime DNA polymerizations that compete with the specific amplification reaction. However, amplimers with phosphorothioate bonds joining their 3'-terminal residues are resistant to degradation and demonstrate greatly enhanced priming specificity. Slight destabilization of base-pairing by phosphorothioate bond-linked residues also diminishes extension of mispaired 3' amplimer termini in Taq polymerasemediated amplifications.
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