Amplimers with 3'-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming.
The 3'-->5' 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 polymerase-mediated amplifications.
de Noronha
C M
CM
Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115.
Mullins
J I
JI
D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005256:Leukemia Virus, Feline / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D013873:Thionucleotides
D001426:Bacterial Proteins; D004279:DNA, Viral; D013873:Thionucleotides; D019914:Taq Polymerase; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase