de Noronha C M, Mullins J I
Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115.
PCR Methods Appl. 1992 Nov;2(2):131-6. doi: 10.1101/gr.2.2.131.
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.
来自嗜热栖热放线菌的耐热聚合酶Vent的3'→5'核酸外切酶活性增强了DNA复制保真度，但也通过降解PCR引物（扩增子）的3'末端，使其偏离靶向扩增。我们证明，具有3个碱基的3'末端错配的扩增子可被Vent有效截短，从而引发与特异性扩增反应竞争的DNA聚合反应。然而，其3'末端残基通过硫代磷酸酯键连接的扩增子对降解具有抗性，并表现出显著增强的引发特异性。硫代磷酸酯键连接的残基导致碱基配对轻微不稳定，这也减少了Taq聚合酶介导的扩增中错配的3'扩增子末端的延伸。
