Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols
Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols
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DOI：
10.1139/o04-134
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发表时间：
2005-04-01
影响因子：
2.9
通讯作者：
Vigneault, F
中科院分区：
文献类型：
--
作者：
Drouin, R;Vigneault, F
建立了一种以滨海热球菌（Thermococcus litoralis）exo-DNA聚合酶（Vent exo-）为模板的连接介导的聚合酶链反应（PCR）技术。确定了引物延伸和PCR扩增步骤中Vent exo-的最佳剂量以及最佳DNA用量。我们表明，Vent exo-可以有效地产生后续接头连接所需的平端末端。Vent exo-被证明比Pyrococcus furiosus exo-（Pfu exo-）更有效地用于该任务。Vent exo-比栖热菌DNA聚合酶（Taq）更好地解析高度富含GC的序列，并且具有与Pfu exo-相似的效率。Vent exo-的DNA/DNA聚合酶活性比显著高于Pfu exo-，这反映在Vent exo-在有效扩增基因组DNA方面的敏感性。此外，Vent exo-的效率范围证明了进行评价性测试以确定使用该聚合酶的最佳剂量以获得成功PCR扩增的重要性。确定了与Vent exo-配合使用的最佳MgSO 4浓度。我们的研究结果表明，Vent exo-DNA聚合酶产生的条带的均匀和强强度，并可以有效地用于分析活细胞中的DNA连接介导的PCR。
An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo- DNA polymerase (Vent exo-) was developed. The optimal dosage of Vent exo- at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo- can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo- proves to be more efficient than Pyrococcus furiosus exo- (Pfu exo-) for this task. Vent exo- resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo-. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo- than for Pfu exo-, which is reflected by the sensibility of Vent exo- in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo- demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo- were established. Our results show that Vent exo- DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.