Optimal conditions to use Pfu exo(–) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(–) DNA polymerase (Pfu exo(–)). The relative efficiency of Pfu exo(–) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(–) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(–), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(–) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(–) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.

连接介导聚合酶链式反应（Ligation-Mediated Polymerase Chain Reaction，LMPCR）是目前可利用基因组DNA在核苷酸分辨率水平绘制单链DNA断裂位点的最灵敏测序技术。LMPCR已被改造用于绘制DNA损伤图谱，并揭示活细胞内的DNA-蛋白质相互作用。然而，序列背景（GC含量）、整体断裂频率以及当前LMPCR中使用的DNA聚合酶组合，均会对实验结果的质量产生影响。本研究开发并优化了一套适配激烈热球菌缺失核酸外切酶活性的DNA聚合酶（Pyrococcus furiosus exo(–) DNA polymerase，Pfu exo(–)）的LMPCR实验方案。在LMPCR的引物延伸步骤中，我们将Pfu exo(–)与T7修饰型DNA聚合酶（T7-modified DNA polymerase，Sequenase 2.0）的相对效率进行了对比；在PCR扩增步骤中，则将其与水生栖热菌DNA聚合酶（Thermus aquaticus DNA polymerase，Taq）进行了比较。在所有测试的断裂频率下，Pfu exo(–)均表现出比Sequenase 2.0更高的催化效率。在引物延伸与PCR扩增两个步骤中，每单位DNA聚合酶对应的DNA分子比例是决定Pfu exo(–)效率的核心因素，而Taq的效率受该比例的影响相对较小。在Taq的PCR反应缓冲液中用NaCl替代KCl，可显著提升该聚合酶的催化活性。Pfu exo(–)在特异性扩增极高GC含量的基因组DNA序列方面，明显优于Taq。本研究结果表明，在LMPCR的引物延伸步骤使用Pfu exo(–)、PCR扩增步骤使用Taq的组合方案，是利用LMPCR开展活体内DNA分析与DNA损伤图谱绘制的最优选择。