Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
PLoS One. 2019 Jan 23;14(1):e0211065. doi: 10.1371/journal.pone.0211065. eCollection 2019.
The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3'-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.
高保真 DNA 聚合酶的出现使得通过 PCR 线性化和扩增整个质粒成为可能，从而大大简化了克隆和诱变方案。市售试剂盒效果很好，但通常使用未公开或专有的成分进行了优化。在这里，我们表明一种突变的 T4 DNA 聚合酶（Y320A）具有较弱的 3' -外切酶活性，比野生型酶更适合以更易于控制的方式产生均匀长度的单链 DNA 突出端，并且可以用于增加克隆和诱变实验中含有正确修饰质粒的菌落产量，当大肠杆菌细胞的相对低competency 时，这特别有用。提供了使用突变 T4 DNA 聚合酶的标准方案，用于序列和连接独立克隆（SLIC）方法和改良的 QuikChange 方法，其中突变酶增强了正确突变质粒的产量，并在 DpnI 消化过程中进一步抑制了亲本质粒。突变 T4 DNA 聚合酶产生的单链 DNA 突出端有助于随后在大肠杆菌中进行质粒环化、退火和连接。
