The Life Science College, Henan Agricultural University, Zhengzhou 450002, China.
The Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China.
Int J Mol Sci. 2020 Mar 2;21(5):1697. doi: 10.3390/ijms21051697.
Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5'- and 3'-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-. The generality was checked by constructing plasmid pET21a- and pET22b- in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.
虽然已经报道了许多方法，但质粒构建会影响转化率（每纳克 DNA 的转化体数量）和质粒准确性（无痕质粒的比率）。一种有效的方法是两步 PCR 进行 DNA 扩增。一种准确的方法是 ExnaseII 克隆进行同源重组 (HR)。我们将 DNA 扩增和 HR 结合起来，通过扩增质粒 DNA 来包含同源 5'和 3'末端，并在体外重组质粒 DNA，从而开发出分子内 HR。以构建质粒 pET20b-为例。通过平行构建质粒 pET21a-和 pET22b-来检查通用性。具有 30-bp 同源臂的 DNA 最适合分子内 HR，其转化产生 14.2 个转化体/ng 和 90%的无痕质粒，优于两步 PCR 和 ExnaseII 克隆。转化率与 HR 产物中缺口环状质粒 DNA 的成分相关，表明体内的缺口修饰导致有缺口的质粒。
