Abstract：
Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don\'t. Their demonstrated short primers spanning the 3\' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.
摘要：
用于创建点突变的定点诱变，有时,在局部产生携带可变数量串联重复序列(VNTRs)的质粒，它们被任意地视为聚合酶链反应(PCR)相关的伪影。这里,据报道，替代的末端连接机制而不是PCR伪影在很大程度上解释了VNTR的形成和扩增。在GPLD1基因上产生点突变的过程中，在pcDNA3.1-GPLD1质粒中观察到使用31bp诱变引物的VNTRs的意外形成作为重复单元。在24.75%的所得克隆中形成31bp的VNTR,拷贝数从2到13变化。所有重复单元以与GPLD1基因相同的方向比对。43.54%的重复连接处有核苷酸突变，而其余的则没有。他们展示的跨越诱变引物3'部分的短引物对于在环状质粒中初始创建2拷贝串联重复序列（TR）至关重要。2拷贝TR的进一步扩增需要以正确的方向通过交替的末端连接进行诱变引物的二聚化。最后,建立了半双启动战略，验证了这些发现，并为在没有连接突变的环状质粒中编码基因上创建VNTRs提供了一种简单的方法。
