Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site‐Directed Mutagenesis and Optimization for Coding Gene Application

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.