Figure 9

The SMLP method can be applied to the mutagenesis of deletion, insertion and point substitution for small plasmids. (A) A diagram showing the pET-30a( +)-hTBP plasmid and the positions of the PCR primers designed for deletion, insertion, and substitution mutations. The red region represents a deletion fragment, the blue short line represents a point mutation site, and the green short line represents the insert bases. (B) Analysis of the small DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer TBPMARP and one of the mutation forward primers, including TBPDFP, TBPINFP, and TBPPMFP. PCR products were detected by agarose gel electrophoresis. TBPMARP represents a TBP mutation-assisting reverse primer; TBPDFP, TBP deletion forward primer; TBPINFP, TBP insertion forward primer; TBPPMFP, TBP point mutation forward primer. (C) Analysis of the large DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer TBPMAFP and one of the mutation reverse primers, including TBPDRP, TBPINRP, and TBPPMRP. PCR products were detected by agarose gel electrophoresis. TBPMAFP represents a TBP mutation-assisting forward primer; TBPDRP, TBP deletion reverse primer; TBPINRP, TBP insertion reverse primer; TBPPMRP, TBP point mutation reverse primer. (D) Analysis of the pET-hTBP plasmids from mutagenesis by the SMLP method using agarose gel electrophoresis. The plasmids from mutagenesis were detected by agarose gel electrophoresis. (E) The results of DNA sequencing for the mutants of TBP gene deletion, insertion and point substitution. The left panels are the DNA sequencing maps for the mutants of TBP deletion, insertion and point substitution. The deletion position is marked with a red triangle (top), whereas the insertion and point mutations were framed by the red rectangle lines (middle and bottom), respectively. The right panels are the DNA fragments from the sequence maps (right panel), where the amino acids flanking each mutation site are indicated. The original and mutated bases were displayed in green and red colours, respectively.