Figure 4

The SMLP method showed consistent results in the mutagenesis of large plasmids. (A) Analysis of the small DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer pcDNAFP and one of the mutation reverse primers, including S149FRP, P207LRP, and E254KRP, where the pcDNA-FLNA plasmids acted as DNA templates. PCR products were monitored by agarose gel electrophoresis. PCRSF represents PCR small fragments. (B) Analysis of the large DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer pcDNARP and one of the mutated forward primers, including S149FFP, P207LFP, and E254KFP, where the pcDNA-FLNA plasmids acted as DNA templates. PCR products were monitored by agarose gel electrophoresis. PCRLF represents PCR large fragments. (C) Analysis of the pcDNA-FLNA plasmids from site-directed mutagenesis by agarose gel electrophoresis. The plasmids from mutagenesis were prepared and analysed by agarose gel electrophoresis. (D) The results of DNA sequencing for S149F, P207L, and E254K mutants. The DNA sequencing maps for the S149F, P207L, and E254K mutants were presented at the left, where the mutated bases were outlined by the red rectangle lines. The right panels are the original and mutated DNA sequences for each mutant, where the bases in the sequencing map were framed by the red rectangle lines. The original and mutated bases were displayed in green and red colours, respectively.