Figure 5

Alteration of the distance between two pairs of primers does not affect the mutagenesis efficacy of large plasmids. (A) A diagram showing the pcDNA-FLNA plasmid and the positions of the PCR primers designed. A1188TFP (or S1199LFP), A1188T (or S1199L) forward primer; A1188TRP (or S1199LRP), A1188T (or S1199L) reverse primer. (B) Analysis of the small DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer pair of pcDNAFP and A1188TRP or the primer pair of pcDNAFP and S1199LRP. PCR products were detected by agarose gel electrophoresis. PCRSF represents PCR small fragments. (C) Analysis of the large DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the primer pair of pcDNARP and A1188TFP or the primer pair of pcDNARP and S1199LFP. PCR products were detected as for (B). PCRLF represents PCR large fragments. (D) Analysis of the plasmids from site-directed mutagenesis by agarose gel electrophoresis. The plasmids derived from site-directed mutagenesis with the SMLP method were directly detected by agarose gel electrophoresis after plasmid preparation. These plasmids displayed in a super-coiled form (S1199L in the last lane) or in a linear form (the 3rd-5th lanes). (E) The results of DNA sequencing for the A1188T and S1199L mutants. The left panels are DNA sequencing maps for the A1188T and S1199L mutants, where the mutated bases are framed by the red rectangle lines. The right panels are the original and mutated DNA fragments for each mutant presented as for Fig. 4D.