Figure 7

The SMLP method has an advantage over the conventional methods in the mutagenesis of large plasmids. (A) Detection of the PCR products from different methods for site-directed mutagenesis by agarose gel electrophoresis. a, PCR with a double-strand DNA fragment; b, PCR with a pair of partially complementary primers; c, PCR with a pair of inverse primers; P1, the small DNA fragments from PCR with the primer pair of pcDNAFP and D1159ARP; P2, the large DNA fragments from PCR with the primer pair of pcDNARP and D1159AFP. (B) Comparison of the transformation efficiency for the different methods for site-directed mutagenesis. The samples used for transformation were generated according to their respective methods as indicated. a, the transformation result from the method based on the PCR with a pair of complementary primers; b, the transformation result from the method based on the PCR with a pair of partially complementary primers; c, the transformation result from the method based on the PCR with a pair of inverse primers; d, the result from the SMLP method. (C) Analysis of the pcDNA-FLNA plasmid derived from site-directed mutagenesis by the SMLP method using agarose gel electrophoresis and DNA sequencing. Lane 1 to 13 in the image represents the plasmids from different colonies generated by the SMLP method, whereas lane 14 represents the pcDNA-FLNA original plasmid (Top panel). The mutated base in the sequencing map (bottom panel) was enclosed with the red rectangle lines as in Fig. 6E. (D) Comparison of the rate of positive clones from different methods for site-directed mutagenesis. a, b, c, and d in the graph represent the results from their respective methods as described in (B). The rate of the positive clones was obtained by calculating the percentage for the number of the mutants in the number of colonies inoculated.