Fig. 1.

Schematic diagram of our modified PCR-based SDM method with introduction of restriction site on the primer. a Design of the mutagenic primers. The desired mutation and silent restriction site mutation can be introduced at the primer-primer complementary sequences or the non-overlapping sequences of the primers as shown in I, II and III. Black and white triangles indicate the locations of the desired mutation and silent restriction site mutation in the primer. b Flow chart of the procedures for mutagenesis and screening. The original plasmid is subjected to PCR using the primer pairs shown in a. The PCR products are transformed into E. coli and the plasmids isolated from the transformed colonies are subjected to silent restriction enzyme (RE) digestion for mutant screening. The mutations present in the plasmid identified by RE digestion are further confirmed by DNA sequencing