Figure 2

Establishment of the method for site-directed mutagenesis of large plasmids (SMLP) based on PCR. (A) A plasmid map showing the locations of the PCR primers designed. The pcDNA-FLNA plasmid is presented in the diagram, and two pairs of PCR primers are designed as indicated. MFP represents a mutation forward primer; MRP represents a mutation reverse primer; MAFP is a mutation-assisting forward primer; MARP is a mutation-assisting reverse primer. The blue or red colours represent partially complementary (overlap) regions between two primers. (B) A diagram showing two independent PCR reactions, PCR I and PCR II. PCR I is performed using the primer pair of MAFP and MPR; whereas PCR II is performed using the primer pair of MFP and MARP. The PCR I and II reaction systems were presented on the right of the panel. (C) A diagram showing the recombinational ligation of the products from the PCR I and PCR II under the Exnase II. The short black arrows represent the places digested by Exnase II; the long black arrows show how two DNA fragments are ligated.