Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Figure 1

Overview of the combined overlap extension PCR (COE-PCR) method. The primary PCR reactions follow a standard PCR reaction, but with limited mutagenic primers. The primary PCR reactions produce double stranded mutagenic DNA fragments which have overlapping ends. In the secondary PCR reaction, the products from the primary PCR reaction are mixed in a 1 : 1 ratio and amplified in a modified PCR reaction consisting of 5–10 cycles to produce full length double stranded mutagenic DNA (FGSP: forward gene specific primer; RGSP: reverse gene specific primer; FMP: forward mutagenic primer; RMP: reverse mutagenic primer).