Figure 5.

Schematic representation of the RE-mediated inverse PCR method. The RE-mediated inverse PCR reaction contains 15 pmol of both of the inverse primers (pink and blue arrows are the sense and antisense primers, respectively) and 0.01 pmol of the 7.4 kb template. The primers are designed in such a way that they contain unique restriction enzyme sites (represented by stars) and anneal to the opposite ends of the desired region to be deleted. The PCR cycle of template degradation for 45 sec at 95°C, primer annealing for 1 min at 56°C and primer extension for 2 min/kb at 68°C, is repeated for 18 cycles followed by a final extension step at 68°C for 2 min/kb. The PCR reaction results in the synthesis of both parental, wild type template DNA (yellow and green in the bottom panel), which is subsequently removed during a DpnI digestion step, as well as linear mutated DNA (pink and blue in the top panel). Digestion with the unique restriction enzyme creates linear DNA with sticky-ends, which improves the ligation efficiency and subsequent circularization of the PCR product containing the deletion mutation.