Figure 1

Outline of the new strategy described in this paper. The first PCR (5 cycles) contains a limiting concentration of the first flanking primer and is followed by a prolonged extension step. The second flanking primer is then added and the entire mutated template is amplified by PCR (25 cycles). The second PCR product is digested with appropriate restriction endonucleases to give desired mutated fragment, which then can be ligated to the expression vector.