Modified QuikChange mutagenesis scheme using a 3’-exonuclease to generate single-stranded 5’ overhangs.

Plasmid DNA is amplified by PCR using a high-fidelity DNA polymerase such as Q5 (New England Biolabs). Forward and reverse primers overlap partially [20]. The red and yellow circles (triangles) mark the 5’ (3’) ends of the primers. The green crosses mark mismatches between the mutation primers and the template DNA. The present work demonstrates the advantage of using an attenuated 3’-exonuclease to generate single-stranded overhangs. Filling in of single-stranded DNA and re-ligation of the circular plasmid is achieved by endogenous E. coli enzymes following transformation.