Half-site editing: An in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site

Abstract Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a bluntend ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place Cla I restriction sites at the 3′ end of the yeast pyruvate kinase promoter and at two positions at the 5′ end of the yeast acetolactate synthase coding sequence.