Site-Directed Mutagenesis Using Double-Stranded Plasmid DNA Templates

In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships, gene expression and vector modification. Several methods for performing this technique have appeared in the literature (1 –5 ). These procedures generally require multiple enzymatic steps, specialized vectors, and convenient restriction sites or subcloning of the sequence of DNA to be mutated into a bacteriophage vector like M13 to produce and recover single-stranded DNA. These manipulations present major limitations to the routine use of these methods because they are time consuming and tedious.