Oligonucleotide-directed mutagenesis using plasmid DNA templates and two primers.

Oligonucleotide-directed mutagenesis using plasmid vectors has been simplified by introducing two changes to the previous method. First, template preparation has been simplified by using the covalently closed circular plasmid DNA directly for mutagenesis, eliminating the need for a wholly or partially single-stranded circular DNA template. Second, two primers are used, eliminating the need for producing the covalently closed molecule during in vitro replication. The advantages of the approach are discussed.