INSULT: a novel mutagenesis method generating high yields of closed circular mutant DNA with one primer per mutant.

INSULT, a novel method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special "ultracompetent" cells. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single-stranded copies of circular mutation-bearing plasmid DNA, addition of a "generic" primer followed by one or more polymerase reaction cycles generates double-stranded circular DNA bearing the desired mutation.