title: INSULT: a novel mutagenesis method generating high yields of closed circular mutant DNA with one primer per mutant.
abstract: 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.
authors.affiliation: Rensselaer Polytechnic Institute, Biology Science Center 1W14, 110 8 Street, Troy, NY 12180 USA.
authors.forename: Eda
authors.initials: E
authors.lastname: Erdogan
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Jones
authors.forename: P
authors.initials: P
authors.lastname: Matzlin
authors.forename: Michael H
authors.initials: MH
authors.lastname: Hanna
authors.forename: Susan M E
authors.initials: SM
authors.lastname: Smith
authors.forename: John C
authors.initials: JC
authors.lastname: Salerno
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010957:Plasmids / Q000737:chemistry; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017384:Sequence Deletion
chemical_list: D017931:DNA Primers