title: Error-prone rolling circle amplification: the simplest random mutagenesis protocol.
abstract: A simple protocol to introduce random mutations, named error-prone rolling circle amplification (RCA), is described. A template plasmid is amplified by RCA in the presence of MnCl2 and used for transformation of a host strain to give a mutant library with three to four random point mutations per kilobase throughout the entire plasmid. The prime advantage of this method is its simplicity. This protocol requires neither the design of specific primers nor the exploration of thermal cycling conditions. It takes just 10 min to prepare the reaction mixture, followed by overnight incubation and transformation of a host strain. This method permits rapid preparation of randomly mutated plasmid libraries, and will enable the wider adoption of random mutagenesis.
authors.affiliation: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Fujii
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Hayashi
mesh_terms: D002712:Chlorides; D015723:Gene Library; D018014:Gene Transfer Techniques; D017895:Manganese Compounds; D016296:Mutagenesis; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids
chemical_list: D002712:Chlorides; D017895:Manganese Compounds; C025340:manganese chloride