title: Error-prone rolling circle amplification greatly simplifies random mutagenesis.
abstract: We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.
authors: Synthetic Chemicals Laboratory, Mitsui Chemicals, Inc., 580-32 Nagaura, Sodegaura, 299-0265, Japan.
authors: Ryota
authors: R
authors: Fujii
authors: Motomitsu
authors: M
authors: Kitaoka
authors: Kiyoshi
authors: K
authors: Hayashi
mesh_terms: D019020:Directed Molecular Evolution; D016296:Mutagenesis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*