One-step random mutagenesis by error-prone rolling circle amplification.
In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3-4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.
Fujii
Ryota
R
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Kitaoka
Motomitsu
M
Hayashi
Kiyoshi
K
D002442:Ceftazidime / Q000494:pharmacology; D004270:DNA, Circular / Q000096:biosynthesis; D019020:Directed Molecular Evolution / Q000379:methods*; D004351:Drug Resistance; D015723:Gene Library; D016296:Mutagenesis; D009154:Mutation; D013379:Substrate Specificity; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000378:metabolism
D004270:DNA, Circular; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1