A rapid, simple, and effective method of constructing a randomly mutagenized plasmid library free from ligation.
The QuikChange site-directed mutagenesis methodology was applied to constructing a randomly mutagenized plasmid library simply by adding manganese to the reaction mixture. This method is superior to the normally employed Pol I-type polymerase-based error-prone PCR in that (i) it does not require a subsequent ligation reaction, and (ii) there is no accumulation of mutations at the same site. alpha-Complementation analysis and subsequent sequence analyses of the lacZ alpha genes in the mutated library revealed that the mutations occurred randomly within the target gene and involved all possible base substitutions.
Koyanagi
Takashi
T
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan.
Yoshida
Erina
E
Minami
Hiromichi
H
Katayama
Takane
T
Kumagai
Hidehiko
H
D001483:Base Sequence; D015723:Gene Library; D008345:Manganese / Q000737:chemistry; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D013269:Stochastic Processes; D013997:Time Factors
D008345:Manganese