A Rapid, Simple, and Effective Method of Constructing a Randomly Mutagenized Plasmid Library Free from Ligation

Abstract

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. α-Complementation analysis and subsequent sequence analyses of the lacZα genes in the mutated library revealed that the mutations occurred randomly within the target gene and involved all possible base substitutions.