Creating Randomized Amino Acid Libraries with the QuikChange ^® Multi Site-Directed Mutagenesis Kit

Abstract

The QuikChange Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse libraries (10(4)-10(5) mutants/reaction) are created consisting of random combinations of mutations at two or three different sites. This method provides a one-day procedure for performing site-directed saturation mutagenesis and, when coupled with a suitable screening assay, should greatly facilitate the process of evaluating alternative amino acid chain substitutions at key residues and evolving protein function.

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

“…The library generated by saturation mutagenesis at positions 93∕94 by using NDT codon degeneracy was found to contain two mutants that catalyze measurable conversion of ketone rac-1d within a reaction time of 24 h as specified in the GC screening process, namely Gln93Val/ Pro94Phe and Gln93Asn/Pro94Asp. Due to amino acid bias inherent in the nature of the saturation mutagenesis protocol (14,31), the possibility that hits were missed cannot be excluded inspite of the high percentage of coverage. Because the double mutant Gln93Asn/Pro94Asp proved to be the much more active of the two identified hits, we subsequently focused all further efforts on this variant.…”

Beyond the Natural Proteome

“…In terms of saturation mutagenesis, the pair of primers need not contain a single mutation, but may instead contain a degenerate codon or indeed take the form of a DNA cassette as described in section 3.1.1. To expand the technology further, the QuikChange® Multi Site-Directed Mutagenesis Kit has been developed to target up to five sites simultaneously (Hogrefe, Cline, Youngblood & Allen, 2002). Target residues have to be at least 5 codons apart and so automatically exclude targeting contiguous sites.…”

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

“…Oligonucleotide-directed SDM methods have been described, including PCR-based methods, and Adachi and Fukuhara, 2012 methods based on plasmid DNA templates and mutant DNA (Hogrefe et al, 2002). Most of these methods are used to introduce only one mutation site at a time.…”