Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit.
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.
Hogrefe
Holly H
HH
Stratagene, La Jolla, CA, USA. holly_hogrefe@stratagene.com
Cline
Janice
J
Youngblood
Geri L
GL
Allen
Ronda M
RM
D019943:Amino Acid Substitution; D018388:Codon, Terminator; D017931:DNA Primers; D004274:DNA, Recombinant / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017354:Point Mutation; D011897:Random Allocation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013698:Templates, Genetic; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics
D018388:Codon, Terminator; D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D001616:beta-Galactosidase