title: All-codon scanning identifies p53 cancer rescue mutations.
abstract: In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure-function analyses and protein design or evolution problems.
authors.affiliation: Institute for Genomics and Bioinformatics, Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA.
authors.forename: Roberta
authors.initials: R
authors.lastname: Baronio
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Danziger
authors.forename: Linda V
authors.initials: LV
authors.lastname: Hall
authors.forename: Kirsty
authors.initials: K
authors.lastname: Salmon
authors.forename: G Wesley
authors.initials: GW
authors.lastname: Hatfield
authors.forename: Richard H
authors.initials: RH
authors.lastname: Lathrop
authors.forename: Peter
authors.initials: P
authors.lastname: Kaiser
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D003062:Codon; D015723:Gene Library; D052138:Genes, Neoplasm; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D003062:Codon