All-codon scanning identifies p53 cancer rescue mutations
| Autor: | Linda V. Hall, G. Wesley Hatfield, Kirsty Anne Lily Salmon, Richard H. Lathrop, Roberta Baronio, Samuel A. Danziger, Peter K. Kaiser |
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| Rok vydání: | 2010 |
| Předmět: |
tumor
Molecular Sequence Data Protein design saturation mutagenesis Mutagenesis (molecular biology technique) in-vitro Biology medicine.disease_cause Polymerase Chain Reaction cassette mutagenesis Cell Line law.invention 03 medical and health sciences 0302 clinical medicine 2nd-site suppressor mutations law Medicine and Health Sciences Genetics medicine Humans Genomic library directed evolution Codon Molecular Biology Gene Gene Library 030304 developmental biology gene synthesis chemistry.chemical_classification 0303 health sciences Mutation Base Sequence mutant p53 Life Sciences Genes p53 Amino acid Amino Acid Substitution chemistry 030220 oncology & carcinogenesis escherichia-coli Suppressor Primer (molecular biology) protein Genes Neoplasm |
| Zdroj: | Baronio, Roberta; Danziger, Samuel A.; Hall, Linda V.; Salmon, Kirsty; Hatfield, G. Wesley; Lathrop, Richard H.; et al.(2010). All-codon scanning identifies p53 cancer rescue mutations. Nucleic Acids Research, 38(20), 7079-7088. UC Irvine: Retrieved from: http://www.escholarship.org/uc/item/81w7m8dj Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkq571 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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