Massively Parallel Single Amino Acid Mutagenesis

Details

• Alternative Title:
  Nat Methods
• Personal Author:
  Kitzman, Jacob O. ; Starita, Lea M. ; Lo, Russell S. ; Fields, Stanley ; Shendure, Jay
• Description:
  Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.
• Keywords:
  Amino Acids DNA-Binding Proteins Gene Library Haplotypes Humans Mutagenesis, Site-Directed Mutation, Missense Oligonucleotide Array Sequence Analysis Saccharomyces Cerevisiae Proteins Transcription Factors Tumor Suppressor Protein P53 Uracil-DNA Glycosidase

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• Source:
  Nat Methods. 12(3):203-206
• Pubmed ID:
  25559584
• Pubmed Central ID:
  PMC4344410
• Document Type:
  Journal Article
• Funding:
  P41 GM103533/GM/NIGMS NIH HHSUnited States/ ; DP1HG007811/DP/NCCDPHP CDC HHSUnited States/ ; P41GM103533/GM/NIGMS NIH HHSUnited States/ ; Howard Hughes Medical InstituteUnited States/ ; DP1 HG007811/HG/NHGRI NIH HHSUnited States/
• Volume:
  12
• Issue:
  3
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:e670878282a4d0a64da5a868adcba15e2662681790fc895fd33ac9cd1370ad8e
• Download URL:
  https://stacks.cdc.gov/view/cdc/30311/cdc_30311_DS1.pdf
• File Type:
  [PDF - 299.10 KB ]