Massively parallel single-amino-acid mutagenesis

Nat Methods. 2015 Mar;12(3):203-6, 4 p following 206. doi: 10.1038/nmeth.3223. Epub 2015 Jan 5.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / genetics
  • DNA-Binding Proteins / genetics*
  • Gene Library
  • Haplotypes
  • Humans
  • Mutagenesis, Site-Directed / methods*
  • Mutation, Missense
  • Oligonucleotide Array Sequence Analysis / methods
  • Saccharomyces cerevisiae Proteins / genetics*
  • Transcription Factors / genetics*
  • Tumor Suppressor Protein p53 / genetics
  • Uracil-DNA Glycosidase / metabolism

Substances

  • Amino Acids
  • DNA-Binding Proteins
  • GAL4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • TP53 protein, human
  • Transcription Factors
  • Tumor Suppressor Protein p53
  • Uracil-DNA Glycosidase

Associated data

  • SRA/PRJNA268398