title: Massively parallel single-amino-acid mutagenesis.
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.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Jacob O
authors.initials: JO
authors.lastname: Kitzman
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Lea M
authors.initials: LM
authors.lastname: Starita
authors.affiliation: 1] Department of Genome Sciences, University of Washington, Seattle, Washington, USA. [2] Howard Hughes Medical Institute, Seattle, Washington, USA.
authors.forename: Russell S
authors.initials: RS
authors.lastname: Lo
authors.affiliation: 1] Department of Genome Sciences, University of Washington, Seattle, Washington, USA. [2] Howard Hughes Medical Institute, Seattle, Washington, USA. [3] Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
mesh_terms: D000596:Amino Acids / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015723:Gene Library; D006239:Haplotypes; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D020125:Mutation, Missense; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C495901:TP53 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051981:Uracil-DNA Glycosidase