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

Massively parallel single-amino-acid mutagenesis.

Author information

1
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
2
1] Department of Genome Sciences, University of Washington, Seattle, Washington, USA. [2] Howard Hughes Medical Institute, Seattle, Washington, USA.
3
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.

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.

PMID:
25559584
PMCID:
PMC4344410
DOI:
10.1038/nmeth.3223
[Indexed for MEDLINE]
Free PMC Article

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