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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.

SUBMITTER: Kitzman JO

PROVIDER: S-EPMC4344410 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Massively parallel single-amino-acid mutagenesis.

Kitzman Jacob O JO Starita Lea M LM Lo Russell S RS Fields Stanley S Shendure Jay J

Nature methods 20150105 3

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 mutationa ...[more]

PMID: 25559584

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