Massively parallel single-amino-acid mutagenesis

Nature Methods 2015 192 cites
2015:62016:82017:112018:132019:152020:172021:182022:202023:202024:212025:212026:22
2015 2026
192

Jacob O. Kitzman, Lea M. Starita, Russell S. Lo, Stanley Fields, Jay Shendure

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.

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