Massively Parallel Single Amino Acid Mutagenesis
By J. Kitzman, Lea M. Starita, Russell S. Lo, Stanley Fields, Jay Shendure. 2015
165 citations recorded by the source corpus.
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.
Subfields: Molecular Biology
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In Molecular Biology
- Kevin Kaichuang Yang
- Zachary Wu
- Stanley Fields
- Douglas M. Fowler
- Jay Shendure
- Lea M. Starita
- Frances H. Arnold
- David L. Young
Citations in the corpus
- Using deep mutational scanning to benchmark variant effect predictors and identify disease mutations (2020, unknown): “Several groups provide both their full fitness scores and a filter for high-quality results (Starita et al , 2015; Mighell et al , 2018).”
- Machine learning in protein engineering (2018, background): “…et al., 2016), influenza nucleoprotein (Ashenberg et al., 2017), influenza hemagglutinin (Doud & Bloom, 2016), PhoQ (Podgornaia & Laub, 2015), GB1 (Wu et al., 2016), the DNA-binding domain of a steroid hormone receptor (Starr et al., 2017), and Gal4 transcription factor (Kitzman et al., 2015).”
- Massively Parallel Functional Analysis of BRCA1 RING Domain Variants (2015, methodology): “(A) We created an allelic series of variants within BRCA1(2-304) with single amino acid substitutions by the method known as Programmed Allelic Series (Kitzman et al. 2015), which uses mutagenic oligonucleotides synthesized on a programmed microarray to create a pool of variants with single codon ch”