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.

Submission Details

ID: 7wvUDLZY3

Submitter: Shu-Ching Ou

Submission Date: July 18, 2018, 5:55 p.m.

Version: 1

Publication Details
Kitzman JO;Starita LM;Lo RS;Fields S;Shendure J,Nat Methods (2015) Massively parallel single-amino-acid mutagenesis. PMID:25559584
Additional Information

Study Summary

Number of data points 7176
Proteins Regulatory protein GAL4
Unique complexes 1196
Assays/Quantities/Protocols Experimental Assay: log2-ratio Enrichment Score
Libraries PALS library for the DNA-binding domain (DBD), selection c (SC medium –uracil --histdine +1.5mM 3-AT, after 64 h) ; PALS library for the DNA-binding domain (DBD), selection c (SC medium –uracil --histdine +1.5mM 3-AT, after 40 h) ; PALS library for the DNA-binding domain (DBD), selection b (SC medium –uracil --histdine +0.5mM 3-AT, after 40 h) ; PALS library for the DNA-binding domain (DBD), selection a (SC medium –uracil --histdine, after 40 h) ; PALS library for the DNA-binding domain (DBD), selection a (SC medium –uracil --histidine , after 24 h) ; PALS library for the DNA-binding domain (DBD), without selection (SC medium –uracil, after 24 h)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Regulatory protein GAL4 P04386 GAL4_YEAST