Site-directed mutant libraries for isolating minimal mutations yielding functional changes
| Autor: | Krishnakumar M. Ravikumar, Sarah C. Potter, Dong hee Chung, Michael D. Toney, Ammon C. Tanomrat |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Aminodeoxychorismate synthase Mutant Mutagenesis (molecular biology technique) Bioengineering medicine.disease_cause Polymerase Chain Reaction Biochemistry 03 medical and health sciences Escherichia coli medicine Site-directed mutagenesis Molecular Biology Transaminases Gene Library Genetics Mutation 030102 biochemistry & molecular biology biology Oligonucleotide Escherichia coli Proteins Directed evolution 030104 developmental biology Mutagenesis Site-Directed biology.protein Anthranilate synthase Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection. 30:347-357 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzx013 |
| Popis: | Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity. |
| Databáze: | OpenAIRE |
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