Codon-Based Combinatorial Alanine Scanning Site-Directed Mutagenesis: Design, Implementation, and Polymerase Chain Reaction Screening
✍ Scribed by J. Chatellier; A. Mazza; R. Brousseau; T. Vernet
- Book ID
- 102969014
- Publisher
- Elsevier Science
- Year
- 1995
- Tongue
- English
- Weight
- 769 KB
- Volume
- 229
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Combinatorial alanine scanning mutagenesis is a powerful tool for the exploration of protein structurefunction relationships. Unfortunately, combinatorial alanine replacement of multiple residues using standard site-directed mutagenesis is restricted to a subset of amino acids. To circumvent this limitation, an efficient procedure for combinatorial site-specific replacement by alanine of any residue in a given protein sequence has been established. The method, which involves simple procedures and commonly used materials, is based upon the use of codon-based mutagenesis. A defined ratio of alanine to wild-type codon was introduced at each predetermined triplet using the "column-splitting" technique during oligonucleotide synthesis. High-throughput genetic screening of mutant libraries was facilitated by the incorporation of diagnostic restriction sites at targeted codons followed by a PCR-based screening procedure. The method was tested on a set of 13 residues located at the interface between the variable domains of a Fab fragment of an antibody. The occurrence of alanine substitutions was found to be comparable to the statistically predicted distribution. 1995 Academic Press, Inc.