BACKGROUND:  Random mutagenesis is frequently used to study the activity, structure and/or function of a protein. Current methods of mutagenesis include: UV, chemical, site-directed, alanine scanning, cysteine scanning, error-prone PCR, and gene shuffling. Common drawbacks using these systems are the inability to control the number of mutations at each round of mutagenesis, and a low mutation rate with high wild type background.

INNOVATION:  Researchers at UCLA have developed a novel mutagenesis method using transposons. Transposon directed base-exchange mutagenesis (TDEM) can substitute up to 11 bases of the target gene with a predetermined sequence of any length. In TDEM, the mutation site is randomly determined by transposon integration, however, the type of mutation (deletion, substitution, insertion) is directed by specially designed mutation inserts. As a proof of principle, the investigators used TDEM to substitute three random bases of the target gene with three bases determined by the mutation insert. One round of TDEM generated a change at a single site of the target gene with 100% efficiency. Repeated rounds of TDEM can be used to accumulate predetermined mutations within the target gene, which is useful in library construction with extremely low wild-type background. Additionally, researchers have developed a vector used during mutagenesis that ensures mutants containing a frame-shift or stop codon are eliminated.

POTENTIAL APPLICATIONS 

• Introduce a controlled number, length and sequence of mutations at a single random position during each round of mutagenesis
• Studying protein evolution, activity, structure and/or function
• Investigating directed evolution or affinity maturation
• Alanine or cysteine scanning mutagenesis
• Stepwise mutation of proteins to gradually achieve desired phenotype
• Mutant library construction with extremely low wild-type background

ADVANTAGES

• Significantly improved mutagenesis system for studying protein evolution
• Controlled number, length and sequence of mutations
• Substitutions, deletions or insertions can be introduced
• Higher mutation rate than other available methods
• Vector available to ensure in-frame mutations
• Extremely low wild-type background
• Ability to generate many types of mutant libraries
• Faster than currently available methodologies

DEVELOPMENT-TO-DATE:  The novel mutagenesis system has been fully tested using LacZα and CcdB.

Reference: UCLA Case No. 2008-618