Method to protect a targeted amino acid residue during random mutagenesis

Abstract:

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C

Nucleic Acids Research.2003:31(16):e91

Division of Chemistry and Chemical Engineering, California Institute of Technology, 210-41, 1200 Eas

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