Umeno, Daisuke and Hiraga, Kaori and Arnold, Frances H. (2003) Method to protect a targeted amino acid residue during random mutagenesis. Nucleic Acids Research, 31 (16). e91. ISSN 0305-1048 http://resolver.caltech.edu/CaltechAUTHORS:UMEnar03
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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 C30 carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C40 synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.
|Additional Information:||© 2003 Oxford University Press Received April 22, 2003; Revised and Accepted June 12, 2003 This research was supported by the U.S. National Science Foundation (BES-0118565) and Maxygen, Inc. K.H. thanks the Japanese Society for Promotion of Science for financial support.|
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|Deposited On:||08 Oct 2005|
|Last Modified:||26 Dec 2012 08:41|
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