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Method to protect a targeted amino acid residue during random mutagenesis
 

Summary: Method to protect a targeted amino acid residue
during random mutagenesis
Daisuke Umeno, Kaori Hiraga and Frances H. Arnold*
Division of Chemistry and Chemical Engineering, California Institute of Technology, 210-41, 1200 East California
Boulevard, Pasadena, CA 91125, USA
Received April 22, 2003; Revised and Accepted June 12, 2003
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 sequen-
ces Żanking 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

  

Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology

 

Collections: Chemistry; Biology and Medicine