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Recombination of polynucleotide sequences using random or defined primers

United States Patent

January 23, 2001
View the Complete Patent at the US Patent & Trademark Office
A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.
Arnold; Frances H. (Pasadena, CA), Shao; Zhixin (Pasadena, CA), Affholter; Joseph A. (Midland, MI), Zhao; Huimin (Pasadena, CA), Giver; Lorraine J. (Pasadena, CA)
California Institute of Technology (Pasadena, CA)
09/ 353,556
July 14, 1999
The U.S. Government has certain rights in this invention pursuant to Grant No. DE-FG02-93-CH10578 awarded by the Department of Energy and Grant No. N00014-96-1-0340 awarded by the Office of Naval Research.