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Title: Recombination of polynucleotide sequences using random or defined primers

Abstract

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.

Inventors:
 [1];  [1];  [2];  [1];  [1]
  1. Pasadena, CA
  2. Midland, MI
Issue Date:
Research Org.:
California Institute of Technology (CalTech), Pasadena, CA (United States)
OSTI Identifier:
873508
Patent Number(s):
6177263
Assignee:
California Institute of Technology (Pasadena, CA)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES
DOE Contract Number:  
FG02-93CH10578
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
recombination; polynucleotide; sequences; random; defined; primers; method; vitro; mutagenesis; based; polymerase-catalyzed; extension; primer; oligonucleotides; disclosed; involves; priming; template; random-sequences; defined-sequence; generate; pool; dna; fragments; level; mutations; subjected; denaturization; followed; annealing; enzyme-catalyzed; polymerization; procedure; repeated; sufficient; times; produce; full-length; genes; comprise; mutants; original; polynucleotides; amplified; polymerase; chain; reaction; cloned; vector; expression; encoded; proteins; chain reaction; nucleotide sequences; nucleotide sequence; method involves; dna fragments; polymerase chain; polynucleotide sequences; dna fragment; sequences based; method involve; vitro mutagenesis; polymerase-catalyzed extension; primer oligonucleotides; defined primers; /435/

Citation Formats

Arnold, Frances H, Shao, Zhixin, Affholter, Joseph A, Zhao, Huimin, and Giver, Lorraine J. Recombination of polynucleotide sequences using random or defined primers. United States: N. p., 2001. Web.
Arnold, Frances H, Shao, Zhixin, Affholter, Joseph A, Zhao, Huimin, & Giver, Lorraine J. Recombination of polynucleotide sequences using random or defined primers. United States.
Arnold, Frances H, Shao, Zhixin, Affholter, Joseph A, Zhao, Huimin, and Giver, Lorraine J. Mon . "Recombination of polynucleotide sequences using random or defined primers". United States. https://www.osti.gov/servlets/purl/873508.
@article{osti_873508,
title = {Recombination of polynucleotide sequences using random or defined primers},
author = {Arnold, Frances H and Shao, Zhixin and Affholter, Joseph A and Zhao, Huimin and Giver, Lorraine J},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2001},
month = {1}
}

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