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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];  [3];  [4]
  1. Pasadena, CA
  2. Midland, MI
  3. San Diego, CA
  4. Sunnyvale, CA
Publication Date:
Research Org.:
California Institute of Technology (CalTech), Pasadena, CA (United States)
OSTI Identifier:
873411
Patent Number(s):
US 6153410
Assignee:
California Institute of Technology (Pasadena, CA)
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 H, and Giver, Lorraine J. Recombination of polynucleotide sequences using random or defined primers. United States: N. p., 2000. Web.
Arnold, Frances H, Shao, Zhixin, Affholter, Joseph A, Zhao, Huimin H, & Giver, Lorraine J. Recombination of polynucleotide sequences using random or defined primers. United States.
Arnold, Frances H, Shao, Zhixin, Affholter, Joseph A, Zhao, Huimin H, and Giver, Lorraine J. 2000. "Recombination of polynucleotide sequences using random or defined primers". United States. https://www.osti.gov/servlets/purl/873411.
@article{osti_873411,
title = {Recombination of polynucleotide sequences using random or defined primers},
author = {Arnold, Frances H and Shao, Zhixin and Affholter, Joseph A and Zhao, Huimin H 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 = {},
url = {https://www.osti.gov/biblio/873411}, journal = {},
number = ,
volume = ,
place = {United States},
year = {Sat Jan 01 00:00:00 EST 2000},
month = {Sat Jan 01 00:00:00 EST 2000}
}

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