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Title: Method for in vitro recombination

Abstract

The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to joinmore » synthetically produced sub-fragments of a gene or genome of interest.« less

Inventors:
;
Issue Date:
Research Org.:
Synthetic Genomics, Inc., San Diego, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1531645
Patent Number(s):
7,776,532
Application Number:
11/502,624
Assignee:
Synthetic Genomics, Inc. (San Diego, CA)
DOE Contract Number:  
FG02-02ER63453
Resource Type:
Patent
Resource Relation:
Patent File Date: 2006-08-11
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Gibson, Daniel Glenn, and Smith, Hamilton O. Method for in vitro recombination. United States: N. p., 2010. Web.
Gibson, Daniel Glenn, & Smith, Hamilton O. Method for in vitro recombination. United States.
Gibson, Daniel Glenn, and Smith, Hamilton O. Tue . "Method for in vitro recombination". United States. https://www.osti.gov/servlets/purl/1531645.
@article{osti_1531645,
title = {Method for in vitro recombination},
author = {Gibson, Daniel Glenn and Smith, Hamilton O.},
abstractNote = {The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2010},
month = {8}
}

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Works referenced in this record:

Process for in vitro creation of recombinant polynucleotide sequences by oriented ligation
patent, January 2006


Methods and kits for recombining nucleic acids
patent, November 2000


Directional cloning
patent, December 1991


Evolution of whole cells and organisms by recursive sequence recombination
patent, April 2002


Stimulation of enzymatic ligation of DNA by high concentrations of nonspecific polymers
patent, April 1986


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