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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):
7776532
Application Number:
11/502,624
Assignee:
Synthetic Genomics, Inc. (San Diego, CA)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES
C - CHEMISTRY C12 - BIOCHEMISTRY C12P - FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {
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:

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