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:

Gibson, Daniel Glenn; Smith, Hamilton O.

Issue Date:

Tue Aug 17 00:00:00 EDT 2010

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 {

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N15/102 - {Mutagenizing nucleic acids}
C12N15/1031 - {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
C12N15/64 - General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
C12N15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation
C12N9/1252 - {DNA-directed DNA polymerase

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 {

C12P19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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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