Title: In vitro recombination method

Abstract

The present invention relates, e.g., to 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 contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

Inventors:

Young, Lei; Smith, Hamilton O.; Gibson, Daniel Glenn

Issue Date:

Tue May 25 00:00:00 EDT 2010

Research Org.:

Synthetic Genomics, Inc., San Diego, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1531636

Patent Number(s):

7723077

Application Number:

11/502,746

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
C12N15/64 - 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
C12N15/902 - {using homologous recombination}
C12N9/1252 - {DNA-directed DNA polymerase
C12N9/22 - Ribonucleases {RNAses, DNAses
C12N9/93 - {Ligases

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

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/62 - involving uric acid
C12Q1/686 - Polymerase chain reaction [PCR]
C12Q1/6862 - Ligase chain reaction [LCR]
C12Q2521/501 - Ligase
C12Q2522/101 - Single or double stranded nucleic acid binding proteins

C - CHEMISTRY C12 - BIOCHEMISTRY C12Y - ENZYMES

C12Y207/07007 - DNA-directed DNA polymerase
C12Y301/11003 - Exodeoxyribonuclease
C12Y605/01001 - DNA ligase

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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