WO2007021944A3 - In vitro recombination method - Google Patents

In vitro recombination method Download PDF

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Publication number
WO2007021944A3
WO2007021944A3 PCT/US2006/031394 US2006031394W WO2007021944A3 WO 2007021944 A3 WO2007021944 A3 WO 2007021944A3 US 2006031394 W US2006031394 W US 2006031394W WO 2007021944 A3 WO2007021944 A3 WO 2007021944A3
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WO
WIPO (PCT)
Prior art keywords
dna
region
joining
dna molecule
sequence identity
Prior art date
Application number
PCT/US2006/031394
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French (fr)
Other versions
WO2007021944A2 (en
Inventor
Lei Young
Hamilton O Smith
Original Assignee
J Craig Venter Inst
Lei Young
Hamilton O Smith
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by J Craig Venter Inst, Lei Young, Hamilton O Smith filed Critical J Craig Venter Inst
Priority to DK06813388.3T priority Critical patent/DK1915446T3/en
Priority to CA2618699A priority patent/CA2618699C/en
Priority to EP06813388.3A priority patent/EP1915446B1/en
Publication of WO2007021944A2 publication Critical patent/WO2007021944A2/en
Publication of WO2007021944A3 publication Critical patent/WO2007021944A3/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12N9/14—Hydrolases (3)
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    • C12P19/28—N-glycosides
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    • C12Y207/07—Nucleotidyltransferases (2.7.7)
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    • C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
    • C12Y301/11003—Exodeoxyribonuclease (lambda-induced) (3.1.11.3)
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    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
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    • C12Q2522/00—Reaction characterised by the use of non-enzymatic proteins
    • C12Q2522/10—Nucleic acid binding proteins
    • C12Q2522/101—Single or double stranded nucleic acid binding proteins

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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' exonuclease; (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.
PCT/US2006/031394 2005-08-11 2006-08-11 In vitro recombination method WO2007021944A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DK06813388.3T DK1915446T3 (en) 2005-08-11 2006-08-11 IN VITRO RECOMBINATION PROCEDURE
CA2618699A CA2618699C (en) 2005-08-11 2006-08-11 In vitro recombination method
EP06813388.3A EP1915446B1 (en) 2005-08-11 2006-08-11 In vitro recombination method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US70716005P 2005-08-11 2005-08-11
US60/707,160 2005-08-11

Publications (2)

Publication Number Publication Date
WO2007021944A2 WO2007021944A2 (en) 2007-02-22
WO2007021944A3 true WO2007021944A3 (en) 2007-04-05

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PCT/US2006/031394 WO2007021944A2 (en) 2005-08-11 2006-08-11 In vitro recombination method

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US (4) US7723077B2 (en)
EP (1) EP1915446B1 (en)
CA (1) CA2618699C (en)
DK (1) DK1915446T3 (en)
MY (1) MY143596A (en)
WO (1) WO2007021944A2 (en)

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