In vitro recombination method
First Claim
1. An in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest, comprising:
- providing a first dsDNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule,contacting the two dsDNA molecules with;
(a) a purified 5′
exonuclease;
(b) a purified DNA polymerase; and
(c) a purified ligase,under conditions whereby;
a 3′
single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;
the two single-stranded overhangs anneal to form a gapped molecule;
the gaps are filled in by the polymerase; and
nicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.
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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.
28 Citations
17 Claims
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1. An in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest, comprising:
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providing a first dsDNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule, contacting the two dsDNA molecules with; (a) a purified 5′
exonuclease;(b) a purified DNA polymerase; and (c) a purified ligase, under conditions whereby; a 3′
single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;the two single-stranded overhangs anneal to form a gapped molecule; the gaps are filled in by the polymerase; and nicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification