Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
First Claim
1. An isolated nucleic acid molecule comprising:
- (a) one or more recombination sites; and
(b) one or more topoisomerase recognition sites and/or one or more topoisomerases.
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Abstract
The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites and/or multiple topoisomerase recognition sites. The methods permit the simultaneous cloning of two or more different nucleic acid molecules. In some embodiments the molecules are fused together while in other embodiments the molecules are inserted into distinct sites in a vector. The invention also generally provides for linking or joining through recombination a number of molecules and/or compounds (e.g., chemical compounds, drugs, proteins or peptides, lipids, nucleic acids, carbohydrates, etc.) which may be the same or different. The invention also provides host cells comprising nucleic acid molecules of the invention or prepared according to the methods of the invention, and also provides kits comprising the compositions, host cells and nucleic acid molecules of the invention, which may be used to synthesize nucleic acid molecules according to the methods of the invention.
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Citations
45 Claims
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1. An isolated nucleic acid molecule comprising:
- (a) one or more recombination sites; and
(b) one or more topoisomerase recognition sites and/or one or more topoisomerases. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 44, 45)
- (a) one or more recombination sites; and
- 12. A vector selected from the group consisting of pcDNAGW-DT(sc), pENTR-DT(sc), pcDNA-DEST41, pENTRID-TOPO, pENTR/SD/D-TOPO, pcDNA3.2/V5/GWD-TOPO and pcDNA6.2/V5/GWD-TOPO.
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16. An in vitro method of cloning a nucleic acid molecule comprising:
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(a) obtaining a first nucleic acid molecule to be cloned;
(b) mixing said first nucleic acid molecule to be cloned in vitro with a second nucleic acid molecule comprising at least a first topoisomerase recognition site flanked by at least a first recombination site, and at least a second topoisomerase recognition site flanked by at least a second recombation site, wherein said first and second recombination sites do not recombine with each other, and at least one topoisomerase; and
(c) incubating said mixture under conditions such that said first nucleic acid molecule to be cloned is inserted into said second nucleic acid molecule between said first and second topoisomerase recognition sites, thereby producing a first product molecule comprising said first nucleic acid molecule to be cloned between said first and second recombination sites. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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28. The method of 17, wherein said vector is an expression vector.
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29. The method of 23, wherein said vector is an expression vector.
Specification