Use of multiple recombination sites with unique specificity in recombinational cloning
View Patent ↗The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites with unique specificity. 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. Such molecules and/or compounds or combinations of such molecules and/or compounds can also be bound through recombination to various structures or supports according to the invention.
1. A composition comprising a solid support having two or more vectors affixed thereon in discrete, defined locations, wherein each of said vectors comprises two copies of a DNA insert that are oriented in opposite orientation relative to each other, and wherein said two insert copies are operably linked to a single promoter.
2. The composition of claim 1 , wherein said vectors are affixed to said solid support via covalent linkage to said support.
3. The composition of claim 1 , wherein said vectors are affixed to said solid support via non-covalent linkage to said support.
4. The composition of claim 1 , wherein said solid support comprises a material selected from the group consisting of nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polyvinyldifluoride and nylon.
5. The composition of claim 1 , wherein said vectors are affixed to said solid support in such a way as to form an array.
6. The composition of claim 1 , wherein said DNA inserts comprise at least one open reading frame.
7. The composition of claim 1 , wherein at least one of said vectors further comprises at least one recombination site.
8. The composition of claim 7 , wherein said recombination site is selected from the group consisting of an att site, a lox site, an Irt site, a dif site, a psi site, a cer site, and mutants, variants and derivatives of these sites.
9. The composition of claim 5 , wherein at least one of said vectors comprises at least one att recombination site located between said promoter and said DNA insert.
10. The composition of claim 9 , wherein said DNA insert is flanked on both ends by at least one att recombination site.
11. The composition of claim 1 , wherein at least one of said first vector and said second vector comprise an origin of replication.
12. The composition of claim 1 , wherein said DNA inserts comprise at least one selectable marker.
13. The composition of claim 1 , wherein said DNA inserts encode an RNA molecule.
14. The composition of claim 1 , wherein said promoter is a regulatable promoter.
15. The composition of claim 1 , wherein said first vector or said second vector or said DNA inserts comprise one or more DNA sequences homologous with DNA sequences in a target nucleic acid molecule such that homologous recombination may occur between said first vector or said second vector or said DNA inserts and said target nucleic acid molecule.
16. The composition of claim 15 , wherein said target molecule is inside of a cell.
17. The composition of claim 1 , wherein said first vector or said second vector is a viral vector.
18. The composition of claim 17 , wherein said viral vector is a retroviral vector.