Use of multiple recombination sites with unique specificity in combinational cloning

Abstract

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 a DNA insert that is operably linked at each end to at least one promoter. 
     
     
         2 . A composition comprising a solid support having two or more vectors affixed thereon in discrete, defined locations, wherein each of said vectors comprises a first copy and a second copy of a DNA insert each of which is operably linked to a promoter, wherein transcription of the first copy of said DNA insert results in the generation of a sense transcript, and wherein transcription of the second copy of said DNA insert results in the generation of an antisense transcript. 
     
     
         3 . (canceled) 
     
     
         4 . A composition comprising a solid support having at least a first vector and a second vector affixed thereon in discrete, defined locations, said first vector comprising a copy of a DNA insert operably linked to a promoter, and said second vector comprising a copy of said DNA insert operably linked to a promoter, wherein transcription of the DNA insert of said first vector results in the generation of a sense transcript, and wherein transcription of the DNA insert of said second vector results in the generation of an antisense transcript. 
     
     
         5 . The composition of any one of  claim 1 ,  2  or  4 , wherein said vectors are affixed to said solid support via covalent linkage to said support. 
     
     
         6 . The composition of any one of  claim 1 ,  2  or  4 , wherein said vectors are affixed to said solid support via non-covalent linkage to said support. 
     
     
         7 . The composition of any one of  claim 1 ,  2  or  4 , wherein said solid support comprises a material selected from the group consisting of nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polyvinyldifluoride and nylon. 
     
     
         8 . The composition of any one of  claim 1 ,  2  or  4 , wherein said vectors are affixed to said solid support in such a way as to form an array. 
     
     
         9 . The composition of any one of  claim 1 ,  2  or  4 , wherein said DNA inserts comprise at least one open reading frame. 
     
     
         10 . The composition of any one of  claim 1 ,  2  or  4 , wherein at least one of said vectors further comprises at least one recombination site. 
     
     
         11 . The composition of  claim 10 , wherein said recombination site is selected from the group consisting of an att site, a lox site, an frt site, a dif site, a psi site, a cer site, and mutants, variants and derivatives of these sites. 
     
     
         12 . The composition of  claim 8 , wherein at least one of said vectors comprises at least one att recombination site located between said promoter and said DNA insert. 
     
     
         13 . The composition of  claim 12 , wherein said DNA insert is flanked on both ends by at least one att recombination site.