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 - 4 . (canceled) 
     
     
         5 . The composition of  claim 1 , wherein said vectors are affixed to said solid support via covalent linkage to said support. 
     
     
         6 . (canceled) 
     
     
         7 - 13 . (canceled) 
     
     
         14 . A method of producing a population of hybrid nucleic acid molecules comprising: (a) mixing at least a first population of nucleic acid molecules comprising one or more recombination sites with at least one target nucleic acid molecule comprising one or more recombination sites; and (b) causing some or all of the nucleic acid molecules of the at least first population to recombine with all or some of the target nucleic acid molecules, thereby forming the population of hybrid nucleic acid molecules. 
     
     
         15 . The method of  claim 14 , wherein the recombination is caused by mixing the first population of nucleic acid molecules and the target nucleic acid molecule with one or more recombination proteins under conditions which favor the recombination. 
     
     
         16 . The method of  claim 15 , wherein the recombination proteins comprise one or more proteins selected from the group consisting of: (a) Cre; (b) Int; (c) IHF; (d) X is; (e) F is; (f) Hin; (g) Gin; (h) Cin; (i) Tn3 resolvase; (j) TndX; (k) XerC; and (l) XerD. 
     
     
         17 . The method of  claim 14 , further comprising mixing the first population of nucleic acid molecules and the target nucleic acid molecule with at least a second population of nucleic acid molecules comprising one or more recombination sites. 
     
     
         18 . The method of  claim 14 , wherein the recombination sites comprise one or more recombination sites selected from the group consisting of: (a) lox sites; (b) psi sites; (c) dif sites; (d) cer sites; (e) frt sites; (f) att sites; and (g) mutants, variants, and derivatives of the recombination sites of (a), (b), (c), (d), (e), or (f) which retain the ability to undergo recombination. 
     
     
         19 . The method of  claim 14 , further comprising selecting for the population of hybrid nucleic acid molecules. 
     
     
         20 . The method of  claim 14 , further comprising selecting for the population of hybrid nucleic acid molecules and against the first population of nucleic acid molecules and against the target nucleic acid molecules. 
     
     
         21 . The method of  claim 20 , further comprising selecting against cointegrate molecules and byproduct molecules. 
     
     
         22 . A method for targeting or mutating a target gene or nucleotide sequence comprising: (a) obtaining at least one first nucleic acid molecule comprising one or more recombination sites and one or more selectable markers, wherein the first nucleic acid molecule comprises one or more nucleotide sequences homologous to the target gene or nucleotide sequence; and (b) contacting the first nucleic acid molecule with one or more target genes or nucleotide sequences under conditions sufficient to cause homologous recombination at one or more sites between the target gene or nucleotide sequence and the first nucleic acid molecule, thereby causing insertion of all or a portion of the first nucleic acid molecule within the target gene or nucleotide sequence. 
     
     
         23 . The method of  claim 22 , wherein the target gene or nucleotide sequence is inactivated. 
     
     
         24 . The method of  claim 22 , further comprising selecting for a host cell containing the target gene or nucleotide sequence. 
     
     
         25 . A method of joining n nucleic acid segments, wherein n is an integer greater than 2, comprising: (a) providing a 1 st  through an n th  nucleic acid segment, each segment flanked by two recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the i th  segment, n i  reacts with one of the recombination sites flanking the n i−1   th  segment and the other recombination site flanking the i th  segment reacts with one of the recombination sites flanking the n i+1   th  segment; and (b) contacting the segments with one or more recombination proteins under conditions causing the segments to join. 
     
     
         26 . The method of  claim 25 , wherein the recombination proteins comprise one or more proteins selected from the group consisting of: (a) Cre; (b) Int; (c) IHF; (d) X is; (e) Fis; (f) Hin; (g) Gin; (h) Cin; (i) Tn3 resolvase; (j) TndX; (k) XerC; and (l) XerD. 
     
     
         27 . The method of  claim 25 , wherein the recombination sites which recombine with each other comprise att sites having identical seven base pair overlap regions. 
     
     
         28 . The method of  claim 25 , further comprising inserting the nucleic acid segments joined in step (b) into a vector. 
     
     
         29 . The method of  claim 25 , wherein the joined nucleic acid segments undergo intramolecular recombination to form a circular molecule. 
     
     
         30 . The method of  claim 25 , wherein one or more of the nucleic acid segments encodes a selectable marker.