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 - 13 . (canceled) 
     
     
         14 . A method of cloning at least one nucleic acid molecule comprising: (a) providing a first population of nucleic acid molecules wherein all or a portion of such molecules are flanked by a first and a second recombination site; (b) providing at least one nucleic acid segment flanked by a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site; (c) conducting a recombination reaction such that all or a portion of the nucleic acid molecules in the population is recombined with the segment to form a second population of nucleic acid molecules; and (d) cloning the second population of nucleic acid molecules. 
     
     
         15 . The method of  claim 14 , wherein second population of nucleic acid molecules encodes a fusion protein. 
     
     
         16 . The method of  claim 14 , wherein the nucleic acid segment encodes a polypeptide selected from the group consisting of: (a) the Fc portion of an immunoglobin; (b) β-glucuronidase; (c) a fluorescent protein; (d) a purification tag; and (e) an epitope tag. 
     
     
         17 . The method of  claim 16 , wherein the nucleic acid segment encodes a fluorescent protein selected from the group consisting of: (a) green fluorescent protein; (b) yellow fluorescent protein; (c) red fluorescent protein; and (d) cyan fluorescent protein. 
     
     
         18 . The method of  claim 16 , wherein the nucleic acid segment encodes a purification tag selected from the group consisting of: (a) an epitope tag; (b) maltose binding protein; (c) a six histidine tag; and (d) glutathione S-transferase. 
     
     
         19 . A nucleic acid molecule produced by the method of  claim 14 . 
     
     
         20 . A method of synthesizing a protein comprising: (a) providing a nucleic acid molecule comprising a coding sequence followed by a stop codon, wherein the nucleic acid molecule is flanked by at least one recombination site; (b) providing a vector comprising at least one recombination site and a coding sequence; (c) causing recombination such that the nucleic acid molecule is inserted into the vector to produce a modified vector with the two coding sequences connected in frame; (d) transforming a host cell which expresses a suppressor tRNA with the modified vector; and (e) causing expression of the two coding sequences such that a fusion protein encoded by at least a portion of both of the coding sequences is produced, wherein either the nucleic acid molecule or the vector comprises at least one suppressible stop codon. 
     
     
         21 . The method according to  claim 20 , wherein the stop codon is selected from the group consisting of amber, opal and ochre codons. 
     
     
         22 . The method according to  claim 20 , wherein the vector comprises a gene which encodes at least one suppressor tRNA molecule. 
     
     
         23 . The method according to  claim 20 , wherein the chromosome of the host cell comprises a gene which encodes at least one suppressor tRNA molecule. 
     
     
         24 . The method according to  claim 20 , further comprising the steps of transforming the host cell with a nucleic acid molecule comprising a gene which encodes at least one suppressor tRNA molecule. 
     
     
         25 . The method according to  claim 20 , wherein the fusion protein comprises an N- or C-terminal tag encoded by at least a portion of the vector. 
     
     
         26 . The method according to  claim 25 , wherein the tag is selected from the group consisting of: (a) glutathione S-transferase; (b) .beta.-glucuronidase; (c) green fluorescent protein; (d) yellow fluorescent protein; (e) red fluorescent protein; (f) cyan fluorescent protein; (g) maltose binding protein; (h) a six histidine tag; and (i) an epitope tag. 
     
     
         27 . A kit for joining, deleting, or replacing nucleic acid segments, the kit comprising (1) one or more recombination proteins or a composition comprising one or more recombination proteins, (2) at least one nucleic acid molecule comprising one or more recombination sites having at least two different recombination specificities, and (3) one or more components selected from the group consisting of: (a) nucleic acid molecules comprising additional recombination sites; (b) one or more enzymes having ligase activity; (c) one or more enzymes having polymerase activity; (d) one or more enzymes having reverse transcriptase activity; (e) one or more enzymes having restriction endonuclease activity; (f) one or more primers; (g) one or more nucleic acid libraries; (h) one or more supports; (i) one or more buffers; (j) one or more detergents or solutions containing detergents; (k) one or more nucleotides; (l) one or more terminating agents; (m) one or more transfection reagents; (n) one or more host cells; and (o) instructions for using the kit components. 
     
     
         28 . The kit of  claim 27 , wherein the recombination sites having at least three different recombination specificities each comprising att sites with different seven base pair overlap regions. 
     
     
         29 . The kit of  claim 27 , wherein the composition comprising one or more recombination proteins is capable of catalyzing recombination between att sites. 
     
     
         30 . The kit of  claim 27 , wherein the composition comprising one or more recombination proteins capable of catalyzing a BP reaction, an LR reaction, or both BP and LR reactions.