Use of multiple recombination sites with unique specificity in recombinational cloning

• US 20040229229A1
• Filed: 08/14/2003
• Published: 11/18/2004
• Est. Priority Date: 12/10/1999
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Temple, Gary F., Hartley, James L., Brasch, Michael A., Cheo, David, Byrd, Devon R. N.
• Granted Patent: US 7,351,578 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

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.

Claims

1-142. -142. (Cancelled).

143. 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.

147. The composition of any one of claims 143-146, wherein said vectors are affixed to said solid support via covalent linkage to said support.

148. The composition of any one of claims 143-146, wherein said vectors are affixed to said solid support via non-covalent linkage to said support.

149. The composition of any one of claims 143-146, wherein said solid support comprises a material selected from the group consisting of nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polyvinyldifluoride and nylon.

150. The composition of any one of claims 143-146, wherein said vectors are affixed to said solid support in such a way as to form an array.

151. The composition of any one of claims 143-146, wherein said DNA inserts comprise at least one open reading frame.

152. The composition of any one of claims 143-146, wherein at least one of said vectors further comprises at least one recombination site.

153. The composition of claim 152, 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.

154. The composition of claim 150, wherein at least one of said vectors comprises at least one att recombination site located between said promoter and said DNA insert.

155. The composition of claim 154, wherein said DNA insert is flanked on both ends by at least one att recombination site.

144. 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.

145. 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.

146. 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.