Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites

• US 8,030,066 B2
• Filed: 12/18/2006
• Issued: 10/04/2011
• Est. Priority Date: 12/11/2000
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Leong, Louis, Brasch, Michael, Gleeson, Martin, Temple, Gray F., Madden, Knut, Byrd, Devon R., Fan, James, Chesnut, Jonathan D., Hartley, James L., Cheo, David, Carrino, John
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US11/612,445
• Publication Number:
  US 20080241889A1
• Time in Patent Office: 1,751 Days
• Field of Search: None
• US Class Current: 435/320.1
• 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 15/64 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 15/902 using homologous recombination
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/70 Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Y 599/01 Other isomerases (5.99.1)

Abstract

The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites and/or multiple topoisomerase recognition sites. 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. The invention also provides host cells comprising nucleic acid molecules of the invention or prepared according to the methods of the invention, and also provides kits comprising the compositions, host cells and nucleic acid molecules of the invention, which may be used to synthesize nucleic acid molecules according to the methods of the invention.

Claims

1. A linear vector comprising:

(a) an origin of replication,

(b) a selection marker,

(c) two att recombination sites which do not recombine with each other, wherein one of the att recombination sites has the seven base pair overlap sequence TTTGTAC and wherein the vector also contains the sequence CCCTT at a terminus.

2. The vector of claim 1, wherein the vector contains the sequence CCCTT at each terminus.

3. The vector of claim 2, wherein each of terminus of the vector is a blunt end.

4. The vector of claim 2, wherein each terminus of the vector has a 3′

overhang.

5. The vector of claim 1, wherein the sequence CCCTT is bound by a type I topoisomerase.

6. The vector of claim 5, wherein the type I topoisomerase is a type IB topoisomerase.

7. The vector of claim 6, wherein the type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.

8. The vector of claim 7, wherein the type IB topoisomerase is a vaccinia topoisomerase.

9. The vector of claim 1, wherein one of the att recombination sites has the sequence GCTTTTTTGTACAAA (SEQ ID No. 83).

10. A linear vector comprising:

(a) an origin of replication,

(b) a selection marker,

(c) two att recombination sites which do not recombine with each other, wherein one of the att recombination sites has the seven base pair overlap sequence CTTGTAC and wherein the vector also contains the sequence CCCTT at a terminus.

11. The vector of claim 10, wherein the vector contains the sequence CCCTT at each terminus.

12. The vector of claim 11, wherein each of terminus of the vector is a blunt end.

13. The vector of claim 11, wherein each terminus of the vector has a 3′

overhang.

14. The vector of claim 10, wherein the sequence CCCTT is bound by a type I topoisomerase.

15. The vector of claim 14, wherein the type I topoisomerase is a type IB topoisomerase.

16. The vector of claim 15, wherein the type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.

17. The vector of claim 16, wherein the type IB topoisomerase is a vaccinia topoisomerase.

18. The vector of claim 10, wherein one of the au recombination sites has the sequence GCTTTCTTGTACAAA (SEQ ID No. 94).