Recombinational cloning using engineered recombination sites

• US 7,223,576 B2
• Filed: 03/10/2004
• Issued: 05/29/2007
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L., Brasch, Michael A.
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/796,868
• Publication Number:
  US 20040219673A1
• Time in Patent Office: 1,175 Days
• Field of Search: 435/6, 435/320.1, 435/462, 435/183, 536/23.1, 536/24.1
• US Class Current: 435/183
• 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 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

Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Claims

1. A composition comprising at least two different isolated recombination proteins, at least one first nucleic acid molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, and at least one second nucleic acid molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other.

2. The composition of claim 1, wherein said first nucleic acid molecule is a circular nucleic acid molecule.

3. The composition of claim 1, wherein said first nucleic acid molecule is a linear nucleic acid molecule.

4. The composition of claim 1, wherein said second nucleic acid molecule is a circular nucleic acid molecule.

5. The composition of claim 1, wherein said second nucleic acid molecule is a linear nucleic acid molecule.

6. The composition of claim 1, wherein said second nucleic acid molecule further comprises (a) at least one toxic gene and (b) at least one selectable marker, wherein said at least one toxic gene and said at least one selectable marker are on different nucleic acid segments, the nucleic acid segments being separated from each other by at least one recombination site.

7. The composition of claim 1, wherein said recombination proteins are selected from the group consisting of γ

δ

, Tn3 resolvase, Hin, Gin, Cin and Flp.

8. The composition of claim 1, wherein said recombination proteins are selected from the group consisting of Int, IHF, Xis and Cre.

9. The composition of claim 1, wherein said recombination proteins are selected from the group consisting of Int, IHF and Xis.

10. The composition of claim 1, wherein at least one of said recombination proteins is Int.

11. The composition of claim 1, wherein at least one of said recombination proteins is encoded by an organism selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.

12. The composition of claim 1, wherein at least one of said recombination proteins is encoded by bacteriophage lambda.

13. The composition of claim 1, wherein at least one of said recombination proteins is encoded by Bacillus thuringiensis.

14. The composition of claim 1, wherein said composition comprises Int and IHF.

15. The composition of claim 14, wherein said composition further comprises Xis.

16. The composition of claim 1, wherein said composition comprises at least three different recombination proteins.

17. A kit comprising at least two different isolated recombination proteins, at least one first nucleic acid molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, and at least one second nucleic acid molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other.

18. The kit of claim 17, wherein said at least one first nucleic acid molecule is a circular nucleic acid molecule.

19. The kit of claim 17, wherein said at least one first nucleic acid molecule is a linear nucleic acid molecule.

20. The kit of claim 17, wherein said at least one second nucleic acid molecule is a circular nucleic acid molecule.

21. The kit of claim 17, wherein said at least one second nucleic acid molecule is a linear nucleic acid molecule.

22. The kit of claim 17, wherein said at least one second nucleic acid molecule further comprises (a) at least one toxic gene and (b) at least one selectable marker, wherein said at least one toxic gene and said at least one selectable marker are on different nucleic acid segments, the nucleic acid segments being separated from each other by at least one recombination site.

23. The kit of claim 17, wherein said recombination proteins are selected from the group consisting of γ

δ

, Tn3 resolvase, Hin, Gin, Cin, and Flp.

24. The kit of claim 17, wherein said recombination proteins are selected from the group consisting of Int, IHF, Xis and Cre.

25. The kit of claim 17, wherein said recombination proteins are selected from the group consisting of Int, IHF and Xis.

26. The kit of claim 17, wherein at least one of said recombination proteins is Int.

27. The kit of claim 17, wherein at least one of said recombination proteins is encoded by an organism selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.

28. The kit of claim 17, wherein at least one of said recombination proteins is encoded by bacteriophage lambda.

29. The kit of claim 17, wherein at least one of said recombination proteins is encoded by Bacillus thuringiensis.

30. The kit of claim 17, wherein said kit comprises Int and IHF.

31. The kit of claim 30, wherein said kit further comprises Xis.

32. The kit of claim 17, wherein said kit comprises at least three different recombination proteins.