RecT or RecET cloning and subcloning method

• US 20030017594A1
• Filed: 07/22/2002
• Published: 01/23/2003
• Est. Priority Date: 07/20/2001
• Status: Abandoned Application

• Current Assignee: Muijrers P.P. Joep, Stewart A. Francis, Zhang Youming
• Original Assignee: Muijrers P.P. Joep, Stewart A. Francis, Zhang Youming
• Inventors: Francis, Stewart A., Joep, Muijrers P. P., Youming, Zhang
• Application Number:
  US10/199,478
• Publication Number:
  US 20030017594A1
• Time in Patent Office: Days
• US Class Current: 435/455
• 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

Abstract

The invention refers to an improved method for DNA cloning and subcloning using Rec T or RecET-mediated homologous recombination. Further, novel reagent kits suitable for carrying out the method are provided.

Claims

1. A method for cloning DNA molecules in cells comprising the steps of:

a) providing means for performing homologous recombination via a RecT dependent mechanism within a host cell and/or in vitro,

b) i) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule under conditions which favour homologous recombination between said first and second DNA molecules and/or

ii) contacting in vitro a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule under conditions which favour homologous recombination between said first and second DNA molecules and introducing recombined DNA molecules into said host cell, and

c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, wherein a chemically competent host cell and a RecA activity are provided.

2. The method of claim 1 wherein means are provided for performing homologous recombination via a RecET-dependent mechanism within a host cell and/or in vitro.

3. The method of claim 1 or 2, wherein the host cell is capable of expressing a recT gene and optionally a recE gene.

4. The method according to any one of claims 1-3, wherein the recE and recT genes are selected from E.coli recE and recT genes or from λ

redα and

redβ

genes.

5. The method according to any one of claims 1-4, wherein the host cell is transformed with at least one vector capable of expressing recE and/or recT genes.

6. The method of any one of claims 1-5, wherein a RecT protein and/or a RecE protein is contacted outside the host cell with the first and/or the second DNA molecule and then introduced into the host cell.

7. The method of any one of claims 1-6, wherein the host cell is capable of expressing a recA gene.

8. The method of any one of claims 1-7, wherein the host cell is transformed with a vector capable of expressing the recA gene.

9. The method of any one of claims 1-8, wherein a RecA protein is contacted outside the host cell with the first and/or the second DNA molecule and then introduced into the host cell.

10. The method of any one of claims 1-9, wherein the expression of the recE, recT and/or recA genes is under control of a regulatable promoter.

11. The method of claim 4, 5 or 10, wherein the recT gene is overexpressed versus the recE gene.

12. The method of any one of the previous claims wherein the host cell is a chemically competent gram-negative or gram-positive bacterial cell.

13. The method of claim 12, wherein the host cell is an Escherichia coli cell.

14. The method of any one of claims 1-13, wherein the host cell has been made competent by a treatment with calcium chloride, rubidium chloride, glycerol, dimethylsulfoxide, or any combination thereof.

15. The method of any one of claims 1-11, wherein the host cell is a chemically competent eukaryotic cell.

16. The method of claim 15, wherein the host cell has been made competent by treatment with calcium phosphate, DEAE dextran, or any combination thereof.

17. The method according to any one of the previous claims wherein a a RecBC inhibitor activity is provided.

18. The method according to claim 17, wherein the host cell is capable of expressing a recBC inhibitor gene.

19. The method of claim 17 or 18, wherein the recBC inhibitor gene is selected from a redy gene.

20. The method according to any one of claims 17-19, wherein the host cell is transformed with a vector expressing the recBC inhibitor gene.

21. The method according to any one of claims 17-20, wherein the expression of the recBC inhibitor gene is under control of a regulatable promoter.

22. The method according to any one of claims 17 to 21, wherein the host cell is a prokaryotic recBC+cell.

23. The method according to any one of claims 17-22, wherein the RecBC inhibitor is contacted outside the host cell with the first and/or second DNA molecule and then introduced into the host cell.

24. The method according to any one of the previous claims wherein the first DNA molecule is circular.

25. The method according to any one of the previous claims wherein the first DNA molecule is an extrachromosomal DNA molecule containing an origin of replication which is operative in the host cell.

26. The method according to claim 24 or 25, wherein the first DNA molecule is selected from plasmids, cosmids, P1 vectors, BAC vectors and PAC vectors.

27. The method according to any one of claims 1-24, wherein the first DNA molecule is a host cell chromosome.

28. The method according to any one of the previous claims wherein the second DNA molecule is linear.

29. The method according to any one of the previous claims wherein the first and/or second DNA molecules are introduced into the host cells by transformation.

30. The method according to any one of claims 1 to 29, wherein the first and second DNA molecules are introduced into the host cell simultaneously by co-transformation.

31. The method according to any one of claims 1 to 29, wherein the second DNA molecule is introduced into a host cell in which the first DNA molecule is already present.

32. A method according to any one of the previous claims wherein the first DNA molecule contains at least one target site for a site specific recombinase and wherein homologous recombination is detected by removal of said target site.

33. Use of chemically competent cells and the RecT protein, the RecA protein and optionally the RecE protein in a cloning method involving homologous recombination.

36. The method of claim 35, wherein means are provided for performing homologous recombination via a RecET-dependent mechanism.

37. The method of claim 35 or 36, wherein the host cell is capable of expressing the recT gene, the recA gene and optionally the recE gene.

40. The method of claim 39, wherein the vector has a temperature-sensitive origin.

34. Use of chemically competent cells capable of expressing the recT gene, the recA gene and optionally the recE gene as a host cell for a cloning method involving homologous recombination.

35. A method for cloning DNA molecules in cells comprising the steps of:

a) providing means for performing homologous recombination via a RecT dependent mechanism within a host cell and/or in vitro,

b) i) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule under conditions which favour homologous recombination between said first and second DNA molecules and/or

ii) contacting in vitro a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule under conditions which favour homologous recombination between said first and second DNA molecules and introducing recombined DNA molecules into said host cell, and

c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, wherein a RecA activity is provided.

38. The method of any one of claims 35-37, wherein the recE, recT and recA genes are under control of a regulatable promoter.

39. The method of any one of claims 35-38, wherein the recE, recT and recA genes are located on at least one extrachromosomal vector.

41. The method of any one of claims 35-40, wherein the RecT protein, the RecA protein and/or the RecE protein are contacted outside the host cell with the first and/or second DNA molecules and then introduced into the host cell.

42. An extrachromosomal vector system capable of expressing recT and recA genes and optionally the recE gene in a host cell.

43. Use of a vector system capable of expressing recT and recA genes and optionally the recE gene in a host cell for a cloning method involving homologous recombination.

44. A reagent kit for cloning comprising

(a) a host cell

(b) means for providing RecT and RecA activity and optionally RecE activity (i) within said host cell, e.g. by expressing recT and recA genes and optionally the recE gene in said host cell and/or (ii) in vitro, e.g. by preincubating at least one of said proteins outside said host cell with DNA molecules, and

(c) a recipient cloning vehicle capable of being replicated in said cell.

45. The reagent kit of claim 44, wherein the host cell is a chemically competent host cell.

46. The reagent kit according to claim 44 or 45, comprising a vector system capable of expressing the recT and recA genes and optionally the recE gene in the host cell.

47. The reagent kit according to any one of claims 44-46 further comprising means for introducing a RecBC inhibitor and/or a site specific recombinase into said host cell, e.g. by expressing a site specific recombinase and/or a recBC inhibitor gene in said host cell, and/or by introducing at least one of said proteins via preincubation with DNA molecules.

48. The reagent kit according to any one of claims 44-47 further comprising adaptor oligonucleotides and/or amplification primers comprising a region of homology to said recipient cloning vehicle.