Enhanced in vitro recombinational cloning of using ribosomal proteins

• US 6,964,861 B1
• Filed: 11/12/1999
• Issued: 11/15/2005
• Est. Priority Date: 11/13/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Gerard, Gary F., Flynn, Elizabeth, Hu, A-Li W.
• Primary Examiner(s): LEFFERS JR, GERALD G
• Application Number:
  US09/438,358
• Time in Patent Office: 2,195 Days
• Field of Search: 435/91.1, 435/91.4, 435/91.42, 435/69.1, 435/320.1, 435/252.3, 435/325, 435/6, 536/23.1, 536/24.2
• US Class Current: 435/91.1
• CPC Class Codes: C07K 14/245 Escherichia (G)
• 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 15/66 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 present invention relates generally to compositions and methods for enhancing recombinational cloning of nucleic acid molecules. In particular, the invention relates to compositions comprising one or more ribosomal proteins and one or more additional protein components required for recombinational cloning. More particularly, the invention relates to such compositions wherein the ribosomal proteins are one or more E. coli ribosomal proteins, still more particularly wherein the ribosomal proteins are selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L20, L21, and L23 through L34, and most particularly S20, L27, and S15. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments. The invention also relates to isolated nucleic acid molecules produced by the methods of the invention, to vectors comprising such nucleic acid molecules, and to host cells comprising such nucleic acid molecules and vectors.

Claims

1. A method for cloning or subcloning one or more desired nucleic acid molecules comprising

(a) forming a mixture by combining in vitro

(i) one or more first nucleic acid molecules comprising one or more desired nucleic acid segments flanked by at least two recombination sites, wherein said recombination sites do not recombine with each other;

(ii) one or more second nucleic acid molecules each comprising at least two recombination sites, wherein said recombination sites do not recombine with each other;

(iii) at least one recombination protein; and

(iv) at least one ribosomal protein that is present in an amount sufficient to enhance recombinational cloning; and

(b) incubating said mixture under conditions sufficient to transfer one or more of said desired segments into one or more of said second nucleic acid molecules, thereby producing one or more desired third nucleic acid molecules.

2. The method of claim 1, further comprising:

(c) forming a mixture by combining in vitro

(i) one or more of said third molecules comprising said desired segments flanked by two or more recombination sites, wherein said recombination sites do not recombine with each other;

(ii) one or more different fourth nucleic acid molecules each comprising two or more recombination sites, wherein said recombination sites do not recombine with each other;

(iii) at least one recombination protein; and

(iv) at least one ribosomal protein that is present in an amount sufficient to enhance recombinational cloning; and

(d) incubating said mixture under conditions sufficient to transfer one or more of said desired segments into one or more different fourth nucleic acid molecules, thereby producing one or more different fifth nucleic acid molecules.

3. The method of claim 1, wherein said ribosomal protein is a prokaryotic ribosomal protein.

4. The method of claim 2, wherein said ribosomal protein is a prokaryotic ribosomal protein.

5. The method of claim 1, further comprising incubating said different third nucleic acid molecules with one or more different fourth nucleic acid molecules under conditions sufficient to transfer one or more of said desired segments into said different fourth nucleic acid molecules.

8. The method of claim 1, wherein said ribosomal protein is an Escherichia coli ribosomal protein.

9. The method of claim 1, wherein said ribosomal protein is a basic ribosomal protein.

10. The method of claim 1, wherein said ribosomal protein has a molecular weight of less than about 14 kilodaltons.

11. The method of claim 8, wherein said E. coli ribosomal protein is selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34.

12. The method of claim 8, wherein said ribosomal protein is S20.

13. The method of claim 8, wherein said ribosomal protein is L27.

14. The method of claim 8, wherein said ribosomal protein is S15.

16. The method of claim 1, wherein said recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF, FIS and HU, and combinations thereof.

17. The method of claim 1, wherein said recombination protein is Int.

51. The method of claim 1, wherein said ribosomal protein is a recombinant ribosomal protein.

55. The method of claim 1, wherein said recombination protein is a recombinant recombination protein.

59. The method of any one of claims 1, 2, 6, 18 and 27, wherein said at least one recombination protein is at least one isolated Int protein and at least one isolated IHF protein.

60. The method of any one of claims 1, 2, 6, 18 and 27, wherein said at least one recombination protein is at least one isolated Int protein, at least one isolated IHF protein and at least one isolated Xis protein.

61. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises at least one isolated FIS protein.

62. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises spermidine.

63. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises Tris-HCl.

64. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises ethylenediamine tetracetic acid (EDTA).

65. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises bovine serum albumin (BSA).

66. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture further comprises at least one additional isolated recombination protein selected from the group consisting of a Cre protein, an FLP protein, a γ

δ

protein, a Tn3 resolvase protein, a Hin protein, a Gin protein, and a Cin protein.

67. The method of any one of claims 1, 2, 6, 18 and 27, wherein said recombination protein is at least one isolated Cre protein.

68. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture comprises at least one isolated Int protein, at least one isolated IHF protein, spermidine, Tris-HCl, EDTA and BSA.

69. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture comprises at least one isolated Int protein, at least one isolated IHF protein, at least one isolated Xis protein, spermidine, Tris-HCl, EDTA and BSA.

70. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture comprises at least one isolated Int protein, at least one isolated IHF protein and spermidine.

71. The method of any one of claims 1, 2, 6, 18 and 27, wherein said mixture comprises at least one isolated Int protein, at least one isolated IHF protein, at least one isolated Xis protein and spermidine.

72. The method of any one of claims 1, 2, 6, 18 and 27, wherein said first or second nucleic acid molecule is an Insert Donor nucleic acid molecule.

73. The method of any one of claims 1, 2, 6, 18 and 27, wherein said first or second nucleic acid molecule is a Vector Donor nucleic acid molecule.

74. The method of claim 2, wherein said fourth nucleic acid molecule is a Vector Donor nucleic acid molecule.

6. A method for cloning or subcloning desired nucleic acid molecules comprising:

(a) forming a mixture by combining in vitro

(i) one or more first nucleic acid molecules comprising one or more nucleic acid segments flanked by two or more recombination sites, wherein said recombination sites do not recombine with each other;

(ii) two or more different second nucleic acid molecules each comprising two or more recombination sites, wherein said recombination sites do not recombine with each other;

(iii) at least one recombination protein; and

(iv) at least one ribosomal protein that is present in an amount sufficient to enhance recombinational cloning; and

(b) incubating said mixture under conditions sufficient to transfer one or more of said desired segments into said different second nucleic acid molecules, thereby producing two or more different third nucleic acid molecules.

7. The method of claim 6, wherein said ribosomal protein is a prokaryotic ribosomal protein.

15. The method of claim 6, wherein said recombination protein is a prokaryotic recombination protein.

52. The method of claim 6, wherein said ribosomal protein is a recombinant ribosomal protein.

56. The method of claim 6, wherein said recombination protein is a recombinant recombination protein.

18. A method for enhancement of recombinational cloning of one or more desired nucleic acid molecules comprising:

(a) forming a mixture by mixing in vitro one or more desired first nucleic acid molecules with one or more second nucleic acid molecules and with at least one ribosomal protein that is present in an amount sufficient to enhance recombinational cloning and an effective amount of at least one recombination protein; and

(b) incubating said mixture under conditions sufficient to transfer said one or more desired first nucleic acid molecules into one or more of said second nucleic acid molecules.

19. The method of claim 18, wherein said desired nucleic acid molecules are obtained from genomic DNA.

20. The method of claim 18, wherein said desired nucleic acid molecules are obtained from cDNA.

21. The method of claim 18, wherein said desired nucleic acid molecules are produced by chemical synthesis.

22. The method of claim 18, wherein said desired nucleic acid molecules are produced by amplification.

23. The method of claim 18, wherein said vector is a prokaryotic or eukaryotic vector.

24. The method of claim 23, wherein said eukaryotic vector replicates in yeast cells, plant cells, fish cells, eukaryotic cells, mammalian cells, or insect cells.

25. The method of claim 18, wherein said prokaryotic vector replicates in bacteria of the genera Escherichia, Salmonella, Bacillus, Streptomyces or Pseudomonas.

26. The method of claim 25, wherein said prokaryotic vector replicates in E. coli.

39. The method of claim 18, wherein said ribosomal protein is a prokaryotic ribosomal protein.

40. The method of claim 18, wherein said ribosomal protein is an Escherichia coli ribosomal protein.

41. The method of claim 18, wherein said ribosomal protein is a basic ribosomal protein.

42. The method of claim 18, wherein said ribosomal protein has a molecular weight of less than about 14 kilodaltons.

43. The method of claim 40, wherein said E. coli ribosomal protein is selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34.

44. The method of claim 40, wherein said E. coli ribosomal protein is S20.

45. The method of claim 40, wherein said E. coli ribosomal protein is L27.

46. The method of claim 40, wherein said E. coli ribosomal protein is S15.

47. The method of claim 18, wherein said recombination protein is a eukaryotic recombination protein.

48. The method of claim 18, wherein said recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF and HU, and combinations thereof.

49. The method of claim 18, wherein said recombination protein is Int.

50. The method of claim 18, wherein said composition further comprises one or more nucleic acid molecules selected from the group consisting of one or more Insert Donor molecules, one or more Vector Donor molecules, one or more Cointegrate molecules, one or more Product molecules and one or more Byproduct molecules.

53. The method of claim 18, wherein said ribosomal protein is a recombinant ribosomal protein.

57. The method of claim 18, wherein said recombination protein is a recombinant recombination protein.

27. A method for enhancement of recombinational cloning, comprising contacting at least a first nucleic acid molecule and at least a second nucleic acid molecule, each comprising at least one recombination site, in vitro with one or more ribosomal proteins that are present in an amount sufficient to enhance recombinational cloning and with one or more recombination proteins to form a mixture, and incubating said mixture under conditions favoring the production of at least one product nucleic acid molecule.

28. The method of claim 27, wherein said ribosomal protein is a prokaryotic ribosomal protein.

29. The method of claim 27, wherein said ribosomal protein is an Escherichia coli ribosomal protein.

30. The method of claim 27, wherein said ribosomal protein is a basic ribosomal protein.

31. The method of claim 27, wherein said ribosomal protein has a molecular weight of less than about 14 kilodaltons.

32. The method of claim 29, wherein said E. coli ribosomal protein is selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34.

33. The method of claim 29, wherein said ribosomal protein is S20.

34. The method of claim 29, wherein said ribosomal protein is L27.

35. The method of claim 29, wherein said ribosomal protein is S15.

36. The method of claim 27, wherein said recombination protein is a prokaryotic recombination protein.

37. The method of claim 27, wherein said recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF, FIS and HU, and combinations thereof.

38. The method of claim 27, wherein said recombination protein is Int.

54. The method of claim 27, wherein said ribosomal protein is a recombinant ribosomal protein.

58. The method of claim 27, wherein said recombination protein is a recombinant recombination protein.