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

• US 7,244,560 B2
• Filed: 12/07/2001
• Issued: 07/17/2007
• Est. Priority Date: 05/21/2000
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Fan, James, Leong, Louis, Temple, Gary F., Hartley, James L., Brasch, Michael A., Cheo, David, Gleeson, Martin, Madden, Knut, Carrino, John, Byrd, Devon R.N., Chestnut, Jonathan D.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US10/005,876
• Publication Number:
  US 20030186233A1
• Time in Patent Office: 2,048 Days
• Field of Search: 435/325.1, 435/252.33, 435/6, 435/91, 435/42, 435/320.1, 536/23.1, 536/24
• US Class Current: 435/6.18
• 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 9/90 Isomerases (5.)

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. An isolated, linear nucleic acid molecule comprising, in order:

(a) at least one first topoisomerase recognition site;

(b) at least one first recombination site;

(c) at least one nucleic acid segment;

(d) at least one second recombination site; and

(e) at least one second topoisomerase recognition site, wherein said first topoisomerase recognition site and said first recombination site are separated from each other by about 0 to about 100 nucleotides.

2. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are the same.

3. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are different.

4. The nucleic acid molecule of claim 1, wherein the separation between said first topoisomerase recognition site and said first recombination site is selected from the group consisting of about 0 to about 50 nucleotides;

about 0 to about 30 nucleotides;

about 0 to about 20 nucleotides; and

about 0 to about 10 nucleotides.

5. The nucleic acid molecule of claim 1, wherein the separation between said first topoisomerase recognition site and said first recombination site is about 0 to about 50 nucleotides.

6. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are selected from the group consisting of:

(a) attB sites;

(b) attP sites;

(c) attL sites;

(d) attR sites;

(e) lox sites;

(f) psi sites;

(g) dif sites;

(h) cer sites; and

(i) frt sites.

7. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are lox sites.

8. The nucleic acid molecule of claim 1, wherein said at least one nucleic acid segment comprises at least one open reading frame.

9. The nucleic acid molecule of claim 8, wherein said open reading frame encodes an antibiotic resistance gene.

10. The nucleic acid molecule of claim 1, wherein said first and/or said second topoisomerase recognition sites are recognized and bound by a type I topoisomerase.

11. The nucleic acid molecule of claim 10, wherein said type I topoisomerase is a type IB topoisomerase.

12. The nucleic acid molecule of claim 11, wherein said type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.

13. The nucleic acid molecule of claim 12, wherein said poxvirus topoisomerase is produced by or isolated from a virus selected from the group consisting of vaccinia virus, Shope fibroma virus, ORE virus, fowlpox virus, molluscum contagiosum virus and Amsacta moorei entomopoxvirus.

14. A vector comprising the nucleic acid molecule of claim 1.

15. The vector of claim 14, wherein said vector is an expression vector.

33. A kit comprising the isolated nucleic acid molecule of claim 1.

35. The kit of claim 33 or claim 34, further comprising one or more components selected from the group consisting of one or more topoisomerases, one or more recombination proteins, one or more vectors, one or more polypeptides having polymerase activity, and one or more host cells.

38. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are att sites.

39. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are mutants, variants, and derivatives of att sites.

40. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are loxP or loxP511 sites.

41. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are selected from the group consisting of attB sites, attP sites, attL sites and attR sites.

42. The nucleic acid molecule of claim 1, wherein said first and second recombination sites are mutants, variants, and derivatives of attB sites, mutants, variants, and derivatives of attP sites, mutants, variants, and derivatives of attL sites, or mutants, variants, and derivatives of attR sites.

16. An isolated, linear nucleic acid molecule comprising:

(a) a first recombination site and a second recombination site; and

(b) a topoisomerase recognition site, wherein the first or second recombination site is separated from the topoisomerase recognition site by about 0 to about 100 nucleotides.

17. The nucleic acid molecule of claim 16, wherein said first or second recombination site is separated from said topoisomerase recognition site by about 0 to about 10 nucleotides.

18. The nucleic acid molecule of claim 16, wherein said first or second recombination site is separated from said topoisomerase recognition site by about 10 to about 30 nucleotides.

19. The nucleic acid molecule of claim 16, wherein said first or second recombination site is separated from said topoisomerase recognition site by about 20 to about 50 nucleotides.

20. The nucleic acid molecule of claim 16, wherein said first or second recombination site is separated from said topoisomerase recognition site by about 40 to about 80 nucleotides.

21. The nucleic acid molecule of claim 16, wherein said first or second recombination site is separated from said topoisomerase recognition site by about 70 to about 100 nucleotides.

22. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are selected from the group consisting of:

(a) an attB site;

(b) an attP site;

(c) an attL site;

(d) an attR site;

(e) a lox site;

(f) a psi site;

(g) a dif site;

(h) a cer site; and

(i) frt sites.

23. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are lox sites.

24. The nucleic acid molecule of claim 16, wherein said topoisomerase recognition site is recognized and bound by a type I topoisomerase.

25. The nucleic acid molecule of claim 24, wherein said type I topoisomerase is a type IB topoisomerase.

26. The nucleic acid molecule of claim 25, wherein said type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.

27. The nucleic acid molecule of claim 26, wherein said poxvirus topoisomerase is produced by or isolated from a virus selected from the group consisting of vaccinia virus, Shope fibroma virus, ORE virus, fowlpox virus, molluscum contagiosum virus and Amsacta moorei entomopoxvirus.

28. A vector comprising the nucleic acid molecule of claim 16.

29. The vector of claim 28, wherein said vector is an expression vector.

30. A host cell comprising the isolated nucleic acid molecule of claim 16.

31. A host cell comprising the vector of claim 28.

32. A host cell comprising the vector of claim 29.

34. A kit comprising the isolated nucleic acid molecule of claim 16.

43. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are att sites.

44. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are mutants, variants, and derivatives of att sites.

45. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are loxP or loxP511 sites.

46. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are selected from the group consisting of attB sites, attP sites, attL sites and attR sites.

47. The nucleic acid molecule of claim 16, wherein said first and second recombination sites are mutants, variants, and derivatives of attB sites, mutants, variants, and derivatives of attP sites, mutants, variants, and derivatives of attL sites, or mutants, variants, and derivatives of attR sites.

36. A vector selected from the group consisting of pcDNAGW-DT(sc), pENTR-DT(sc), pcDNN-DEST41, pENTR/D-TOPO, pENTRISD/D-TOPO, pcDNA3.2/V5/GWD-TOPO and pcDNA6.2/V5/GWD-TOPO.

37. A host cell comprising the vector of claim 36.

48. An isolated nucleic acid molecule comprising, in order:

(a) at least one first topoisomerase recognition site;

(b) at least one first recombination site;

(c) at least one nucleic acid segment;

(d) at least one second recombination site; and

(e) at least one second topoisomerase recognition site, wherein said first topoisomerase recognition site and said first recombination site are separated from each other by about 0 to about 100 nucleotides and wherein said irst and second recombination sites are different.

49. The nucleic acid molecule of claim 48, wherein said first and second recombination sites are selected from the group consisting of:

(a) attB sites;

(b) attP sites;

(c) attL sites;

(d) attR sites;

(e) lox sites;

(f) psi sites;

(g) dif sites;

(h) cer sites; and

(i) frt sites.

50. The nucleic acid molecule of claim 48, wherein said first and/or said second topoisomerase recognition sites are recognized and bound by a type I topoisomerase.

51. The nucleic acid molecule of claim 50, wherein said type I topoisomerase is a type IB topoisomerase.

52. The nuclic acid molecule of claim 51, wherein said type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.

53. The nucleic acid molecule of claim 52, wherein said poxvirus topoisomerase is produced by or isolated from a virus selected from the group consisting of vaccina virus, Shope fibroma virus, ORF virus, fowlpox virus, molluscum contagiosum virus and Amsacta moorei entomopoxvirus.

54. A host cell comprising the isolated nucleic acid molecule of claim 48.

55. A host cell comprising a vector, the vector further comprising the nucleic acid molecule of claim 48.

56. The host cell of claim 55 wherein the vector is an expression vector.