Direct Cloning

• US 20130210681A1
• Filed: 06/10/2011
• Published: 08/15/2013
• Est. Priority Date: 06/10/2010
• Status: Active Grant

• Current Assignee: Gene Bridges GmbH
• Original Assignee: Gene Bridges GmbH
• Inventors: Zhang, Youming, Fu, Jun, Stewart, Adrian Francis
• Granted Patent: US 10,443,051 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/16 Exonucleases active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters (3.16)

Abstract

A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology. A method for improving the efficiency of homologous recombination.

Claims

1. A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology, wherein the method comprises bringing the first nucleic acid molecule into contact with the second nucleic acid molecule in the presence of a 5′

to 3′

exonuclease and an annealing protein;

wherein the 5′

to 3′

exonuclease comprises a region having 5′

to 3′

exonuclease activity and at least;

i) amino acids 564-587 of SEQ ID NO;

1;

or

ii) a 24 amino acid sequence having at least 70% identity to amino acids 564-587 of SEQ ID NO;

1 over the entire length of the 24 amino acid sequence.

2. A method according to claim 1, wherein the 5′

to 3′

exonuclease is RecE.

3. A method according to claim 1 or claim 2, wherein the RecE comprises or consists of amino acids 564-866 of SEQ ID NO:

1 or a variant thereof comprising or consisting of a sequence 303 amino acids in length that has at least 70% sequence identity to SEQ ID NO;

1 over the entire length of the 303 amino acid sequence.

4. The method of any one of claims 1 to 3, wherein the RecE comprises or consists of a sequence selected from the group consisting of amino acids 1-866, 141-866, 423-866 or 564-866 of SEQ ID NO:

1 or a variant of a sequence from this group, wherein the variant has at least 70% sequence identity to SEQ ID NO;

1 over the entire length of the sequence.

5. The method of any one of the preceding claims, wherein the RecE has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the RecE provided in SEQ ID NO:

1.

6. The method of any one of the preceding claims, wherein the RecE is full-length RecE.

7. The method of any one of claims 2 to 6, wherein the annealing protein is RecT.

8. The method of any one of the preceding claims wherein the first and second nucleic acid molecules are linear nucleic acid molecules.

9. The method of any one of the preceding claims wherein the homologous recombination is carried out in a host cell.

10. The method of claim 9 wherein the 5′

to 3′

exonuclease is expressed from heterologous DNA.

11. The method of any one of claims 1-9 wherein the 5′

to 3′

exonuclease is RecE and is expressed from the RecE gene of an integrated prophage, and wherein expression of RecE is driven by a heterologous promoter.

12. The method of claim 10 or claim 11, wherein expression of the 5′

to 3′

exonuclease is driven by an inducible promoter such as an arabinose inducible promoter or a rhamnose inducible promoter.

13. The method of any one of the preceding claims, wherein the second nucleic acid is a linearised cloning vector, such as a linearised BAC, a linearised p15A origin based vector, a linearised pBR322 origin based vector, a linearised fosmid, a linearised pUC origin based vector or a linearised ColE1 origin based vector.

14. The method of any one of claims 1 to 12, wherein the first and second nucleic acid molecules are linear and the method further comprises bringing a third nucleic acid molecule into contact with the first and second nucleic acid molecules in the presence of the 5′

to 3′

exonuclease and the annealing protein, wherein the first nucleic acid molecule shares a region of homology with the second nucleic acid molecule and shares a different region of homology with the third nucleic acid molecule, wherein the second nucleic acid molecule shares a region of homology with the first nucleic acid molecule and shares a different region of homology with the third nucleic acid molecule and wherein the third nucleic acid molecule shares a region of homology with the second nucleic acid molecule and shares a different region of homology with the first nucleic acid molecule.

15. The method of any one of claims 1 to 12, wherein first and second nucleic acid molecules are linear and the method further comprises bringing a third nucleic acid molecule and a fourth nucleic acid molecule into contact with the first and second nucleic acid molecules in the presence of the 5′

to 3′

exonuclease and the phage annealing protein, wherein the wherein the first nucleic acid molecule shares a region of homology with the second nucleic acid molecule and shares a different region of homology with the fourth nucleic acid molecule, wherein the second nucleic acid molecule shares a region of homology with the first nucleic acid molecule and shares a different region of homology with the third nucleic acid the second nucleic acid molecule and shares a different region of homology with the fourth nucleic acid molecule, and wherein the fourth nucleic acid molecule shares a region of homology with the third nucleic acid molecule and shares a different region of homology with the first nucleic acid molecule.

16. The method of claim 15, wherein the first nucleic acid molecule comprises a sequence of interest, the second and fourth nucleic acid molecules are short oligonucleotides and the third nucleic acid molecule is a cloning vector.

17. The method of any one of the preceding claims, wherein the first nucleic acid molecule comprises a sequence of interest of 2 kb or more in length.

18. The method of any one of the preceding claims, wherein the first nucleic acid molecule comprises a sequence of interest which is a gene cluster.

19. The method of claim 18 wherein the gene cluster encodes a secondary metabolite pathway or a fatty acid synthesis pathway.

20. The method of any one of the preceding claims wherein the first nucleic acid molecule is a fragment of genomic DNA.

21. The method of any one of the preceding claims, wherein the first nucleic acid molecule is a linearised BAC and the method is used to subclone a sequence of interest from the BAC into a cloning vector.

22. The method of any one of the preceding claims, wherein the at least first and second nucleic acid molecules are linear and the method further comprises using the product of the linear to linear homologous recombination reaction between the at least first and second nucleic acid molecules in a second step of linear to circular homologous recombination in the presence of Redalpha and Redbeta or truncated RecE and RecT.

23. The method of claim 22, wherein the linear to linear homologous recombination is carried out in vitro in the presence of full length RecE and RecT, and wherein the method further comprises bringing the product of the linear to linear homologous recombination reaction into contact with a further nucleic acid molecule in a host cell and carrying out linear to circular homologous recombination in vivo in the presence of Redalpha and Redbeta and preferably also Redgamma.

24. The method of any one of the previous claims, wherein the first nucleic acid molecule is linear and comprises a phosphorothioation proximal to its 5′

end and a phosphorothioation proximal to its 3′

end.

25. The method of claim 24, wherein the second nucleic acid molecule is linear and comprises a phosphorothioation proximal to its 3′

end but does not comprise a phosphorothioation proximal to its 5′

end.

26. The method of any one of the preceding claims for generating a cDNA library.

27. A method according to any one of claims 1 to 25, wherein the first and second nucleic acid molecules are linear and wherein the method further comprises bringing the first and second nucleic acid molecules into contact with one or more additional nucleic acid molecules in the presence of the 5′

to 3′

exonuclease and the annealing protein to produce a linear product.

28. A method according to claim 27, wherein the linear product is a gene, an operon, a chromosome or an entire genome.

29. A host cell that expresses a 5′

to 3′

exonuclease as described in any one of claims 1 to 6.

30. The host cell of claim 29, which additionally comprises genes encoding Redalpha and Redbeta, wherein the 5′

to 3′

exonuclease is under the control of a different promoter from Redalpha and Redbeta.

32. A kit comprising a nucleic acid encoding a 5′

to 3′

exonuclease as recited in any one of claims 1 to 6 for use in a method of homologous recombination.

33. A kit comprising a 5′

to 3′

exonuclease as recited in any one of claims 1 to 6 for use in a method of homologous recombination

41. The method of any one of claims 34-39, wherein the method of homologous recombination is a method according to any one of claims 1 to 28.

44. A kit according to any one of claim 32, 33 or 43, wherein the kit comprises a host cell which comprises a nucleic acid encoding a RecE.

45. A kit according to claim 44, wherein the host cell is a cell as described in any one of claims 24-26.

47. A kit according to claim 44 or claim 45, wherein the host cell additionally comprises a nucleic acid sequence encoding one or more of Red gamma, RecA, Redalpha and Redbeta.

48. A kit according to any one of claims 32, 33 and 43-47, wherein the kit additionally comprises one or more linearised cloning vectors.

53. The method of any one of claims 49 to 52, wherein the method of homologous recombination is a method according to any one of claims 1 to 28 or 34 to 42.

54. A host cell according to any one of claims 29-31, further comprising a rare-cutting sequence specific DNA cleaving enzyme as recited in claim 50 or claim 51.

56. A kit for use in performing a method of homologous recombination comprising a host cell according to claim 54.

31. A host cell comprising a recE gene on an integrated prophage, wherein the recE gene is under the control of a heterologous promoter.

34. A method for improving the efficiency of homologous recombination by performing homologous recombination in the presence of at least one single stranded oligonucleotide that has no sequence homology to the nucleic acid molecules undergoing homologous recombination, wherein the efficiency of homologous recombination is improved relative to when homologous recombination is performed in the absence of the at least one single stranded oligonucleotide.

35. The method of claim 34, wherein the at least one single stranded oligonucleotide comprises or consists of DNA.

36. The method of claim 34 or 35 wherein the at least one single stranded oligonucleotide is 10-100 nucleotides in length.

37. The method of claim 36, wherein the at least one single stranded oligonucleotide is about 40 nucleotides in length.

38. The method of any one of claims 34 to 37, wherein the at least one single stranded oligonucleotide is used at a concentration of 1-200 pmol for each electroporation.

39. The method of any one of claims 34 to 38, wherein the homologous recombination is mediated by RecE and RecT.

40. The method of claim 39, wherein the RecE is truncated RecE.

42. The method of any one of claims 34 to 38, wherein the homologous recombination is mediated by Red alpha and Red beta.

43. A kit comprising at least one single stranded oligonucleotide as recited in any one of claims 34 to 37 for use in performing homologous recombination.

46. A kit according to claim 43, wherein the kit comprises a host cell that expresses Redalpha and Redbeta.

49. A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology, comprising, prior to performing homologous recombination in vivo, the step of linearising at least one circular nucleic acid molecule in vivo using a rare-cutting sequence specific DNA cleaving enzyme to generate the first and/or the second nucleic acid molecule.

50. The method of claim 49, wherein the rare-cutting sequence specific DNA cleaving enzyme is selected from a homing endonuclease, a zinc finger nuclease (ZFN) or transcription activation-like effector nuclease (TALEN).

51. The method of claim 50, wherein the homing endonuclease is selected from the group consisting of I-SceI, I-CeuI, I-CreI, I-ChuI, I-CsmI, I-DmoI, I-PanI, I-SceII, I-SceIII, I-SceIV, F-SceI, F-SceII, PI-AaeI, PI-ApeI, PICeuI, PI-CirI, PI-CtrI, PI-DraI, PI-MavI, PI-MflI, PI-MgoI, PI-MjaI, PI-MkaI, PI-MleI, PI-MtuI, PI-MtuHI, PI-PabIII, PI-PfuI, PI-PhoI, PI-PkoI, PI-PspI, PI-RmaI, PI-SceI, PI-SspI, PI-TfuI, PI-TfuII, PI-TliI, Pi-TliII. PI-TspI, PI-TspII, PI-BspI, PI-MchI, PI-MfaI, PI-MgaI, PI-MgaII, PI-MinI, PI-MmaI, Pi-MshI, PI-MsmII, PI-MthI, PI-TagI, PI-ThyII, I-NcrI, I-NcrII, I-PanII, I-TevI, I-PpoI, I-DirI, I-HmuI, I-HmuII, I-TevII, I-TevIII, F-SceI, F-SceII (HO), F-SuvI, F-TevI, and F-TevII.

52. The method of any one of claims 49 to 51, wherein the homologous recombination is mediated by RecE and RecT.

55. A kit for use in performing a method of homologous recombination comprising at least one rare-cutting sequence specific DNA cleaving enzyme as recited in claim 50 or claim 51.