Method for in vitro recombination

• US 20070037196A1
• Filed: 08/11/2006
• Published: 02/15/2007
• Est. Priority Date: 08/11/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: J. Craig Venter Institute
• Inventors: Smith, Hamilton, Gibson, Daniel
• Granted Patent: US 7,776,532 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.

Claims

1. An in vitro method, using isolated proteins, for joining two or more double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity, comprising

(a) treating the DNA molecules with an enzyme having an exonuclease activity, under conditions effective to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence homology to hybridize specifically to the region of sequence homology of its pair;

(b) incubating the treated DNA molecules of (a) under conditions effective to achieve specific annealing of the single-stranded overhanging portions; and

(c) treating the incubated DNA molecules in (b) under conditions effective to fill in remaining single-stranded gaps and to seal the nicks thus formed,

wherein the region of sequence identity comprises at least 20 non-palindromic nucleotides (nt).

2. The method of claim 1, wherein the region of sequence identity comprises at least about 30 non-palindromic nt.

3. The method of claim 1, wherein a crowding agent, which increases molecular crowding, is present in the reaction mixture at each of steps (a), (b) and (c).

4. The method of claim 3, wherein the crowding agent is PEG.

5. The method of claim 4, wherein the concentration of the PEG is about 5% PEG.

6. The method of claim 1, wherein, in (c), the DNA molecules are treated with Taq DNA polymerase and a compatible ligase.

7. The method of claim 6, wherein the compatible ligase is Taq ligase.

8. The method of claim 1, wherein about 5% PEG is present in the reaction mixture at each of steps (a), (b) and (c);

and in (c) the mixture is treated with Taq DNA polymerase and a compatible ligase.

9. The method of claim 8, wherein the compatible ligase is Taq ligase.

10. The method of claim 1 wherein the enzyme in (a) has a 3′

→

5′

exonuclease activity.

11. The method of claim 10, wherein the enzyme having a 3′

→

5′

exonuclease activity is exonuclease III.

13. The method of claim 10, wherein the enzyme having a 3′

→

5′

exonuclease activity is a DNA polymerase which exhibits exonuclease activity when it is incubated in the absence of dNTPs.

15. The method of claim 13, wherein the enzyme in (a) is one of the following DNA polymerases:

T4 DNA polymerase, T7 DNA polymerase, DNA polymerase I, Klenow DNA polymerase, Phi 29 DNA polymerase, Pfu polymerase, Phusion™

High-Fidelity polymerase.

16. The method of claim 15, wherein in (a) the enzyme is T4 polymerase.

17. The method of claim 1, wherein in (b), the treated DNA molecules of (a) are incubated under conditions effective to separate the strands of the overhangs which have annealed and, optionally, to inactivate the enzyme, and then are slowly cooled to about 24°

C. or less, under conditions effective to allow the single-stranded overlaps to anneal.

18. The method of claim 17, wherein the treated DNA molecules of (a) are incubated at 75°

C. plus or minus about 5°

C.

19. The method of claim 1, wherein specific annealing of the single-stranded overhanging portions is achieved by including in the treating step in (a) a protein that enhances the binding of the single-stranded overhanging portions.

20. The method of claim 19, wherein the protein that enhances the binding of the single-stranded overhanging portions is recA, E. Coli single-stranded binding protein (SSB), T7 SSB (T7 gene 2.5 product), or T4 gene 32 protein.

21. The method of claim 1, wherein in (c), the conditions effective to fill in remaining single-stranded gaps and to seal the nicks comprise incubating the annealed DNA molecules with a DNA polymerase in the presence of dNTPs and a compatible ligase.

22. The method of claim 21, wherein in (c)

(i) the DNA polymerase is T4, T7, E. coli Poll, Klenow, Taq, Phusion™

or Pfu polymerase;

the ligase is T4, E. coli or Taq DNA ligase or Ampligase; and

the treatment is performed at about 37°

C.;

or

(ii) the DNA polymerase is Taq, Phusion™

or Pfu DNA polymerase;

the ligase is Taq DNA ligase or Ampligase; and

the treatment is performed at about 45°

C.

23. The method of claim 22, wherein in (c)

(i) the DNA polymerase is T4 DNA polymerase;

the ligase is T4 DNA ligase; and

the treatment is performed at about 37°

C.;

or

(ii) the DNA polymerase is Taq DNA polymerase;

the ligase is Taq DNA ligase; and

the treatment is performed at about 45°

C.

28. The method of claim 1, comprising joining at least about 4 double-stranded DNA molecules, wherein for each pair of molecules to be joined, the distal region of one DNA molecule comprises a region of sequence homology to the proximal region of the other DNA molecule, and each set of distal and proximal regions of homology is unique for each pair of DNA molecules to be joined.

30. The method of claim 28, wherein at least about 8 double-stranded DNA molecules are joined.

31. The method of claim 1, wherein the DNA molecules to be joined are at least about 5 kb.

33. The method of claim 31, wherein the molecules to be joined are at least about 140 kb.

34. The method of claim 31, wherein the molecules to be joined are at least about 500 kb.

36. The method of claim 1, wherein the region of sequence identity comprises at least about 40 non-palindromic nucleotides.

38. The method of claim 36, wherein the region of sequence identity comprises at least about 300 nt.

41. The method of claim 1, which is carried out in a single vessel, and wherein

the reactions in (a) and (b) are carried out in a solution that comprises about 0.2 M Tris-Cl, pH 7.5 and about 5% PEG;

and

when the reactions in (a) and (b) are complete, the reaction mixture is diluted 1;

4;

more PEG is added to a final concentration of about 5%; and

the reaction in (c) is allowed to proceed.

46. The method of claim 1, wherein the DNA molecules are generated synthetically and comprise adjacent portions of a gene or genome of interest;

the DNA molecules are synthesized so as to comprise overlapping regions of sequence identity at their ends; and

the DNA molecules are joined to form part or all of a synthetic gene or genome.

52. The method of claim 1, further comprising repeating the method to join a second set of two or more ds DNA molecules of interest to one another, and then repeating the method again to join the first and the second set of DNA molecules of interest.

61. The method of claim 1, wherein

each of the DNA molecules of interest comprises, at the free end of the region of sequence identity, a restriction enzyme cleavage site that is not present elsewhere in the DNA molecules of interest;

the DNA molecules of interest are cleaved with the restriction enzyme; and

during steps (b) and (c), the restriction enzyme cleavage site is removed from the joined molecules.

62. The method of claim 61, wherein the restriction enzyme is Not I.

12. (canceled)

14. (canceled)

24. An in vitro method, using isolated proteins, for joining at least two ds DNA molecules of interest, each of about 5-6 kilobases (kb), wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a unique region of sequence identity, comprising

(a) treating approximately equimolar amounts of the DNA molecules with T4 DNA polymerase at about 37°

C., in a solution comprising about 0.2 M Tris at about pH 7.5, in the absence of added dNTPs, under conditions effective to chew-back at least the regions of sequence identity in each molecule, thereby forming single-stranded overhanging ends of sufficient length to hybridize specifically to overhangs having the complement of the shared region of sequence identity;

(b) annealing the treated DNA molecules in (a) by incubating them at 75°

C. plus or minus about 5°

C. for about 20 minutes, and slow cooling them to about 24°

C. or less, under conditions effective to anneal the single-stranded DNA regions which were generated during (a); and

(c) incubating the cooled DNA molecules in (b) with Taq DNA polymerase and Taq DNA ligase at about 45°

C., in the presence of added dNTPs, under conditions effective to fill in the gaps and seal the nicks,

wherein about 5% PEG is present throughout the joining procedure.

25. An in vitro method, using isolated proteins, for joining at least two ds DNA molecules of interest, each of about 5-6 kilobases (kb), wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a unique region of sequence identity, comprising

(a) incubating approximately equimolar amounts of the DNA molecules with:

T4 DNA polymerase;

a protein that enhances annealing of single-stranded DNAs; and

a ligase that is compatible with the polymerase, at about 37°

C., in the absence of added dNTPs, under conditions effective to chew-back at least the regions of sequence identity in each molecule, thereby forming single-stranded overhanging ends of sufficient length to hybridize specifically to overhangs having the complement of the shared region of sequence identity, and to allow hybridization of the single-stranded overhangs, thereby forming gapped molecules; and

(b) incubating the incubated DNA molecules in (a) with a sufficient amount of dNTPs, under conditions effective to allow filling in of the gaps, generation of nicks, and sealing of the nicks,

wherein the method is carried out in a single vessel.

27. The method of claim 25, wherein the protein that enhances annealing of single-stranded DNAs is recA, E. coli single-stranded binding protein (SSB), T7 SSB (T7 gene 2.5 product), or T4 gene 32 protein.

26. (canceled)

29. (canceled)

32. (canceled)

35. (canceled)

37. (canceled)

39-40. -40. (canceled)

42-45. -45. (canceled)

47-50. -50. (canceled)

51. An in vitro method, using isolated protein reagents, for joining a plurality of double-strand (ds) DNA molecules of interest, wherein for each pair of molecules to be joined, the distal region of one DNA molecule comprises a region of sequence identity to the proximal region of the other DNA molecule, and each set of distal and proximal regions of identity is unique for each pair of DNA molecules to be joined, comprising generating 5′

single-strand overhangs at both ends of the DNA molecules;

annealing the regions of sequence identity in the single-stranded overhangs, filling in the gaps formed; and

sealing the nicks,

wherein each region of sequence identity comprises at least 20 non-palindromic nucleotides (nt).

53. (canceled)

54. A kit for in vitro joining a plurality of dsDNA molecules, comprising

(a) an isolated enzyme having a 3′

or 5′

exonuclease activity;

(b) an isolated non strand-displacing DNA polymerase;

(c) a ligase which is compatible with the polymerase; and

, optionally,

(d) solution, or components for making the solution which, when combined with the exonuclease and the dsDNA molecules to be joined, comprises about 5% PEG and/or about 0.2 M Tris, at about pH7.5.

55-57. -57. (canceled)

58. A kit for in vitro joining a plurality of dsDNA molecules, comprising

(a) a vessel containing purified T4 DNA polymerase;

a protein that enhances annealing of single-stranded DNAs; and

a ligase that is compatible with the polymerase; and

, optionally

(b) a solution, or components for making the solution, that, when combined with an aliquot of the protein mixture in (a) and a plurality of suitable DNA molecules containing regions of sequence identity at their termini, is effective to allow chew-back of regions of sequence identity of the DNA molecules, the formation of single-stranded overhangs containing the regions of sequence identity, and hybridization of the single-stranded overhangs, thereby forming gapped molecules; and

, optionally

(c) a concentrated solution of dNTPs that, when added in a suitable volume to the solution in (b) which contains gapped molecules, and incubated with that solution under suitable conditions, is effective to allow filling in of the gaps.

59-60. -60. (canceled)

63. (canceled)