Method for in vitro recombination

• US 7,776,532 B2
• Filed: 08/11/2006
• Issued: 08/17/2010
• Est. Priority Date: 08/11/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Smith, Hamilton O., Gibson, Daniel Glenn
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US11/502,624
• Publication Number:
  US 20070037196A1
• Time in Patent Office: 1,467 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 for joining a first set of double-stranded (ds) DNA molecules, comprising:

(a) providing two or more dsDNA molecules to be joined in a reaction mixture, wherein, for each pair of dsDNA molecules to be joined, a distal region of a first DNA molecule and a proximal region of a second DNA molecule share a region of sequence homology;

(b) treating the provided dsDNA molecules with a substantially purified enzyme having 3′

-5′

exonuclease activity, whereby a single-stranded overhanging portion is generated in each of the dsDNA molecules by 3′

-5′

exonuclease digestion, wherein each overhanging portion contains the region of homology or a portion thereof sufficient to specifically anneal to the overhanging portion in the other molecule of the pair;

(c) incubating the DNA molecules generated in step (b), under conditions whereby they anneal through the regions of homology or portions thereof; and

(d) treating the annealed molecules with a substantially purified polymerase and a substantially purified compatible ligase, under conditions whereby remaining single-stranded gap(s) are filled in by the polymerase and nicks are sealed by the ligase;

thereby joining the dsDNA molecules, wherein a crowding agent is present in the reaction mixture during each of steps (b), (c), and (d).

2. The method of claim 1, wherein the crowding agent is PEG.

3. The method of claim 2, wherein the concentration of PEG in the reaction mixture is about 5% PEG.

4. The method of claim 1, wherein the polymerase in step (d) is Taq DNA polymerase.

5. The method of claim 1, wherein the compatible ligase in step (d) is Taq ligase.

6. The method of claim 1, wherein the enzyme having 3′

-5′

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

7. The method of claim 1, wherein the enzyme having 3′

-5′

exonuclease activity is selected from the group consisting of T4 DNA polymerase, T7 DNA polymerase, DNA polymerase I, Klenow DNA polymerase, Phi 29 DNA polymerase, Pfu polymerase, Phusion™

High-Fidelity polymerase, exonuclease III, Vent_R®

, Deep Vent_R® and

9°

N_m™

DNA polymerase.

8. The method of claim 1, wherein the enzyme having 3′

-5′

exonuclease activity is T4 DNA polymerase.

9. The method of claim 1, wherein the incubating in step (c) is carried out by subjecting the molecules to conditions effective to separate any single-stranded portions that have annealed and, optionally, to inactivate the enzyme having 3′

-5′

exonuclease activity, followed by slowly cooling the molecules to about 24°

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

10. The method of claim 9, wherein subjecting the molecules to conditions effective to separate any annealed portions and, optionally, to inactivate the enzyme having 3′

-5′

exonuclease activity, includes heating said molecules to 75°

C. plus or minus 5°

C.

11. The method of claim 1, wherein the treating in step (d) is performed at between 45°

C. and 55°

C.

12. The method of claim 1, wherein the incubation in step (c) is carried out in the presence of a protein that enhances the binding of single-stranded overhanging portions containing homologous regions or portions thereof.

13. The method of claim 12, 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.

14. The method of claim 1, wherein at least four dsDNA molecules are joined and each shared region of sequence homology is unique for each pair of DNA molecules joined.

15. The method of claim 14, wherein at least eight dsDNA molecules are joined.

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

17. The method of claim 16, wherein the DNA molecules to be joined are at least about 140 kb in length.

18. The method of claim 17, wherein the DNA molecules to be joined are at least about 500 kb in length.

19. The method of claim 1, wherein, for at least one pair of dsDNA molecules to be joined, the region of sequence homology comprises at least about 20 non-palindromic nucleotides in length.

20. The method of claim 1, wherein, for at least one pair of dsDNA molecules to be joined, the region of sequence homology comprises at least about 300 nucleotides in length.

21. The method of claim 1, wherein steps (b) through (d) are carried out in a single reaction vessel.

22. The method of claim 1, further comprising:

(i) joining a second set of dsDNA molecules by performing steps (a) through (d); and

(ii) performing a second stage assembly, comprising steps (a) through (d), wherein the dsDNA molecules provided in step (a) comprise a product produced by joining the first set and a product produced by joining the second set.

23. The method of claim 1, wherein the method is automated and high-throughput.

24. The method of claim 1, wherein a PCI clean-up procedure is not carried out following the exonuclease digestion.

25. The method of claim 1, wherein the overhanging portions are generated without the use of a restriction enzyme.

26. The method of claim 1, wherein the dsDNA molecules are joined in a predefined order and orientation.

27. The method of claim 1, wherein steps (b) and (c) are performed in the same reaction mixture, containing the same buffer and reaction components.

28. The method of claim 1, wherein steps (b) and (c) are performed in the same reaction vessel and the vessel is not opened between steps (b) and (c).

29. The method of claim 1, wherein the crowding agent is a polymer selected from the group consisting of PEG, Ficoll, and dextran.

30. The method of claim 2, wherein the size of the PEG is within the range of PEG 4,000 to PEG 20,000.

31. The method of claim 30, wherein the PEG is PEG 8,000.

32. The method of claim 1, wherein the treating in step (d) is performed at between 37°

C. and 75°

C.

33. The method of claim 1, wherein the treating in step (d) is performed at 37°

C.