METHODS FOR MULTIPART, MODULAR AND SCARLESS ASSEMBLY OF DNA MOLECULES

• US 20140038240A1
• Filed: 07/10/2013
• Published: 02/06/2014
• Est. Priority Date: 07/10/2012
• Status: Abandoned Application

• Current Assignee: Pivot Bio, Inc.
• Original Assignee: Pivot Bio, Inc.
• Inventors: Temme, Karsten, Tamsir, Alvin
• Application Number:
  US13/939,110
• Publication Number:
  US 20140038240A1
• Time in Patent Office: Days
• US Class Current: 435/91.53
• 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
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention consists of methods for joining DNA molecules (parts) together to form larger DNA molecules (assemblies) of specified sequence and organization. The invention exhibits three necessary characteristics. Firstly, the invention enables 2 or more parts to be joined in a single reaction. Secondly, the seam between joined parts is scarless, producing no residual sequence dependencies like restriction enzyme recognition sites. Thirdly, parts are modular and can easily be reused in novel assemblies without modification. Prior technologies have exhibited no more than two of the three necessary characteristics, limiting their utility in synthesizing and editing DNA molecules of arbitrary sequence.

Claims

1. A method for scarless assembly of two or more DNA molecules, said method comprising:

generating a first DNA molecule having a single stranded terminus,

generating a second DNA molecule having a single stranded terminus,

ligating the first and second DNA molecules such that the ligation product corresponds to the combined sequence of the first and second DNA molecules.

2. The method of claim 1, wherein the following reactions are performed:

a. generating a first DNA molecule having a 5′

single stranded overhang;

b. generating a second DNA molecule having a 3′

single stranded overhang;

c. providing a short oligonucleotide staple linker containing perfect or near perfect complementarity to the 5′ and

3′

-overhangs; and

d. ligating the first DNA molecule, the second DNA molecule, and the staple linker.

3. The method of any of the preceding claims, wherein the 5′

or 3′

single stranded overhangs are generated with a restriction enzyme.

4. The method of any of the preceding claims, wherein a Type IIs restriction enzyme generates DNA with 3′

single stranded overhangs.

5. The method of any of the preceding claims, wherein a Type IIb restriction enzyme generates DNA with 3′

single stranded overhangs.

6. The method of any of the preceding claims, wherein a Type IIp restriction enzyme generates DNA with 3′

single stranded overhangs.

7. The method of any of the preceding claims, wherein a Type IIs restriction enzyme generates DNA with 3′

single stranded overhangs, and the Type IIs restriction enzyme is optionally RleAI.

8. The method of any of the preceding claims, wherein a Type IIb restriction enzyme generates DNA with 3′

single stranded overhangs, and the Type IIb restriction enzyme is optionally BsaXI.

9. The method of any of the preceding claims, wherein a Type IIp restriction enzyme generates DNA with 3′

single stranded overhangs, and the Type IIp restriction enzyme is optionally BstXI.

10. The method of any of the preceding claims, wherein the Type IIs restriction enzyme generates DNA with 5′

single stranded overhangs.

11. The method of any of the preceding claims, wherein the Type IIs restriction enzyme generates DNA with 5′

single stranded overhangs, and the Type IIs restriction enzyme is optionally selected from EarI, BspMI, BsaI, BbsI, or BsmBI.

12. The method of any of the preceding claims, wherein the single stranded DNA terminus with a 3′

overhang is generated through the action of an exonuclease.

13. The method of any of the preceding claims, wherein the exonuclease digests DNA that was produced by PCR using oligos containing phosphorothioate bonds.

14. The method of any of the preceding claims, wherein the exonuclease is selected from T7 exonuclease, T5 exonuclease, or Lambda exonuclease.

15. The method of any of the preceding claims, wherein the single stranded DNA terminus with a 3′

-overhang is generated through the action of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII.

16. The method of any of the preceding claims, wherein the staple linker contains a defined sequence capable of binding with perfect or near perfect complementarity to the single stranded DNA termini of the first and second DNA molecules.

17. The method of any of the preceding claims, wherein the staple linker binds to both a single stranded terminus with a 3′

-overhang and a single stranded terminus with a 5′

-overhang.

18. The method of any of the preceding claims, wherein the single stranded terminus with a 3′

-overhang and the single stranded terminus with a 5′

-overhang are ligated together with the staple linker by a DNA ligase, and the DNA ligase enzyme is optionally selected from T4 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

19. The method of any of the preceding claims, wherein the staple linker is an oligonucleotide of DNA, RNA, or modified DNA and RNA molecules between 4 and 20 nucleotides in length.

20. The method of any of the preceding claims, wherein the staple linker contains single stranded DNA, double stranded DNA, or combination thereof.

21. The method of any of the preceding claims, wherein the ligating step involves a single stranded terminus adapter containing a degenerate sequence or a defined sequence.

22. The method of any of the preceding claims, wherein the single stranded terminus adapter contains dsDNA.

23. The method of any of the preceding claims, wherein the single stranded terminus adapter is between 5 and 100 nucleotides in length.

24. The method of any of the preceding claims, wherein the single stranded terminus adapter duplicates the terminal sequence of the second DNA molecule.

25. The method of any of the preceding claims, wherein the single stranded terminus adapter includes a single stranded DNA terminus of defined sequence.

26. The method of any of the preceding claims, wherein the single stranded terminus adapter and/or the second DNA molecule are modified via the action of an exonuclease.

27. The method of any of the preceding claims, wherein the single stranded terminus adapter contains a degenerate sequence capable of binding to a single stranded DNA terminus complementary to the single stranded DNA terminus.

28. The method of any of the preceding claims, wherein the single stranded terminus adapter is between 5 and 100 nucleotides in length.

29. The method of any of the preceding claims, wherein the single stranded DNA terminus of the single stranded terminus adapter and second DNA molecule are annealed and ligated.

30. A reaction mixture capable of generating a 3′

single stranded DNA terminus overhang according to the method of any of the preceding claims.

31. A reaction mixture capable of generating a 5′

single stranded DNA terminus overhang according to the method of any of the preceding claims.

32. A reaction mixture comprising enzymes capable of generating 3′

, 5′

, and/or combination of 3′ and

5′

single stranded DNA terminus overhang in a single reaction, according to the method of any of the preceding claims.

33. The reaction mixture of any of the preceding claims, wherein the restriction enzyme is a Type IIs, Type IIb or Type IIp restriction enzyme.

34. The reaction mixture of any of the preceding claims, wherein the Type IIs, Type IIb or Type IIp restriction enzyme is selected from BsaXI, RleAI, and TstI and the restriction enzyme generates single stranded terminus with a 3′

-overhang.

35. The reaction mixture of any of the preceding claims, wherein the Type IIs restriction enzyme is selected from EarI, BspMI, BsaI, BbsI, and BsmBI and the restriction enzyme generates single stranded terminus with a 5′

-overhang.

36. A reaction mixture for performing the method of any of the preceding claims.

37. The method of any of the preceding claims, in which the product of scarless assembly method is circular DNA.

38. The method any of the preceding claims, in which the product of scarless assembly method can be transformed or transfected into cells.

39. The method or reaction mixture of any of the preceding claims, wherein more than two DNA molecules are simultaneously ligated together.