Methods and Devices for Nucleic Acid Synthesis

• US 20130296194A1
• Filed: 11/10/2011
• Published: 11/07/2013
• Est. Priority Date: 11/12/2010
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Kung, Li-yun A., Ramu, Senthil, Schindler, Daniel, Wilson, Andrew Kirk, Hudson, Mike
• Granted Patent: US 9,295,965 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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 15/1031 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/343 Abzyme
• CPC Class Codes: C12Q 2521/50 Other enzymatic activities
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.

Claims

1. A method for producing at least one polynucleotide having a predefined sequence, the method comprising:

a. providing at least a first and a second pluralities of support-bound single-stranded oligonucleotides, wherein each first and second pluralities of oligonucleotides has a predefined sequence and is bound to a discrete feature of the support, each first plurality of oligonucleotides comprising a sequence region at its 3′

end complementary to a sequence region of a 3′

end of the second plurality of oligonucleotides;

b. generating at least a first and a second pluralities of construction oligonucleotides complementary to the first and second pluralities of support-bound oligonucleotides in a chain extension reaction;

c. providing a plurality support-bound anchor single-stranded oligonucleotides at a selected feature, wherein the 5′

end of the plurality of a first anchor oligonucleotide is the same as a sequence region of the first plurality of support-bound oligonucleotides;

d. hybridizing the at least first and second pluralities of construction oligonucleotides to the of anchor oligonucleotides; and

e. ligating the at least first and second pluralities of construction oligonucleotides, thereby generating the at least one polynucleotide.

2. The method of claim 1 wherein the at least first and second pluralities of construction oligonucleotides are dissociated from the at least first and second plurality of support-bound oligonucleotides.

3. The method of claim 2 further comprising transferring the first plurality of construction oligonucleotides from a first feature to a selected feature and transferring the second plurality of construction oligonucleotides from a second feature to the selected feature, wherein the selected feature comprises the plurality support-bound anchor single-stranded oligonucleotides.

4. The method of claim 3 wherein the selected feature is on the same support than the first and the second features.

5. The method of claim 3 wherein the selected feature is on a different support than the first and second features.

6. The method of claim 1 further:

providing a third plurality of support-bound single-stranded oligonucleotides wherein each third plurality of oligonucleotides has a predefined sequence and is bound to a third discrete feature of the support, each third plurality of oligonucleotides comprising a sequence region at its 3′

end that is complementary to a sequence region of a 3′

end of the second plurality of oligonucleotides;

generating a third plurality of construction oligonucleotides complementary to the third plurality of support-bound oligonucleotides in a chain extension reaction;

hybridizing the at least first, second and third pluralities of construction oligonucleotides to the plurality of anchor oligonucleotides at the selected feature; and

ligating the at least first, second and third pluralities of construction oligonucleotides thereby producing the at least one polynucleotide.

7. The method of claim 1 further sequentially repeating steps b) through e) using at least a third plurality of oligonucleotides, wherein each plurality of oligonucleotides comprises a sequence region at its 3′

end that is complementary to a sequence region of a 3′

end of a next plurality of oligonucleotides, thereby producing the at least one polynucleotide.

8. The method of claim 1 wherein each plurality of support-bound oligonucleotides has a primer binding site at its 3′

end.

9. The method of claim 8 wherein the primer binding site is a universal primer binding site.

10. The method of claim 8 further comprising annealing a primer to the at least first and second pluralities of support-bound oligonucleotides under conditions promoting extension of the primer, thereby forming extension product duplexes.

11. The method of claim 10 wherein the primer sequence comprises at least one Uracil.

12. The method of claim 1 wherein the plurality of support-bound oligonucleotides is synthesized or spotted on the solid support.

13. The method of claim 1 wherein the plurality of support-bound oligonucleotides are immobilized on the support through their 3′

end.

14. The method of claim 1 wherein the support is a microarray device.

15. The method of claim 10 wherein at least one feature is subjected to conditions promoting primer extension.

16. The method of claim 1 comprising:

providing N pluralities of predefined support-bound single-stranded oligonucleotides wherein the first plurality of oligonucleotides comprises at its 3′

end a sequence region that is complementary to a sequence region at the 3′

end of a second oligonucleotide, wherein a plurality of oligonucleotides N comprises at its 3′

end a sequence region that is complementary to a sequence region of a plurality of oligonucleotides (N−

1); and

providing a plurality of anchor oligonucleotides comprising at its 5′

end a sequence that is the same as a sequence region of the first plurality of support-bound oligonucleotides.

17. The method of claim 16 wherein N pluralities of construction oligonucleotides complementary to the support-bound single-stranded oligonucleotides are generated and wherein the N pluralities of construction oligonucleotides span the entire sequence of the polynucleotide without gaps.

18. The method of claim 16 wherein the 3′

end sequence region of the first plurality of support-bound oligonucleotides is identical to the 5′

end region of the anchor oligonucleotides.

19. The method of claim 10 further dissociating the extension duplexes thereby releasing the at least a first and a second pluralities of construction oligonucleotides.

20. The method of claim 11 further removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

21. A method of generating a polynucleotide having a predefined sequence, the method comprising:

a. synthesizing a plurality of support-bound double-stranded polynucleotides comprising a free single-stranded overhang, the plurality of polynucleotide sequences comprising the predefined polynucleotide sequence, wherein the single-stranded overhang comprises the sequence of a terminal construction oligonucleotide N;

b. providing a stem-loop oligonucleotide comprising a single-stranded overhang wherein the single-stranded overhang is complementary to the terminal construction oligonucleotide sequence N;

c. hybridizing the stem-loop oligonucleotide to the free overhang of the polynucleotide having predefined sequence;

d. ligating the stem-loop oligonucleotide to the free overhang of the polynucleotide having predefined sequence; and

e. degrading polynucleotide sequences that do not comprise the terminal construction oligonucleotide sequence N.

22. The method of claim 21 comprising hybridizing a pool of oligonucleotides comprising N pluralities of oligonucleotides to an anchor support-bound single-stranded oligonucleotide, wherein the first plurality of oligonucleotides in the pool comprises at its 5′

end a sequence region that is complementary to a sequence region at the 5′

end of the anchor oligonucleotide, and wherein a plurality of oligonucleotides N comprises at its 3′

end a sequence complementary to a sequence region of the plurality of oligonucleotides (N−

1).

23. The method of claim 21 further degrading the polynucleotide sequences that do not comprise the terminal oligonucleotide sequence using a single strand specific exonuclease.

24. The method of claim 23 wherein the exonuclease is selected from the group consisting of a single strand-specific 3′

exonuclease, a single strand-specific endonuclease, and a single strand-specific 5′

exonuclease.

25. The method of claim 21 wherein the stem-loop oligonucleotide comprises a type II restriction site and further removing the stem-loop oligonucleotide using a type II restriction endonuclease.

26. The method of claim 21 wherein the stem-loop oligonucleotide comprises at least one Uracil nucleotide and further removing the stem-loop oligonucleotide a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

27. The method of claim 21 further amplifying the predefined polynucleotide sequence.

28. The method of claim 21 wherein in the step of synthesizing the plurality of support-bound double-stranded polynucleotides is synthesized on a support by polymerase chain extension.

29. The method of claim 21 further releasing the polynucleotide from the support.

30. The method of claim 29 wherein the polynucleotides are released using a Type II restriction enzyme.

31. The method of claim 29 wherein the polynucleotides are released using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

32. A method of synthesizing a polynucleotide having a predefined sequence, the method comprising:

a. providing a support with (i) a first plurality of support-bound anchor oligonucleotides wherein the 5′

end of the first plurality of anchor oligonucleotide is complementary to the 5′

end of a first plurality of oligonucleotides and with (ii) a second plurality of support-bound anchor oligonucleotides wherein the 5′

end of the second anchor oligonucleotide is complementary to a terminal construction oligonucleotide N;

b. synthesizing a plurality of support-bound double-stranded polynucleotides comprising a 5′

single-stranded overhang, the plurality of polynucleotide sequences comprising the predefined polynucleotide sequence, wherein the single-stranded 5′

overhang of the predefined polynucleotide sequence comprises the terminal construction oligonucleotide N sequence and the single stranded 3′

end of the polynucleotide sequence comprises the first oligonucleotide sequence;

c. hybridizing the plurality of synthesized polynucleotides to the first anchor oligonucleotides; and

d. subjecting the synthesized polynucleotides to hybridization conditions, thereby hybridizing the 5′

overhang of the polynucleotide having terminal construction oligonucleotide N to the 5′

end of the second plurality of anchor oligonucleotides, thereby selecting the polynucleotides having the predefined sequence.

33. The method of claim 32 further degrading the polynucleotide sequences having a free 3′

or 5′

end.

34. The method of claim 33 wherein the polynucleotide sequences are degraded using an exonuclease.

35. The method of claim 32 further releasing the polynucleotides having the predefined sequence from the support.

36. The method of claim 35 wherein the polynucleotides are released using a Type II endonuclease.

37. The method of claim 35 wherein the polynucleotides are released using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

38. The method of claim 32 wherein the first plurality of anchor oligonucleotides is separated from the second plurality of anchor oligonucleotides by a distance corresponding to the length of the predefined polynucleotide.

39. The method of claim 38 wherein the support further comprises support-bound spacer single-stranded oligonucleotides to set the distance between the first and second anchor oligonucleotides.

40. The method of claim 39 wherein the distance between the first and second anchor oligonucleotides is a function of a concentration of the first and second anchor oligonucleotides and of the concentration of the spacer oligonucleotide.

41. The method of claim 32 wherein the plurality of support-bound double-stranded polynucleotides is synthesized by polymerase chain extension using a plurality of support-bound single-stranded oligonucleotides as templates.

42. A nucleic acid array comprising:

a. a solid support;

b. a plurality of discrete features associated with the solid support wherein each feature comprises a plurality of support-bound oligonucleotides having a predefined sequence wherein the first plurality of oligonucleotides comprises at its 5′

end a sequence region that is complementary to a sequence region at the 5′

end of a second oligonucleotide, wherein a plurality of oligonucleotides N comprises at its 5′

end a sequence complementary to a 5′

end sequence region of a plurality of oligonucleotides (N−

1); and

c. at least a first plurality of anchor oligonucleotides comprising at its 5′

end a sequence that is identical to a sequence region of the first plurality of support bound oligonucleotides.

43. The nucleic acid array of claim 42 further comprising a second plurality of support-bound anchor oligonucleotide wherein the 5′

end of the second anchor oligonucleotide is identical to the 5′

end of the plurality of oligonucleotides N.

44. The nucleic acid of claim 42 further comprising a plurality of support bound oligonucleotides having a sequence that is not identical to the plurality of the plurality of support bound oligonucleotides.

45. A method for producing a plurality of polynucleotides having a predefined sequence, the method comprising:

a. providing a first and second supports having a plurality of features, wherein each feature comprises a plurality of different support-bound oligonucleotides having a different predefined sequence;

b. generating a first and second pluralities of different construction oligonucleotides having different predefined sequences using the plurality of support-bound oligonucleotides as templates, the first and second pluralities of construction oligonucleotides having 3′

end complementary sequences;

c. providing a support comprising a plurality of features, wherein each feature comprises a plurality support-bound anchor single-stranded oligonucleotides, wherein the 5′

end of each of the plurality of the anchor oligonucleotides is complementary to the 5′

end the first plurality of construction oligonucleotides;

d. hybridizing the first plurality of construction oligonucleotides to the anchor oligonucleotides;

e. hybridizing the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides;

f. optionally sequentially hybridizing a third plurality of construction oligonucleotides to the second plurality of construction oligonucleotides; and

g. ligating the pluralities of construction oligonucleotides.

46. The method of claim 45 wherein each plurality of construction oligonucleotides defining each predefined polynucleotide is synthesized on a different support.

47. The method of claim 45 wherein the plurality of different polynucleotides are assembled at different features of the support comprising the support-bound anchor oligonucleotides.

48. The method of claim 45 wherein the step of generating the plurality of first construction oligonucleotides comprises annealing a primer sequence having at least one uracil to the first plurality of support-bound oligonucleotides under conditions promoting extension of the primer and removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

49. A method for synthesizing a plurality of polynucleotides having a predefined sequence, the method comprising:

a. providing a first support comprising a plurality of features, wherein each feature comprises a plurality support-bound anchor single-stranded oligonucleotides, wherein the 5′

end of each of the plurality of the anchor oligonucleotides is complementary to the 5′

end of a first plurality of construction oligonucleotides;

b. providing a second support having a plurality of features, wherein each feature comprises a plurality of single-stranded support-bound template oligonucleotides, each plurality of support-bound oligonucleotides having a different predefined sequence;

c. generating a first plurality of construction oligonucleotides by polymerase extension, using the plurality of support-bound oligonucleotides as templates;

d. positioning the first and the second support such as each feature of the second support is aligned to a corresponding feature of the first support;

e. releasing the first plurality of construction oligonucleotides in a solution under conditions promoting hybridization of the first plurality of oligonucleotides to the plurality of corresponding anchor oligonucleotides; and

f. optionally repeating steps b-e with a third support comprising a second plurality of construction oligonucleotides, the second and the third plurality of construction oligonucleotides having 3′

end complementary sequences.

50. The method of claim 49 wherein the step of generating the plurality of first construction oligonucleotides comprises annealing a primer sequence having at least one uracil to the first plurality of support-bound oligonucleotides under conditions promoting extension of the primer and removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

51. The method of claim 49 further ligating the anchor oligonucleotides and second plurality of construction oligonucleotides.

52. The method of claim 49 wherein the second support is positioned above and facing the first support.

53. The method of claim 52 wherein step of releasing the first plurality of construction oligonucleotides in solution allows for the diffusion of the first plurality of oligonucleotides towards the anchor oligonucleotides.

54. The method of claim 53 wherein the step of releasing the first plurality of construction oligonucleotides in solution is in presence of a porous membrane allowing for a substantial vertical diffusion of the construction oligonucleotides towards the anchor oligonucleotides.

55. The method of claim 54 wherein the porous membrane decrease the lateral diffusion of construction oligonucleotides.

56. The method of claim 49 wherein each feature of the second support comprises a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises at least two populations of oligonucleotides having different predefined sequences, the at least two populations of oligonucleotides having complementary sequences.

57. The method of claim 56 wherein the two populations of oligonucleotides comprise 3′

end complementary sequences.

58. The method of claim 56 wherein the step of releasing the two populations of oligonucleotides in solution allows for the hybridization of the first population of construction oligonucleotides to the second population of construction oligonucleotides and for the hybridization of the first population of oligonucleotides to the anchor oligonucleotide.

59. The method of claim 58 wherein the solution comprises a ligase.

60. The method of claim 49 wherein the stoichiometry of the first plurality of construction oligonucleotides is higher than the stoichiometry of the anchor oligonucleotides.

61. The method of claim 49 further comprising exposing the plurality of polynucleotides to a mismatch recognizing and cleaving component under conditions suitable for cleavage of double-stranded polynucleotides containing a mismatch nucleotide.

62. The method of claim 61 wherein the plurality of polynucleotides is immobilized on the support or is in solution.

63. The method of claim 62 the mismatch recognizing and cleaving component comprises a mismatch endonuclease.

64. The method of claim 63 wherein the mismatch specific endonuclease is a CEL I enzyme.

65. The method of claim 49 wherein the third support comprises a plurality of polynucleotides immobilized by hybridization to a plurality of anchor oligonucleotides.