Methods and devices for nucleic acid synthesis

• US 9,295,965 B2
• Filed: 11/10/2011
• Issued: 03/29/2016
• 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., Wilson, Andrew Kirk, Ramu, Senthil, Schindler, Daniel, Hudson, Mike
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/884,463
• Publication Number:
  US 20130296194A1
• Time in Patent Office: 1,601 Days
• Field of Search: None
• US Class Current: 1/1
• 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 target polynucleotide having a predefined sequence, the method comprising:

a. providing at least a first and a second pluralities of support-bound, single-stranded, template oligonucleotides, wherein each of the first and second pluralities of template oligonucleotides has a predefined sequence and is bound to a first and second feature of one or more planar support, respectively, each of the first plurality of template oligonucleotides comprising a sequence region at its 5′

end complementary to a sequence region of a 5′

end of the second plurality of template oligonucleotides;

b. generating at least a first and a second pluralities of construction oligonucleotides complementary to the first and second pluralities of template oligonucleotides in a chain extension reaction, each of the first plurality of construction 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 construction oligonucleotides;

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

end of each anchor oligonucleotide is complementary to the 5′

end of the first plurality of construction oligonucleotides; and

d. transferring the first plurality of construction oligonucleotides from the first feature to the selected feature on the planar anchor support, transferring the second plurality of construction oligonucleotides from the second feature to the selected feature on the planar anchor support, and hybridizing the at least first and second pluralities of construction oligonucleotides to the plurality of anchor oligonucleotides on the planar anchor support.

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 template oligonucleotides.

3. The method of claim 1 wherein the selected feature is on the same planar support than the first and the second features.

4. The method of claim 1 wherein the selected feature is on a different planar support than the first and second features.

5. The method of claim 1 further comprising:

(i) providing a third plurality of support-bound, single-stranded, template oligonucleotides wherein each of the third plurality of template oligonucleotides has a predefined sequence and is bound to a third feature of the one or more planar support, each of the third plurality of template 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 template oligonucleotides;

(ii) generating a third plurality of construction oligonucleotides complementary to the third plurality of template oligonucleotides in a chain extension reaction;

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

(iv) ligating the first and third pluralities of construction oligonucleotides thereby producing the at least one target polynucleotide.

6. The method of claim 5 further comprising sequentially repeating steps (i) through (iii) till a Nth plurality of construction oligonucleotides.

7. The method of claim 1 wherein each plurality of template oligonucleotides has a primer binding site at its 3′

end.

8. The method of claim 7 wherein the primer binding site is a universal primer binding site.

9. The method of claim 7 further comprising annealing a primer to the at least first and second pluralities of template oligonucleotides and synthesizing the first and second plurality of construction oligonucleotides by chain extension from the primer, thereby forming extension product duplexes.

10. The method of claim 9 wherein the primer comprises at least one Uracil.

11. The method of claim 1 wherein the at least first and second pluralities of template oligonucleotides are synthesized or spotted on the one or more planar support.

12. The method of claim 1 wherein the at least first and second pluralities of template oligonucleotides are immobilized on the one or more planar support at their 3′

end.

13. The method of claim 1 wherein the one or more planar support is a microarray device.

14. The method of claim 9 wherein at least one of the first and second feature is subjected to chain extension conditions.

15. The method of claim 1 comprising:

providing N pluralities of support-bound, single-stranded, template oligonucleotides thereby generating the first plurality of construction oligonucleotides comprising at its 3′

end sequence complementarity to the 3′

end of the second plurality of construction oligonucleotides, and a Nth plurality of construction oligonucleotides comprising at its 3′

end sequence complementarity to the 3′

end of a (N−

1)th plurality of construction oligonucleotides.

16. The method of claim 15 wherein the N pluralities of construction oligonucleotides span the entire sequence of the at least one target polynucleotide without gaps.

17. The method of claim 9 further comprising dissociating the extension product duplexes thereby releasing the first and second pluralities of construction oligonucleotides.

18. The method of claim 10 further comprising removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

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

(a). providing a first planar support having a plurality of first features and a second planar support having a plurality of second features, wherein each first or second feature comprises a plurality of different support-bound, template oligonucleotides;

(b). generating a first and second pluralities of construction oligonucleotides using the template oligonucleotides as templates in a chain extension reaction, the first and second pluralities of construction oligonucleotides having sequence complementarity to each other at their 3′

end;

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

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

end of the first plurality of construction oligonucleotides;

(d). transferring the first plurality of construction oligonucleotides to preselected features on the planar anchor support, and hybridizing the first plurality of construction oligonucleotides to the anchor oligonucleotides, wherein the preselected features correspond to the first features on the first planar support; and

(e). transferring the second plurality of construction oligonucleotides to the preselected features on the planar anchor support, and hybridizing the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides, wherein the preselected features correspond to the second features on the second planar support.

20. The method of claim 19 wherein each plurality of construction oligonucleotides is synthesized on a different planar support.

21. The method of claim 19 wherein each of the plurality of target polynucleotides is assembled at different preselected features of the planar anchor support.

22. The method of claim 19 wherein step (b) comprises annealing a primer having at least one uracil to the first plurality of template oligonucleotides and synthesizing the first plurality of construction oligonucleotides by chain extension from the primer and removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

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

(a). providing a first planar support comprising a plurality of first features, wherein each first feature comprises a support-bound, single-stranded, anchor oligonucleotide having a 5′

end complementary to the 5′

end of one of a first plurality of construction oligonucleotides;

(b). providing a second planar support having a plurality of second features, wherein each second feature comprises a single-stranded, support-bound, template oligonucleotide, for serving as template for synthesizing one of the first plurality of construction oligonucleotides in a polymerase based chain extension reaction;

(c). generating the first plurality of construction oligonucleotides by polymerase based chain extension from the template oligonucleotides;

(d). positioning the first planar support and the second planar support such that each second feature of the second planar support is aligned to a corresponding first feature of the first planar support;

(e). releasing the first plurality of construction oligonucleotides from each second feature of the second planar support in a solution and hybridizing the first plurality of construction oligonucleotides to the anchor oligonucleotides on the corresponding first feature of the first planar support;

(f). generating a second plurality of construction oligonucleotides using a third planar support comprising a second template oligonucleotide on each feature that serves as template for synthesizing the second plurality of construction oligonucleotides, wherein the second plurality of construction oligonucleotides each have a 3′

end that is complementary to the 3′

end of one of the first plurality of construction oligonucleotides, positioning the third planar support and the first planar support such that each feature of the third planar support is aligned to a corresponding first feature of the first planar support, releasing the second plurality of construction oligonucleotides from the third planar support, and hybridizing the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides on said corresponding first feature of the first planar support;

(g). performing step (f) by one or more times to produce N pluralities of construction oligonucleotides hybridized on the first planar support, wherein N is no less than 2; and

(h). assembling the N pluralities of construction oligonucleotides, thereby generating the plurality of target polynucleotides.

24. The method of claim 23 wherein step (c) comprises annealing a primer having at least one uracil to the template oligonucleotides, synthesizing the first plurality of construction oligonucleotides by chain extension from the primer, and removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

25. The method of claim 23 wherein step (h) comprises ligating the N pluralities of construction oligonucleotides and the anchor oligonucleotides.

26. The method of claim 23 wherein the second planar support is positioned above and facing the first planar support.

27. The method of claim 23 wherein step (e) comprises diffusing the first plurality of construction oligonucleotides towards the anchor oligonucleotides.

28. The method of claim 27 wherein said diffusing is in the presence of a porous membrane allowing for a substantial vertical diffusion of the first plurality of construction oligonucleotides towards the anchor oligonucleotides.

29. The method of claim 28 wherein the porous membrane decreases lateral diffusion of the first plurality of construction oligonucleotides.

30. The method of claim 23 wherein each second feature of the second planar support comprises a plurality of template oligonucleotides having different predefined sequences, and each having a 5′

end sequence that is complementary to the 5′

end of the second template oligonucleotide.

31. The method of claim 23 wherein the solution comprises a ligase.

32. The method of claim 23 wherein the first plurality of construction oligonucleotides have a higher stoichiometry than the anchor oligonucleotides.

33. The method of claim 23 further comprising exposing the plurality of target polynucleotides to a mismatch recognizing and cleaving component for cleavage of double-stranded polynucleotides containing a mismatch nucleotide.

34. The method of claim 33 wherein the plurality of target polynucleotides is immobilized on the first planar support.

35. The method of claim 34 wherein the mismatch recognizing and cleaving component comprises a mismatch endonuclease.

36. The method of claim 35 wherein the mismatch specific endonuclease is a CEL I enzyme.

37. The method of claim 23 wherein the plurality of target polynucleotides are immobilized by hybridization to the anchor oligonucleotides.

38. The method of claim 23 wherein the N pluralities of construction oligonucleotides span each predefined sequence of the plurality of target polynucleotides.

39. The method of claim 23, wherein on the first planar support, each first feature further comprises a second anchor oligonucleotide having a 5′

end that is complementary to the 5′

end of a terminal, Nth plurality of construction oligonucleotide.

40. The method of claim 39, further comprising:

(i). subjecting the assembled polynucleotides from step (h) to hybridization conditions, wherein each assembled polynucleotide on a discrete first feature has a 5′

single-stranded overhang that is complementary to the 5′

end of the second anchor oligonucleotide on said discrete first feature; and

(j). hybridizing the 5′

single-stranded overhang of each target polynucleotide to the 5′

end of the second anchor oligonucleotide on said discrete first feature, thereby selecting the target polynucleotides having the predefined sequence.

41. The method of claim 40 further comprising degrading the assembled polynucleotides having a free 3′

or 5′

end that is not hybridized to the second anchor oligonucleotide.

42. The method of claim 41 further comprising degrading using an exonuclease.

43. The method of claim 40 further comprising releasing the target polynucleotides having the predefined sequence from the first planar support.

44. The method of claim 43 further comprising releasing the target polynucleotides using a Type II endonuclease.

45. The method of claim 43 further comprising releasing the target polynucleotides using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

46. The method of claim 40 wherein the first anchor oligonucleotide is separated from the second anchor oligonucleotide by a distance corresponding to a length of the predefined sequence.

47. The method of claim 46 wherein the first planar support further comprises a support-bound, single-stranded, spacer oligonucleotide to set the distance between the first and second anchor oligonucleotides.

48. The method of claim 47 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 a concentration of the spacer oligonucleotide.