Assembly of High Fidelity Polynucleotides

• US 20130059761A1
• Filed: 01/06/2011
• Published: 03/07/2013
• Est. Priority Date: 01/07/2010
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Chu, Larry Li-Yang
• Granted Patent: US 9,217,144 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

Abstract

Methods and apparatus relate to the synthesis of high fidelity polynucleotides and to the reduction of sequence errors generated during synthesis of nucleic acids on a solid support. Specifically, design of support-bound template oligonucleotides is disclosed. Assembly methods include cycles of annealing, stringent wash and extension of polynucleotides comprising a sequence region complementary to immobilized template oligonucleotides. The error free synthetic nucleic acids generated therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.

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 plurality of support-bound single-stranded oligonucleotides, wherein each first and second plurality of support-bound oligonucleotides has a predefined sequence and is bound to a discrete feature of the support, each first plurality of support-bound oligonucleotides comprising a sequence region at its 5′

end that is the same as a sequence region of a 3′

end of the second plurality of support-bound oligonucleotides;

b) providing a plurality of first input single-stranded oligonucleotides, wherein the 3′

end of the plurality of the first input oligonucleotide is complementary to the 3′

end of the first plurality of support-bound oligonucleotides;

c) hybridizing the plurality of first input oligonucleotides to the first plurality of support-bound oligonucleotides at a first feature;

d) generating a first plurality of complementary oligonucleotides in a chain extension reaction, thereby forming an extension product duplex;

e) dissociating the extension product duplex, thereby producing a first plurality of complementary oligonucleotides;

f) transferring the first plurality of complementary oligonucleotides from the first feature to a second feature, thereby bringing into contact the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides;

g) annealing the first plurality of complementary oligonucleotides to the second plurality of support-bound single-stranded oligonucleotides at the second feature, wherein the annealing of the first plurality of complementary oligonucleotides to the second plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby producing the polynucleotide; and

h) optionally amplifying the polynucleotide.

3. The method of claim 1 further sequentially repeating steps c) through g) using at least a third plurality of support-bound oligonucleotides, wherein each plurality of oligonucleotides comprises a sequence region at its 3′

end that is the same as a sequence region of a 5′

end of a next plurality of oligonucleotides, thereby producing a longer polynucleotide.

12. The method of claim 1 wherein steps b) through e) are performed within a first droplet volume at the first feature thereby releasing the first plurality of complementary oligonucleotides in the first droplet volume and transferring the first droplet volume to the second feature comprising the second plurality of support-bound oligonucleotides.

13. The method of claim 12 wherein the droplet volumes are transferred by electrowetting, temperature gradients, wettability gradients, mechanical force or any combination thereof.

40. A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising:

a) synthesizing a polynucleotide on a discrete feature of a support according to the method of claim 1;

b) providing at least a first plurality of support-bound oligonucleotides, wherein the at least first plurality of support-bound oligonucleotides has a predefined sequence and is bound to a first discrete feature of the support, each first plurality of support-bound oligonucleotides comprising a primer binding sequence at its 3′

end and a sequence region at its 5′

end substantially identical to a 5′

end of the polynucleotide;

c) annealing the primer to the first plurality of support-bound oligonucleotides at the first discrete feature, wherein the annealing of the primer to the first plurality of support-bound oligonucleotides serves as a primer for extension of the first plurality of complementary oligonucleotides, thereby generating a first extension product duplex;

d) removing the primer from the first extension product duplex;

e) dissociating the first extension product duplex thereby producing a first plurality of complementary oligonucleotides;

f) transferring the first plurality of complementary oligonucleotides to a discrete feature comprising the polynucleotide thereby bringing into contact the first plurality of oligonucleotides with the polynucleotide, wherein the first plurality of complementary oligonucleotides is complementary to the 5′

end of the polynucleotide;

g) annealing the first plurality of complementary oligonucleotides to the polynucleotide, wherein the annealing of the oligonucleotides serves as a primer for extension of the polynucleotide, thereby producing a double-stranded polynucleotide.

41. The method of claim 40 wherein the polynucleotide comprises a 3′

terminal sequence region complementary to a 5′

region of an oligonucleotide at a discrete feature and a 5′

terminal region that is not complementary to the oligonucleotide.

42. The method of claim 40 wherein the primer sequence comprises at least one Uracil and wherein the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.

56. A method for producing at least one double-stranded polynucleotide having a predefined sequence, the method comprising

a) synthesizing a polynucleotide at a first discrete feature according to claim 1;

b) synthesizing a complementary oligonucleotide at a second discrete feature, wherein the 3′

terminal region of the complementary oligonucleotide is complementary to the 5′

terminal region of the polynucleotide;

c) transferring the complementary oligonucleotide to the first feature; and

d) hybridizing the complementary oligonucleotide to the polynucleotide.

2. (canceled)

4-11. -11. (canceled)

14-16. -16. (canceled)

17. A method for producing at least one high fidelity target polynucleotide having a predefined sequence, the method comprising:

a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, each first and second plurality of support-bound oligonucleotides having a predefined sequence and each first and second plurality of support-bound oligonucleotides being bound to a discrete feature of the support,

wherein each first plurality of support-bound oligonucleotides has a sequence region at its 5′

end that is the same as a sequence region of the 3′

end of the second plurality of support-bound oligonucleotides,

wherein each first plurality of support-bound oligonucleotides has a 3′

end that is complementary to a 3′

end of an input single-stranded polynucleotide;

b) hybridizing a plurality of first input polynucleotides with the first plurality of support-bound oligonucleotides at a first feature under hybridizing conditions thereby forming duplexes;

c) subjecting the duplexes to stringent melt conditions to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides;

d) removing error-containing polynucleotides;

e) generating a first plurality of complementary polynucleotides in a chain extension reaction under conditions promoting extension of the input polynucleotides, thereby forming extension product duplexes;

f) dissociating the extension product duplexes, thereby releasing a plurality of second input polynucleotides;

g) annealing the plurality of second input polynucleotides to a second plurality of support-bound single-stranded oligonucleotides at a second feature of the support wherein each second plurality of support-bound oligonucleotides has a 3′

end that is complementary to a 3′

end of the second input single-stranded polynucleotide; and

h) optionally repeating the cycles of stringent melt, extension, dissociation and annealing until the target polynucleotide is synthesized.

23. The method of claim 17

wherein steps b) through d) are performed within a first droplet volume at the first feature,

wherein step d) is performed by removing the first droplet volume,

wherein steps e) and f) are performed within a second droplet volume at the first feature, thereby releasing the plurality of second input polynucleotides in the second droplet volume, and

wherein step h) comprises transferring the second droplet volume to the second feature comprising the second plurality of support-bound oligonucleotides.

25. The method of claim 23 wherein the first droplet is subjected first to annealing conditions and second to stringent melt conditions and wherein the second droplet is subjected to conditions promoting primer extension.

18-22. -22. (canceled)

24. (canceled)

26. (canceled)

27. A method for producing at least one high fidelity target polynucleotide having a predefined sequence, the method comprising:

a) providing at least a first and a second plurality of support-bound single-stranded oligonucleotides, each plurality of support-bound oligonucleotides having a predefined sequence and each plurality of support-bound oligonucleotides being bound to a discrete feature of a solid support,

wherein each first plurality of support-bound oligonucleotides has at least three sequence regions, a 5′

end sequence region N, at least two sequence regions (N−

1) and (N−

2) that are complementary to the 3′

end of an input polynucleotide, and a 3′

end sequence region,

wherein each first plurality of support-bound oligonucleotides has a 3′

end that is complementary to a 3′

end of a plurality of an input single-stranded polynucleotide;

b) providing a plurality of first input polynucleotides at a first feature comprising the first plurality of support-bound oligonucleotides, wherein the plurality of first input polynucleotides comprises sequences regions complementary at least in part to the two sequences regions (N−

1) and (N−

2);

c) hybridizing the plurality of first input polynucleotides with the first plurality of support-bound oligonucleotides under hybridizing conditions wherein the 3′

end of the plurality of first input polynucleotides hybridize to the at least two sequence regions (N−

1) and (N−

2) of the first plurality of support-bound oligonucleotides, thereby forming duplexes;

d) subjecting the duplexes to stringent melt conditions sufficient to denature duplexes having at least one mismatch in a complementary region without denaturing the duplexes that do not comprise a mismatch in the complementary region, thereby releasing a population of error-containing input polynucleotides;

e) removing error-containing polynucleotides;

f) generating a first plurality of complementary oligonucleotides by template-dependent synthesis under condition promoting extension of the input polynucleotides, thereby forming extension product duplexes;

g) dissociating the extension product duplexes, thereby releasing a plurality of second input polynucleotides;

h) annealing the plurality of second input polynucleotides to the second plurality of support-bound single-stranded oligonucleotides; and

i) optionally repeating the cycles of stringent melt, extension, dissociation and annealing until the target polynucleotide is synthesized.

29. The method of claim 27 wherein in the step of providing the at least first and second plurality of support-bound single-stranded oligonucleotides, the (N−

1) sequence region is adjacent to the 5′

end sequence region and the (N−

2) sequence region is adjacent to the (N−

1) sequence region.

30. The method of claim 27 wherein in the step of providing the at least first and second plurality of support-bound single-stranded oligonucleotides, the support-bound oligonucleotides comprise at least three different sequences regions (N−

1), (N−

2) and (N−

3) between the 5′

end and the 3′

end sequence regions, and wherein the input polynucleotide hybridize to the (N−

1), (N−

2) and (N−

3) sequences regions of the support-bound oligonucleotides.

31. The method of claim 27 wherein the input polynucleotide is a product of at least two consecutive extension chain reactions using the sequences (N−

2) and (N−

1) as templates.

33. The method of claim 27 wherein the extension duplexes are subjected to a shuffling process before undergoing a next cycle of extension.

34. The method of claim 33 wherein the shuffling process comprises:

a) denaturing the extension duplexes, thereby releasing single-stranded extension products in solution;

b) re-annealing single-stranded extension products to the support-bound oligonucleotides, thereby producing re-annealed duplexes;

c) subjecting the re-annealed duplexes to stringent melt conditions sufficient to dissociate error-containing duplexes;

d) removing error-containing single-stranded extension products; and

f) dissociating error-free duplexes, thereby releasing error-free extension products in solution.

35. The method of claim 27 wherein in the step of providing the at least first and second plurality of support-bound single-stranded oligonucleotides, the 3′

end sequence region of the support-bound oligonucleotides is a spacer sequence and wherein the spacer sequence comprises a primer binding site.

37. The method of claim 27 wherein in the step of generating, each plurality of support-bound oligonucleotides is designed to serve as a template to a different polymerase extension reaction, thereby forming pluralities of extension duplexes, wherein each plurality of extension duplexes has a substantially identical melting temperature.

38. The method of claim 37 wherein the difference of melting temperature between the plurality of duplexes is less than 10°

C.

28. (canceled)

32. (canceled)

36. (canceled)

39. (canceled)

43-55. -55. (canceled)

57-59. -59. (canceled)