Assembly of high fidelity polynucleotides

• US 9,925,510 B2
• Filed: 11/20/2015
• Issued: 03/27/2018
• 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
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/947,655
• Publication Number:
  US 20160144333A1
• Time in Patent Office: 858 Days
• Field of Search: 435 912, 435 61
• 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 a target polynucleotide, the method comprising:

a) providing a first feature having a first bound oligonucleotide and a second feature having a second bound oligonucleotide, wherein the first and second features are addressable and located at predetermined locations of one or more support, wherein the first and second bound oligonucleotides are designed to have an overlapping sequence region;

b) synthesizing a first complementary oligonucleotide at the first feature using the first bound oligonucleotide as template;

c) transferring in droplet volume the first complementary oligonucleotide from the first feature to the second feature; and

d) producing a second complementary oligonucleotide using the second bound oligonucleotide as template.

2. The method of claim 1, wherein in the providing step, each feature comprises a plurality of bound oligonucleotides.

3. The method of claim 1, wherein in the providing step, the first feature and the second feature are provided on the same solid support.

4. The method of claim 3, wherein the first and second bound oligonucleotides are synthesized on the solid support.

5. The method of claim 3, wherein the first and second bound oligonucleotides are immobilized on the solid support.

6. The method of claim 3, wherein the solid support is a microarray device.

7. The method of claim 1, wherein in the providing step, the first and second bound oligonucleotides each comprises a junction interrogation sequence.

8. The method of claim 1, further comprising synthesizing a clonal population of the first complementary oligonucleotides.

9. The method of claim 1, further comprising synthesizing the first complementary oligonucleotide in a chain extension reaction.

10. The method of claim 1, wherein in the transferring step, the droplet volume is a microvolume.

11. The method of claim 1, wherein in the transferring step, the droplet volume is transferred by electrowetting, temperature gradients, wettability gradients, mechanical force or any combination thereof.

12. The method of claim 1, further comprising producing a clonal population of the second complementary oligonucleotides.

13. The method of claim 1, further comprising producing the second complementary oligonucleotide in a chain extension reaction.

14. The method of claim 1, wherein the second complementary oligonucleotide is a target polynucleotide.

15. The method of claim 1, further comprising:

(i) providing on the one or more support a Nth feature having a Nth bound oligonucleotide and a (N+1)th feature having a (N+1)th bound oligonucleotide, wherein N is an integer no less than 3, wherein the Nth and (N+1)th bound oligonucleotides are designed to have an overlapping sequence;

(ii) synthesizing a Nth complementary oligonucleotide at the Nth feature using the Nth bound oligonucleotide as template;

(iii) transferring in droplet volume the Nth complementary oligonucleotide from the Nth feature to the (N+1)th feature; and

(iv) producing a (N+1)th complementary oligonucleotide using the (N+1)th bound oligonucleotide as template, wherein the (N+1)th complementary oligonucleotide is a target polynucleotide.

16. The method of claim 1, further comprising providing a first non-immobilized input oligonucleotide having sequence complementarity with the first bound oligonucleotide, annealing the first non-immobilized input oligonucleotide with the first bound oligonucleotide, and extending from the first non-immobilized input oligonucleotide in a chain extension reaction to form the first complementary oligonucleotide.

17. The method of claim 16, wherein the first non-immobilized input oligonucleotide is a universal primer.

18. The method of claim 16, wherein the first non-immobilized input oligonucleotide is a primer unique to the first bound oligonucleotide.

23. The method of claim 17 wherein the primer comprises at least one Uracil.

24. The method of claim 23 further comprising removing the primer using a mixture of Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII.

19. A method for producing a target polynucleotide, the method comprising:

a. providing a first feature having a first template oligonucleotide bound thereto and a second feature having a second template oligonucleotide bound thereto, wherein the first and second features are addressable and located at predetermined locations of one or more support, wherein the first template oligonucleotide comprises sequence complementarity to the second template oligonucleotide;

b. generating a first and a second construction oligonucleotides complementary to the first and second template oligonucleotides, respectively, in a chain extension reaction, wherein the first construction oligonucleotide comprises at sequence complementarity to the second construction oligonucleotide;

c. providing an anchor oligonucleotide at an assembly feature, wherein the anchor oligonucleotide comprises sequence complementarity to the first construction oligonucleotide;

d. transferring in droplet volume the first construction oligonucleotide from the first feature to the assembly feature, and transferring in droplet volume the second construction oligonucleotide from the second feature to the assembly feature; and

e. hybridizing the first and second construction oligonucleotides to the anchor oligonucleotide.

20. The method of claim 19 wherein the first and the second features are on the same support.

21. The method of claim 19 wherein the assembly feature is on the same support as the first and the second features.

22. The method of claim 19, wherein the first and second template oligonucleotides each comprises a region for primer binding in the chain extension reaction.

25. A method for producing a target polynucleotide, the method comprising:

a) providing a solid support comprising an assembly feature having an anchor oligonucleotide immobilized thereon;

b) hybridizing a first construction oligonucleotide with the anchor oligonucleotide, wherein the first construction oligonucleotide and the anchor oligonucleotide has sequence complementarity; and

c) hybridizing a second construction oligonucleotide with the first construction oligonucleotide, wherein the first construction oligonucleotide and the second construction oligonucleotide has sequence complementarity;

wherein the first construction oligonucleotide and the second construction oligonucleotide together form a target polynucleotide.

26. The method of claim 25, further comprising providing a first and a second template oligonucleotides bound to a first and a second feature of a support, respectively, and generating the first and second construction oligonucleotides complementary to the first and second template oligonucleotides, respectively, in a chain extension reaction.

27. The method of claim 25, wherein the second construction oligonucleotide is a stem-loop oligonucleotide having an overhang complementary to a region of the first construction oligonucleotide.

28. The method of claim 27, further comprising ligating the first construction oligonucleotide with the second construction oligonucleotide.