Methods and Devices for Nucleic Acid Synthesis

• US 20110217738A1
• Filed: 03/02/2011
• Published: 09/08/2011
• Est. Priority Date: 03/03/2010
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph
• Granted Patent: US 8,716,467 B2
• Time in Patent Office: Days
• US Class Current: 435/91.5
• CPC Class Codes: C12M 1/00 Apparatus for enzymology or microbiology
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Disclosed are devices and methods to synthesize polynucleotides and libraries of polynucleotides such as libraries of oligonucleotides. In exemplary embodiments, the device includes a support having a plurality of features. Each feature contains a plurality of oligonucleotides. Within each feature, each of the plurality of oligonucleotides includes an identical predetermined subunit sequence of X nucleosides and a degenerate sequence of Y nucleosides. A predetermined combination of a subset of the features can be used to produce a polynucleotide having a predetermined sequence of Z nucleosides.

Claims

1. A device for synthesizing at least one polynucleotide having a predetermined sequence, the device comprising:

a solid support comprising a plurality of spots, wherein each of the plurality of spots comprises a plurality of oligonucleotides having a predetermined subunit sequence, wherein the plurality of oligonucleotides are covalently linked at the 3′

end via a plurality of binding sequences on the solid support;

a microfluidic member for providing a droplet to a first spot having a plurality of first oligonucleotides having a first predetermined subunit sequence, wherein the droplet comprises one or more reagents that allow one or more of annealing, denaturing, chain extension, ligation, and digestion reaction to produce a first product comprising the first predetermined subunit sequence; and

a member for advancing microfluidic communication between the first spot and a second spot, wherein the second spot has a second plurality of oligonucleotides having a second predetermined subunit sequence, whereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second spot to produce a second product comprising the first and the second predetermined subunit sequences.

2. The device of claim 1 wherein the plurality of binding sequences comprises degenerate sequences having a length of N1 nucleosides, wherein the plurality of binding sequences comprises up to 4^N1 different sequences and wherein N1 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

3. The device of claim 1 wherein the subunit sequence has a length of N2 nucleosides and is one of 4^N2 possible sequences, wherein the solid support comprises 4^N2 spots or a subset or superset thereof and wherein N2 is 3, 4, 5, 6, 7, 8, 9, or 10.

4. The device of claim 1, further comprising means for controlling a temperature of the droplet at each of the plurality of spots on the solid support, wherein the temperature is controlled at 1°

C., 2°

C., 3°

C., 4°

C., 5°

C., 6°

C., 7°

C., 8°

C., 9°

C., 10°

C. or 20°

C. above a predetermined temperature which corresponds to an average annealing temperature of the plurality of binding sequences.

5. The device of claim 4 wherein the temperature is controlled at above the predetermined temperature such that duplexes stabilized by a polymerase or a subunit thereof remain substantially annealed at said temperature.

6. The device of claim 1 wherein the droplet comprises a primer that at least partially binds to at least one of the plurality of the binding sequences on the first spot, a polymerase or a subunit thereof, and dNTPs, thereby allowing chain extension of the primer using the first predetermined subunit sequence as a template to produce the first product comprising the first predetermined subunit sequence.

7. The device of claim 1 wherein the first product serves as a primer for chain extension at the second spot using the second predetermined subunit sequence as a template to produce the second product comprising the first and the second predetermined subunit sequences.

8. The device of claim 1 wherein the first and the second predetermined subunit sequences are the same.

9. The device of claim 1 wherein the first and the second predetermined subunit sequences are complementary.

10. A method for synthesizing at least one polynucleotide having a predetermined sequence, the method comprising:

(a) providing a solid support having a plurality of spots thereon, wherein each of the plurality of spots comprises a plurality of oligonucleotides having a predetermined subunit sequence, wherein the plurality of oligonucleotides is covalently linked at the 3′

end via a plurality of binding sequences to the solid support;

(b) providing a droplet to a first spot comprising a first plurality of oligonucleotides having a first predetermined subunit sequence, wherein the droplet comprises one or more reagents that allow one or more of annealing, denaturing, chain extension, ligation, and digestion reaction to produce a first product comprising the first predetermined subunit sequence; and

(c) advancing microfluidic communication between the first spot and a second spot having a second plurality of oligonucleotides having a second predetermined subunit sequence, thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second spot to produce a second product comprising the first and the second predetermined subunit sequences.

11. The method of claim 10 wherein the plurality of binding sequences comprises degenerate sequences having a length of N1 nucleosides, wherein the plurality of binding sequences comprises up to 4^N1 different sequences and wherein N1 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

12. The method of claim 10 wherein the subunit sequence has a length of N2 nucleosides and is one of 4^N2 possible sequences, wherein the solid support comprises 4^N2 spots or a subset or superset thereof and wherein N2 is 3, 4, 5, 6, 7, 8, 9, or 10.

13. The method of claim 10, further comprising controlling a temperature of the droplet at each of the plurality of spots on the solid support, wherein the temperature is controlled at 1°

C., 2°

C., 3°

C., 4°

C., 5°

C., 6°

C., 7°

C., 8°

C., 9°

C., 10°

C. or 20°

C. above a predetermined temperature which corresponds to an average annealing temperature of the plurality of binding sequences.

14. The method of claim 13 wherein the temperature is controlled at above the predetermined temperature such that duplexes stabilized by a polymerase or a subunit thereof remain substantially annealed at said temperature.

15. The method of claim 10 further comprising providing in the droplet a primer that at least partially binds to at least one of the plurality of the binding sequences on the first spot, a polymerase or a subunit thereof, and dNTPs, thereby allowing chain extension of the primer using the first predetermined subunit sequence as a template to produce the first product comprising the first predetermined subunit sequence.

16. The method of claim 15 further comprising cleaving the primer to remove unwanted primer sequences from the predetermined sequence.

17. The method of claim 15 further comprising using the first product as a primer for chain extension at the second spot and using the second predetermined subunit sequence as a template to produce the second product comprising the first and the second predetermined subunit sequences.

18. A device for synthesizing at least one polynucleotide having a predetermined sequence, the device comprising:

a support having a plurality of features, each feature comprising a plurality of different oligonucleotides immobilized via their 3′

end, wherein each plurality of oligonucleotides has a different predetermined subunit sequence of X nucleosides at its 5′

end and has a degenerate sequence of Y nucleosides at its 3′

end;

wherein the device has at least 4^X different features,

wherein a subset of the plurality of the features comprises oligonucleotides having different predetermined subunit sequences that together represent a polynucleotide having a predetermined sequence of Z nucleosides

19. The device of claim 18 wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of all possible combinations (4^Z) of the polynucleotide having the predetermined sequence of Z nucleosides are represented on the device.

20. The device of claim 18 wherein X is 3, 4, 5, 6, 7, 8, 9, or 10 and Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.