Methods and devices for nucleic acid synthesis

• US 8,716,467 B2
• Filed: 03/02/2011
• Issued: 05/06/2014
• 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
• Primary Examiner(s): Gussow, Anne
• Assistant Examiner(s): Brown, Mindy G
• Application Number:
  US13/038,714
• Publication Number:
  US 20110217738A1
• Time in Patent Office: 1,161 Days
• Field of Search: None
• US Class Current: 536/25.3
• 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 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, each plurality of oligonucleotides having a different predetermined subunit sequence at its 5′

end and a degenerate binding sequence at its 3′

end, wherein the plurality of oligonucleotides is covalently linked at the 3′

end via a plurality of binding sequences to the solid support and wherein the predetermined subunit sequences together comprise the at least one polynucleotide;

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

(c) advancing a droplet comprising the first extension product from the first spot to a predetermined second spot, the second spot having a second plurality of oligonucleotides comprising a degenerate binding sequence and a second predetermined subunit sequence different than the first predetermined sequence; and

(d) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second spot to produce a second extension product comprising the first and the second predetermined subunit sequences.

2. The method of claim 1 wherein the degenerate sequences have 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 method 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 method of claim 1, further comprising individually 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 method 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 method of claim 1 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 extension product comprising the first predetermined subunit sequence.

7. The method of claim 6 further comprising cleaving the primer to remove unwanted primer sequences from the extension product comprising the predetermined sequence.

8. The method of claim 6 further comprising using the first extension product as a primer for chain extension at the second spot comprising the second plurality of oligonucleotides having the degenerate binding sequence and the second predetermined subunit sequence and using the second predetermined subunit sequence as a template to produce the second extension product comprising the first and the second predetermined subunit sequences.

9. The method of claim 1 wherein the droplet is advanced by electrical, magnetic or mechanical action.

10. The method of claim 1 wherein a predetermined combination of a subset of spots is used to produce the at least one polynucleotide.

11. The method of claim 1 wherein the degenerate sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long.

12. The method of claim 1 further repeating steps c and d to produce the at least one polynucleotide having the predetermined sequence.

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

(a) providing a solid support having a plurality of distinct features thereon, each feature comprising a plurality of oligonucleotides bound to the support through their 3′

end, each oligonucleotide having a sequence complementary to a predetermined subunit sequence at its 5′

end and a degenerate binding sequence at its 3′

end, wherein the predetermined subunit sequences together comprise the at least one polynucleotide;

(b) providing a droplet to a first predetermined feature comprising a first plurality of oligonucleotides, wherein the droplet comprises a sequence capable of annealing to the degenerate sequence of the first plurality of oligonucleotides;

(c) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction;

(d) generating within a droplet a first extension product comprising the first predetermined subunit sequence;

(e) advancing the droplet comprising the first extension product from the first feature to a predetermined second feature, the second feature having a second plurality of oligonucleotides comprising a degenerate binding sequence and a sequence complementary to a second predetermined subunit sequence different than the first predetermined sequence;

(f) providing one or more reagents thereby allowing one or more of annealing, denaturing, chain extension, ligation, and digestion reaction at the second feature; and

(h) generating a second extension product comprising the first and the second predetermined subunit sequences.