Methods and devices for nucleic acid synthesis

• US 9,388,407 B2
• Filed: 03/31/2014
• Issued: 07/12/2016
• 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): Joike, Michele K
• Assistant Examiner(s): Brown, Mindy G
• Application Number:
  US14/230,683
• Publication Number:
  US 20150065393A1
• Time in Patent Office: 834 Days
• Field of Search: None
• US Class Current: 1/1
• 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 a target polynucleotide having a predetermined sequence, the method comprising

(a) providing a solid support having a first feature and a second feature thereon, the first feature having a first plurality of immobilized oligonucleotides each comprising a first predetermined subunit sequence and a first degenerate binding sequence, the second feature having a second plurality of immobilized oligonucleotides each comprising a second predetermined subunit sequence and a second degenerate binding sequence, wherein the first predetermined subunit sequence and the second predetermined subunit sequence together comprise a target polynucleotide;

(b) generating, in a chain extension reaction using an input oligonucleotide as primer that anneals with the first degenerate binding sequence, a first product comprising the first predetermined subunit sequence at the first feature;

(c) advancing, via microfluidic communication, the first product to the second feature; and

(d) generating, in a chain extension reaction using the first product as primer that anneals with the second degenerate binding sequence, a second product comprising the first and the second predetermined subunit sequences at the second feature.

2. The method of claim 1 further comprising

(i) providing a first solution at the first feature, wherein the first solution comprises the input oligonucleotide and one or more reagents that allow one or more of annealing, denaturing, chain extension, ligation, and digestion reaction to produce the first product;

and

(ii) providing a second solution to the second feature, wherein the second solution comprises one or more reagents that allow one or more of annealing, denaturing, chain extension reaction, ligation, and digestion reaction to produce the second product.

3. The method of claim 1 wherein the first and second degenerate binding sequences are different and each have a length of N1 nucleosides, wherein each degenerate sequence is one of 4^N1 possible sequences.

4. The method of claim 3 wherein N1 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

5. The method of claim 1 wherein the first and second predetermined subunit sequences each have a length of N2 nucleosides and are one of 4^N2 possible sequences, wherein the solid support comprises 4^N2 spots or a subset or superset thereof.

6. The method of claim 5 wherein N2 is an integer between 2 and 50.

7. The method of claim 1 further comprising individually controlling a temperature at each feature on the solid support, above a predetermined temperature which corresponds to an average annealing temperature of the degenerate binding sequences.

8. The method of claim 7 further comprising controlling the temperature at above the predetermined temperature such that duplexes stabilized by a polymerase or a subunit thereof remain substantially annealed at said temperature.

9. The method of claim 2 further comprising providing in the first solution a polymerase or a subunit thereof, and dNTPs, thereby allowing chain extension of the input oligonucleotide using the first predetermined subunit sequence as a template to produce the first product.

10. The method of claim 9 further comprising cleaving the input oligonucleotide to remove unwanted primer sequences.

11. The method of claim 1 further comprising repeating steps (c) and (d) one or more times until a longer polynucleotide is produced.

12. The method of claim 1 wherein the solid support is positioned in a microfluidic channel.

13. The method of claim 1 wherein the solid support has a plurality of features, a predetermined combination of a subset of which features is used to produce the target polynucleotide.

14. The method of claim 5 wherein N2 is 3, 4, 5, 6, 7, 8, 9, or 10.

15. The method of claim 3 wherein N1 is an integer between 5 and 100.

16. The method of claim 9 wherein the input oligonucleotide contains a site for cleavage by a type IIs restriction endonuclease.

17. The method of claim 16 wherein the type IIs restriction endonuclease is one or more of Bsr I, Bsm I, BstF5 I, BsrD I, Bts I, Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I, Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I, Psr I, BsmA I, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I, BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I, Mly I or Btr I.

18. The method of claim 1 wherein the first and second plurality of immobilized oligonucleotides are immobilized via a linker.