Methods and devices for de novo oligonucleic acid assembly

• US 10,669,304 B2
• Filed: 02/03/2016
• Issued: 06/02/2020
• Est. Priority Date: 02/04/2015
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Indermuhle, Pierre F., Marsh, Eugene P., Fernandez, Andres, Banyai, William, Peck, Bill J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/015,059
• Publication Number:
  US 20160229884A1
• Time in Patent Office: 1,581 Days
• 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 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C23C 16/455 characterised by the method used for introducing gases into reaction chamber or for modifying gas flows in reaction chamber

Abstract

Methods and devices are provided herein for surfaces for de novo nucleic acid synthesis which provide for low error rates. In addition, methods and devices are provided herein for increased nucleic acid mass yield resulting from de novo nucleic acid synthesis.

Claims

1. A method for preparing a surface for polynucleotide synthesis, the method comprising:

depositing a first population of a first plurality of molecules on a surface of a solid support at a first region, wherein the first region comprises a plurality of loci, and wherein the first plurality of molecules comprises a plurality of non-coupling molecules that bind to the surface and lack a reactive group capable of binding to a nucleoside; and

depositing a mixture on the solid support at the first region, wherein the mixture comprises a second population of the first plurality of molecules and a second plurality of molecules, and wherein the second plurality of molecules comprises a plurality of coupling molecules that bind to the solid support and comprise the reactive group that is capable of binding to the nucleoside, and wherein the first plurality of molecules and the second plurality of molecules are present in the mixture in a molar ratio of 10;

1 to 2500;

1.

2. The method of claim 1, wherein each of the plurality of coupling molecules is a silane.

3. The method of claim 1, wherein each of the plurality of coupling molecules is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

4. The method of claim 1, wherein the second molecule each of the plurality of coupling molecules is 3-glycidoxypropyltrimethoxysilane.

5. The method of claim 2, wherein the silane is an aminosilane.

6. The method of claim 1, wherein each of the plurality of non-coupling molecules is propyltrimethoxysilane.

7. The method of claim 1, wherein each of the plurality of non-coupling molecules is a fluorosilane.

8. The method of claim 7, wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

9. The method of claim 1, wherein the mixture comprises the plurality of non-coupling molecules and the plurality of coupling molecules present in a molar ratio of about 100:

1 to about 2500;

1.

10. The method of claim 1, wherein the mixture comprises the plurality of non-coupling molecules and the plurality of coupling molecules present in a molar ratio of about 2000:

1.

11. The method of claim 1, wherein the plurality of coupling molecules lacks a free hydroxyl, amino, or carboxyl group.

12. The method of claim 1, wherein the mixture or the first population is in a gaseous state when deposited on the surface.

13. The method of claim 1, wherein the surface comprises a layer of silicon oxide.

14. The method of claim 1, further comprising synthesizing a plurality of polynucleotides, wherein each polynucleotide of the plurality of polynucleotides comprises at least 10 bases in length.

15. The method of claim 14, wherein the plurality of polynucleotides encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to predetermined sequences.

16. The method of claim 15, wherein the aggregate deletion error rate is achieved without correcting errors.

17. The method of claim 14, wherein the plurality of polynucleotides synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to predetermined sequences without correcting errors.

18. The method of claim 14, wherein each polynucleotide of the plurality of polynucleotides is 50 to 120 bases in length.

19. The method of claim 14, wherein the plurality of polynucleotides comprises at least 5,000 polynucleotides.

20. The method of claim 14, wherein the plurality of polynucleotides comprises at least 30,000 polynucleotides.