DEVICES AND METHODS FOR OLIGONUCLEIC ACID LIBRARY SYNTHESIS

• US 20160310927A1
• Filed: 04/21/2016
• Published: 10/27/2016
• Est. Priority Date: 04/21/2015
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: MARSH, Eugene P., BANYAI, William, PECK, Bill James, FERNANDEZ, Andres, CHEN, Siyuan, INDERMUHLE, Pierre
• Granted Patent: US 9,981,239 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00522 in a multiple parallel arrangement
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 3/50857 using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Devices and methods for de novo synthesis of large and highly accurate libraries of oligonucleic acids are provided herein. Devices include structures having a main channel and microchannels, where the microchannels have a high surface area to volume ratio. Devices disclosed herein provide for de novo synthesis of oligonucleic acids having a low error rate.

Claims

1. A device for synthesizing oligonucleic acids, comprising:

a plate;

a plurality of main channels, wherein each main channel extends vertically into the plate from an opening on a top side of the plate; and

for each main channel, a plurality of microchannels connected thereto, wherein each microchannel of the plurality of microchannels extends vertically from an opening on a bottom side of the plate to said main channel, and wherein each microchannel of the plurality of microchannels has a surface area to volume ratio of greater than 0.2 (1/um).

2. The device of claim 1, wherein the surface area to volume ratio provides for rapid exchange of chemical exposure during de novo synthesis of oligonucleic acids.

3. The device of claim 1, wherein the surface area to volume ratio is about 0.4 (1/um).

6. The device of claim 1, wherein each microchannel of the plurality of microchannels has a surface area greater than 10,000 um².

8. The device of claim 6, wherein each microchannel of the plurality of microchannels has a surface area of about 13,000 um².

9. The device of claim 1, wherein the plurality of microchannels comprises 50 to 500 microchannels.

11. The device of claim 1, wherein a ratio of width to depth of a narrowest segment of each microchannel is from 0.5 to 0.01.

13. The device of claim 1, wherein each microchannel of the plurality of microchannels has a total width of 30 um to 100 um.

15. The device of claim 1, wherein each microchannel of the plurality of microchannels has a depth of 10 to 500 um.

17. The device of claim 1, wherein each main channel has a width from 0.5 to 2 mm.

20. The device of claim 1, wherein the plurality of main channels comprises more than 250 main channels.

21. The device of claim 20, wherein the plurality of main channels comprises about 10,000 main channels.

22. The device of claim 1, further comprising a plurality of first molecules, wherein each first molecule is bound to an interior surface of the plurality of microchannels and comprises a reactive group capable of binding to a nucleoside phosphoramidite.

23. The device of claim 22, wherein each first molecule is a silane or a siloxane.

24. The device of claim 23, wherein each first molecule is an aminosilane.

25. The device of claim 22, wherein each first molecule is 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

26. The device of claim 22, further comprising a plurality of second molecules, wherein each second molecule is bound to an interior surface of the main channel and lacks the reactive group capable of binding to the nucleoside phosphoramidite.

27. The device of claim 26, wherein each second molecule is a fluorosilane.

28. The device of claim 27, wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

29. The device of claim 26, wherein the first molecule has a higher surface energy than the second molecule.

30. The device of claim 1, wherein the plate is a silicon plate.

60. A method for de novo oligonucleic acid synthesis, comprising:

(a) providing predetermined sequences for a plurality of non-identical oligonucleic acids;

(b) providing the device of claim 1;

(c) adding a droplet of fluid comprising an extension reaction reagent specific to a microchannel;

(d) allowing sufficient time for an extension reaction step to occur; and

(e) repeating steps (c) and (d) until the plurality of non-identical oligonucleic acids are synthesized, wherein each oligonucleic acid at least 10 bases in length and attached to an inside region of the microchannel.

63. The method of claim 60, wherein the synthesized non-identical oligonucleic acids encode sequences with an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences.

64. The method of claim 60, further comprising washing the surface with a washing reagent, and wherein washing removes greater than 95% of unincorporated extension reaction reagent.

65. The method of claim 64, wherein washing removes greater than 99% of unincorporated extension reaction reagent.

69. The method of claim 60, wherein the synthesized plurality of non-identical oligonucleic acids collectively encode for at least 200 genes at least 1 kb in length.

70. The method of claim 60, further comprising:

releasing the plurality of non-identical oligonucleic acids from the surface; and

subjecting the plurality of non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes.

71. The method of claim 70, wherein the at least 200 genes have an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences without correcting errors.

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33. A device for synthesizing oligonucleic acids, comprising:

a silicon plate;

a plurality of main channels, wherein each main channel extends vertically into the silicon plate from an opening on a top side of the silicon plate, and wherein each main channel has a width of 0.5 to 2 mm; and

for each main channel, a plurality of microchannels connected thereto, wherein each microchannel of the plurality of microchannels extends vertically from an opening on a bottom side of the silicon plate to said main channel, and wherein each microchannel of the plurality of microchannels comprises a total width that is less than 100 um, a microchannel surface area greater than 10,000 um, and a maximum width for the narrowest segment of the microchannel of 10 um.

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