De novo synthesized gene libraries

• US 10,272,410 B2
• Filed: 10/16/2015
• Issued: 04/30/2019
• Est. Priority Date: 08/05/2013
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Banyai, William, Peck, Bill James, Fernandez, Andres, Chen, Siyuan, Indermuhle, Pierre
• Primary Examiner(s): Gordon, Brian R
• Application Number:
  US14/885,965
• Publication Number:
  US 20160089651A1
• Time in Patent Office: 1,292 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 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00623 Immobilisation or binding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00709 Type of synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/74 : Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/117 : incorporating modified base
• CPC Class Codes: C12Q 2525/186 : incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/00 : Methods of creating libraries, e.g. combinatorial synthesis
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 50/18 : using a particular method of attachment to the solid support

Abstract

Provided herein are devices for the manufacturing of high-quality oligonucleic acids. Longer nucleic acids, e.g., genes, can be synthesized in parallel using microfluidic assemblies described herein. Devices described herein include silicon plates having a plurality of channels in fluid communication with a plurality of microchannels. The number of microchannels and dimensions of the microchannels provide for rapid exchange of chemical exposure during de novo synthesis of oligonucleic acids.

Claims

1. A device for synthesizing oligonucleotides, the device comprising:

a silicon plate having formed therein;

a plurality of first channels, wherein each first channel extends vertically to a depth into the silicon plate from an opening on a top side of the silicon plate, and wherein each first channel has a width of 0.5 mm to 1.5 mm; and

for each first channel, 50 to 500 second channels in fluid communication with the first channel, wherein each second channel extends vertically to a height from an opening on a bottom side of the silicon plate to the first channel, wherein each second channel has a width of 1 um to 100 um, wherein a ratio of width to height of each second channel is from 1 to 0.1, wherein each second channel comprises a plurality of oligonucleotides bound to a first interior surface of said second channel, wherein each of the oligonucleotides comprises at least 80 nucleotides in length, and wherein the device provides for rapid exchange of chemical exposure during de novo synthesis of oligonucleotides, and

wherein the first interior surface of said second channel comprises a first set of molecules, wherein each molecule of the first set of molecules is an aminosilane or N-(3-triethoxvsilvlpropvD-4-hydroxybutvramide.

2. The device of claim 1, wherein the aminosilane comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, or (3-aminopropyl)triethoxysilane.

3. The device of claim 1, wherein each first channel comprises a second interior surface, wherein the second interior surface comprises a second set of molecules, wherein each molecule of the second set of molecules is a passive functionalization agent, bound to the second interior surface and lacks a reactive group capable of binding to a nucleoside phosphoramidite.

4. The device of claim 3, wherein the second set of molecules comprises a fluorosilane.

5. The device of claim 4, wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

6. The device of claim 1, wherein the plurality of first channels comprises 2 to 250 first channels.

7. The device of claim 1, wherein a total number of the second channels in the silicon plate is at least 15,000 second channels.

8. The device of claim 1, wherein a total number of the second channels in the silicon plate is at least 30,000 second channels.

9. The device of claim 1, wherein each first channel of the plurality of first channels has a width of 1 mm to 1.5 mm.

10. The device of claim 9, wherein each first channel of the plurality of first channels has a width of 1.15 mm.

11. The device of claim 1, wherein each of the 50 to 500 second channels has a width of 4 um to 70 um.

12. The device of claim 11, wherein each of the 50 to 500 second channels has a width of about 60 um.

13. The device of claim 1, wherein the first interior surface of each of the 50 to 500 second channels is coated with a layer of silicon dioxide.

14. The device of claim 1, wherein:

the first interior surface comprises an aminosilane bound thereto; and

each second channel comprises a second interior surface, wherein the second interior surface comprises a fluorosilane bound thereto.

15. The device of claim 1, wherein the ratio of width to height is 0.2.

16. The device of claim 1, wherein the ratio of width to height is 0.1.

17. The device of claim 1, wherein the ratio of width to height is 0.5.

18. The device of claim 1, wherein the 50 to 500 second channels comprise 100 to 200 second channels.

19. The device of claim 1, wherein each of the 50 to 500 second channels has a height of 10 um to 100 um.