De novo synthesized gene libraries

• US 9,889,423 B2
• Filed: 08/23/2016
• Issued: 02/13/2018
• 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:
  US15/245,054
• Publication Number:
  US 20160354752A1
• Time in Patent Office: 539 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 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• 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

De novo synthesized large libraries of nucleic acids are provided herein with low error rates. Further, devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Longer nucleic acids can be synthesized in parallel using microfluidic assemblies. Further, methods herein allow for the fast construction of large libraries of long, high-quality genes. Devices for the manufacturing of large libraries of long and high-quality nucleic acids are further described herein.

Claims

1. A device for synthesis of nucleic acids, the device comprising:

a plate;

a plurality of clusters, wherein each cluster is located on a top surface of the plate, wherein a horizontal cross-sectional shape of each cluster is defined by a plurality of loci, wherein each cluster has a net surface energy that is higher than a surface energy for a region of the top surface of the plate adjacent to and surrounding each cluster, wherein the plurality of loci located within each cluster comprise 50 to 500 loci, and wherein each locus has a total width of up to 500 um; and

a plurality of posts located within each locus, wherein each post extends from the top surface of the plate, has a horizontal cross-section that is rectangular or circular in shape, is separated from a neighboring post, has a width of up to 1 um, and has a height from the top surface of the plate of up to 1 um, wherein a ratio of the width to the height of each post is from 0.1 to 1, wherein each post comprises a plurality of oligonucleotides bound thereto, wherein each of the oligonucleotides comprises at least 80 nucleotides in length, and wherein the plurality of posts provide for rapid exchange of chemical exposure during de novo synthesis of oligonucleotides.

2. The device of claim 1, wherein the ratio of the width to the height of each post is from 0.4 to 0.8.

3. The device of claim 1, wherein each of the posts has rounded edges.

4. The device of claim 1, wherein each locus has a total width of 1 um to 500 um.

5. The device of claim 1, wherein each locus has a total width of 1 um to 100 um.

6. The device of claim 1, wherein a total number of the loci on the surface comprises 700,000 loci.

7. The device of claim 1, wherein the width of each locus is up to 300 times larger than the width of each of the posts.

8. The device of claim 1, wherein each cluster provides for a water contact angle that is up to 90 degrees greater than a water contact angle of the region of the top surface adjacent to and surrounding each cluster.

9. The device of claim 1, wherein each cluster provides for a water contact angle that is up to 20 degrees greater than a water contact angle of the region of the top surface adjacent to and surrounding each cluster.

10. The device of claim 1, wherein each cluster has a square or round shape.

11. The device of claim 1, wherein each cluster has a width of 0.5 mm to 1.5 mm.

12. The device of claim 1, wherein each locus comprises a first plurality of molecules, and wherein the first plurality of molecules are each bound to the top surface of the plate and comprise a reactive group capable of binding to a nucleoside.

13. The device of claim 12, wherein the first plurality of molecules comprises a silane or a siloxane.

14. The device of claim 12, wherein the first plurality of molecules comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, or (3-aminopropyl)triethoxysilane or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

15. The device of claim 12, wherein a region surrounding each locus comprises a second plurality of molecules, wherein the second plurality of molecules are each bound to the top surface of the plate and lack a reactive group capable of binding to a nucleoside.

16. The device of claim 15, wherein the second plurality of molecules comprises a fluorosilane.

17. The device of claim 16, wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

18. The device of claim 1, wherein each locus comprises a plurality of first molecules, wherein each first molecule is bound to the top surface of the plate and comprises a reactive group capable of binding to a nucleoside, and wherein a region adjacent to and surrounding each locus comprises a plurality of second molecules, wherein each second molecule is bound to the top surface of the plate and lacks the reactive group capable of binding to the nucleoside, and wherein each locus has a higher surface energy than the region adjacent to and surrounding each locus.

19. The device of claim 1, wherein the plate comprises nylon, nitrocellulose, or polypropylene.

20. The device of claim 1, wherein the plate comprises glass, silica, silicon, polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, gold, platinum, agarose, dextran, polyacrylamides, or polydimethylsiloxane (PDMS).

21. The device of claim 1, wherein the plate comprises a silicon layer.

22. The device of claim 1, wherein each of the oligonucleotides comprises at least 100 nucleotides in length.