De novo synthesized gene libraries

• US 9,403,141 B2
• Filed: 10/16/2015
• Issued: 08/02/2016
• Est. Priority Date: 08/05/2013
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Chen, Siyuan, Banyai, William, Peck, Bill James, Fernandez, Andres, Indermuhle, Pierre
• Primary Examiner(s): LEVKOVICH, NATALIA A
• Application Number:
  US14/885,963
• Publication Number:
  US 20160096160A1
• Time in Patent Office: 291 Days
• Field of Search: 422/552, 422/551
• 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 2219/00313 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 nucleic acid assembly, comprising:

(i) a first silicon plate, the first silicon plate comprising;

a base layer of the first silicon plate;

an insulator layer on top of the base layer, wherein the insulator layer comprises silicon dioxide; and

one or more wells, wherein each well of the one or more wells comprises;

a side wall that extends vertically from the insulator layer, wherein the side wall has a length from 0.1 to 0.6 mm;

an interior region of the well having a width from 0.8 to 2.0 mm; and

an exterior region of the well, wherein the exterior region of the well has a lower surface energy than the interior region of the well; and

(ii) a second silicon plate, wherein the second silicon plate comprises a plurality of clusters, wherein each cluster comprises a plurality of loci, wherein each locus comprises a physically distinct coating comprising molecules capable of binding to a nucleoside phosphoramidite, and wherein each cluster is vertically aligned to a single well of the one or more wells.

2. The device of claim 1, wherein each well of the one or more wells comprises an aqueous liquid, wherein the aqueous liquid has a surface tension from 12 to 80 millinewtons per meter.

3. The device of claim 1, wherein the width of the interior region of the well is 1.15 mm, and wherein the length of the side wall is 0.45 mm.

4. The device of claim 1, wherein the device further comprises an enclosure, wherein the first silicon plate and second silicon plate are both within the enclosure.

5. The device of claim 1, wherein each well of the one or more wells comprises a sealing means located at a distal end of the side wall of said well.

6. The device of claim 1, wherein the interior region of each well of the one or more wells comprises a silane.

7. The device of claim 6, wherein the silane is methoxyethoxyundecyltrichlorosilane, methacryloxypropyltrimethoxysilane, ethyltrimethoxysilane, propyltrimethoxysilane, trifluoropropyltrimethoxysilane, 3-(2-aminoethyl)aminopropyltrimethoxysilane, p-tolyltrimethoxysilane, cyanoethyltrimethoxysilane, aminopropyltriethoxysilane, acetoxypropyltrimethoxysilane, phenyltrimethoxysilane, chloropropyltrimethoxysilane, mercaptopropyltrimethoxysilane, trichloro-octodecyl-silane, methyltrimethoxysilane, butyl-aldehyde-trimethoxysilane, heptadecafluorodecyltrimethoxysilane, or glycidoxypropyltrimethoxysilane.

8. The device of claim 1, wherein the interior region of each well of the one or more wells comprises an aminosilane.

9. The device of claim 1, wherein the exterior region of each well of the one or more wells comprises a fluorosilane.

10. The device of claim 9, wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

11. The device of claim 1, wherein each cluster of the plurality of clusters of the second silicon plate has a lower surface energy than the interior region of each well the plurality of wells of the first silicon plate.

12. The device of claim 1, where each locus of the plurality of loci comprises a microchannel.