DE NOVO SYNTHESIZED GENE LIBRARIES

• US 20160096160A1
• Filed: 10/16/2015
• Published: 04/07/2016
• 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
• Granted Patent: US 9,403,141 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 2219/00313 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

1-20. -20. (canceled)

21. A device for nucleic acid assembly, comprising:

a silicon dioxide structure comprising a plurality of wells, wherein each well of the plurality of wells has height from 0.1 mm to 0.6 mm, and wherein each well of the plurality of wells has a cross-section from 0.8 to 2.0 mm;

an inner region of each well of the plurality of wells, wherein the inner region comprises a high surface energy coating;

an outer region of each well of the plurality of wells, wherein the outer region comprises a low surface energy coating, and wherein a difference in water contact angle between the inner region and the outer region is at least 10 degrees as measured on a planar surface.

22. The device of claim 21, wherein each well of the plurality of wells comprises an aqueous liquid, wherein the aqueous liquid has a surface tension from 12 to 80 millinewtons per meter.

23. The device of claim 22, wherein the aqueous liquid is water.

24. The device of claim 22, wherein the aqueous liquid comprises a DNA polymerase and dNTPs.

25. The device of claim 21, wherein the difference in water contact angle between the inner region and outer region is at least 20 degrees as measured on a planar surface.

26. The device of claim 21, wherein the difference in water contact angle between the inner region and outer region is at least 75 degrees as measured on a planar surface.

27. The device of claim 21, wherein each well of the plurality of wells has an interior diameter of 1.15 mm, and wherein the height of each well is 0.45 mm.

28. The device of claim 21, wherein the silicon dioxide structure comprises 2 to 250 wells.

29. The device of claim 21, wherein a surface of the silicon dioxide structure comprises silicon oxide located at a base of each well of the plurality of wells.

30. The device of claim 21, wherein the plurality of wells are configured for alignment to a plurality of chambers of an oligonucleic acid synthesizer.

31. The device of claim 30, wherein each well of the plurality of wells has a diameter that is the same as a diameter for each chamber of the plurality of chambers.

32. The device of claim 30, wherein the plurality of wells are configured for alignment to the plurality of chambers of the oligonucleic acid synthesizer within an enclosure.

33. The device of claim 30, wherein the outer region of each well of the plurality of wells comprises a sealing reagent.

34. A device for nucleic acid assembly, comprising:

a structure comprising a plurality of wells, wherein each well of the plurality of wells has a height from 0.1 mm to 0.6 mm, and wherein each well of the plurality of wells has a cross-section from 0.8 to 2.0 mm;

an inner region of each well of the plurality of wells, wherein the inner region comprises a hydrophilic coating;

an outer region of each well of the plurality of wells, wherein the outer region comprises a hydrophobic coating, and wherein the hydrophobic coating has a surface energy lower than a surface energy of the hydrophilic coating; and

an aqueous liquid comprising nucleic acid assembly reagents, wherein the aqueous liquid has a surface tension from 12 to 80 millinewtons per meter.

35. The device of claim 34, wherein the surface tension of the aqueous liquid is from 40 to 80 millinewtons per meter.

36. The device of claim 34, wherein the surface tension of the aqueous liquid is from 20 to 35 millinewtons per meter.

37. The device of claim 34, wherein the hydrophilic coating comprises a silane.

38. The device of claim 37, 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-aldehydr-trimethoxysilane. heptadecafluorodecyltrimethoxysilane, or glycidoxypropyltrimethoxysilane.

39. The device of claim 34, wherein the hydrophilic coating comprises an aminosilane.

40. The device of claim 34, wherein the hydrophobic coating comprises a fluorosilane.

41. The device of claim 40, wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

42. The device of claim 34, wherein the plurality of wells are configured for alignment to a plurality of chambers of an oligonucleic acid synthesizer.

43. The device of claim 42, wherein each well of the plurality of wells has a diameter that is the same as a diameter for each chamber of the plurality of chambers.

44. The device of claim 42, wherein the plurality of wells are configured for alignment to the plurality of chambers of the oligonucleic acid synthesizer within an enclosure.

45. The device of claim 42, wherein the outer region of each well of the plurality of wells comprises a sealing reagent.

46. The device of claim 34, wherein each well of the plurality of wells has an interior diameter of 1.15 mm, and wherein the height of each well is 0.45 mm.

47. The device of claim 34, wherein the structure comprise silicon dioxide.

48. The device of claim 47, wherein a surface of the structure comprises silicon oxide at a base of each well of the plurality of wells.

49. The device of claim 34, wherein the structure comprises 2 to 250 wells.

50. The device of claim 34, wherein the nucleic acid assembly reagents comprise a DNA polymerase and dNTPs.