Methods and devices for de novo oligonucleic acid assembly

Patent metadata

Field	Value
Publication number	US-2016229884-A1
Application number	US-201615015059-A
Country	US
Kind code	A1
Filing date	Feb 3, 2016
Priority date	Feb 4, 2015
Publication date	Aug 11, 2016
Grant date	—

Abstract

Methods and devices are provided herein for surfaces for de novo nucleic acid synthesis which provide for low error rates. In addition, methods and devices are provided herein for increased nucleic acid mass yield resulting from de novo nucleic acid synthesis.

First claim

1 . A method for preparing a surface for oligonucleic acid synthesis, the method comprising:
(a) providing a silicon wafer comprising a surface; (b) depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; (c) depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci, wherein each locus is surrounded by the first region, and wherein the second molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and (d) depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and is capable of binding nucleoside phosphoramidite, and wherein the second molecule and the third molecule are present in the mixture in a molar ratio of 10:1 to about 2500:1.

2 . The method of claim 1 , wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule.

3 . The method of claim 2 , wherein a difference in water contact angle between the first region and the second region is at least 10 degrees.

4 . The method of claim 1 , wherein the third molecule is a silane.

5 . The method of claim 1 , wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxy silane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

6 . (canceled)

7 . The method of claim 4 , wherein the silane is an aminosilane.

8 . The method of claim 1 , wherein the second molecule is propyltrimethoxysilane.

9 . The method of claim 1 , wherein the first molecule is a fluorosilane.

10 . The method of claim 9 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

11 . The method of claim 1 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 50:1 to 2500:1.

12 . (canceled)

13 . The method of claim 1 , wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1.

14 . The method of claim 1 , wherein the first molecule or the second molecule lacks a free hydroxyl, amino, or carboxyl group.

15 . (canceled)

16 . The method of claim 1 , wherein the mixture or the first molecule is in a gaseous state when deposited on the surface.

17 . The method of claim 1 , wherein the surface comprises a layer of silicon dioxide.

18 . The method of claim 1 , comprising:
coating the surface with a light-sensitive material; exposing the first region of the surface to a light source to remove a portion of the light-sensitive material coated on the surface; depositing the first molecule on the surface; removing a remaining portion of the light-sensitive material coated on the surface to expose the second region; depositing the second molecule on the surface at the second region; and depositing the mixture on the surface at the second region.

19 - 35 . (canceled)

36 . The method of claim 18 , further comprising applying oxygen plasma to the surface prior to coating the surface with the light-sensitive material or after exposing predetermined regions of the surface to light.

37 . A method for oligonucleic acid synthesis, the method comprising:
(a) providing predetermined sequences for at least 20,000 non-identical oligonucleic acids; (b) providing a silicon wafer comprising a patterned surface, wherein the patterned surface is generated by:
depositing a first molecule on the surface at a first region, wherein the first region comprises a plurality of loci, and wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and
depositing a mixture on the surface at the first region, wherein the mixture comprises the first molecule and a second molecule, wherein the second molecule binds to the surface and is capable of binding nucleoside phosphoramidite, and wherein the first molecule and the second molecule are present in the mixture in a molar ratio of 10:1 to about 2500:1; and
(c) synthesizing the at least 20,000 non-identical oligonucleic acids each at least 10 bases in length, wherein the at least 20,000 non-identical oligonucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences, and wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus.

38 . The method of claim 37 , further comprising depositing a third molecule on the surface at a second region, wherein the third molecule binds to the surface and lacks a reactive group that binds to nucleoside phosphoramidite, wherein the first molecule and the second molecule both have a higher surface energy than a surface energy of the third molecule.

39 . The method of claim 38 , wherein a difference in water contact angle between the first region and the second region is at least 10 degrees.

40 . The method of claim 37 , wherein the second molecule is a silane.

41 . The method of claim 37 , wherein the second molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxy silane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.

42 . The method of claim 40 , wherein the silane is an aminosilane.

43 . (canceled)

44 . The method of claim 37 , wherein the first molecule is propyltrimethoxysilane.

45 . The method of claim 38 , wherein the third molecule is a fluorosilane.

46 . The method of claim 45 , wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

47 . The method of claim 37 , wherein the mixture comprises the first molecule and the second molecule present in a molar ratio of 50:1 to 2500:1.

48 . (canceled)

49 . The method of claim 37 , wherein the mixture comprises the first molecule and second present in a molar ratio of 2000:1.

50 - 54 . (canceled)

55 . The method of claim 37 , wherein each of the at least 20,000 non-identical oligonucleic acids is 10 bases to 1 kb in length.

56 . The method of claim 55 , wherein each of the at least 20,000 non-identical oligonucleic acids is about 50 to about 120 bases in length.

57 . The method of claim 37 , wherein the aggregate deletion error rate of the at least 20,000 non-identical oligonucleic acids is less than about 1 in 1700 bases compared to the predetermined sequences.

58 . The method of claim 62 , wherein the aggregate deletion error rate of the assembled at least 200 preselected nucleic acids is achieved without correcting errors.

59 - 61 . (canceled)

62 . The method of claim 37 , further comprising:
(d) releasing the at least 20,000 non-identical oligonucleic acids from

Assignees

• Twist Bioscience Corp

Inventors

• Indermuhle Pierre F
• Marsh Eugene P
• Fernandez Andres
• Banyai William
• Peck Bill J

Classifications

• B01J2219/00711

  Light-directed synthesis · CPC title

• B01J2219/00432

  Photolithographic masks · CPC title

• B01J2219/00659

  Two-dimensional arrays · CPC title

• B01J2219/00529

  DNA chips · CPC title

• B01J2219/00722

  Nucleotides · CPC title