De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-9409139-B2
Application number	US-201414452429-A
Country	US
Kind code	B2
Filing date	Aug 5, 2014
Priority date	Aug 5, 2013
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

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.

First claim

What is claimed is:

1. A method of synthesizing nucleic acids, comprising:
a) providing predetermined sequences for at least 200 preselected nucleic acids;
b) providing a structure comprising a patterned surface, wherein the patterned surface comprises predetermined regions coated with a first molecule, wherein the first molecule comprises a silane, a siloxane, or is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, and wherein each of the predetermined regions coated with the first molecule is surrounded by a region coated with a second molecule, wherein the second molecule binds to the patterned surface and lacks an available reactive group that binds to a nucleoside phosphoramidite;
c) synthesizing at least 20,000 non-identical polynucleotides attached to the patterned surface, wherein the at least 20,000 non-identical polynucleotides are each at least 80 bases long;
d) releasing the at least 20,000 non-identical polynucleotides from the patterned surface;
e) suspending the at least 20,000 non-identical polynucleotides in a solution; and
f) subjecting the solution comprising the at least 20,000 non-identical polynucleotides to a polymerase chain assembly reaction to assemble the at least 200 preselected nucleic acids, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences without correcting errors in the at least 200 assembled preselected nucleic acids, and wherein the patterned surface provides for the aggregate error rate of less than 1 in 2000 bases.

2. The method of claim 1 , wherein the at least 20,000 non-identical polynucleotides attached to the patterned surface are clustered, wherein each cluster is separated from another cluster by a structural barrier on the patterned surface, and wherein each cluster includes a subset of the at least 20,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

3. The method of claim 2 , wherein each cluster comprises 100 to 150 loci.

4. The method of claim 3 , wherein each locus is separated from another locus by a structural barrier on the patterned surface.

5. The method of claim 3 , wherein each locus is separated from another locus by a chemical barrier on the patterned surface.

6. The method of claim 2 , wherein each subset of the at least 20,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 1.0 to 1.5 mm in diameter.

7. The method of claim 1 , wherein the at least 20,000 non-identical polynucleotides released from the patterned surface are clustered, wherein each cluster includes a subset of the at least 20,000 non-identical polynucleotides, and wherein the subset of the at least 20,000 non-identical nucleotides collectively encodes for a single nucleic acid.

8. The method of claim 1 , wherein the at least 20,000 non-identical polynucleotides are released by gas cleavage.

9. The method of claim 1 , wherein steps a) to f) are completed in less than one week.

10. The method of claim 1 , wherein steps a) to f) are completed in less than 48 hours.

11. The method of claim 1 , wherein the assembled at least 200 preselected nucleic acids are at least 1 kb long on average.

12. The method of claim 1 , wherein the second molecule is a fluorosilane.

13. The method of claim 12 , wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

14. The method of claim 1 , wherein the first molecule is an aminosilane.

15. The method of claim 1 , wherein the first molecule is 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, or (3-aminopropyl)triethoxysilane.

16. The method of claim 1 , further comprising treating the assembled at least 200 preselected nucleic acids with an error correction enzyme.

17. A method of synthesizing nucleic acids, comprising:
a) providing predetermined sequences for at least 6,000 preselected nucleic acids;
b) providing a structure comprising a patterned surface, wherein the patterned surface comprises predetermined regions coated with a first molecule, wherein the first molecule comprises a silane, a siloxane, or is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, and wherein each predetermined region coated with the first molecule is surrounded by a region coated with a second molecule, wherein the second molecule binds to the patterned surface and lacks an available reactive group that binds to a nucleoside phosphoramidite;
c) synthesizing at least 800,000 of non-identical polynucleotides attached to the patterned surface, wherein the at least 800,000 non-identical polynucleotides are each at least 125 bases long and extend from the patterned surface at the predetermined regions coated with the first molecule;
d) releasing the at least 800,000 non-identical polynucleotides from the patterned surface;
e) suspending the at least 800,000 non-identical polynucleotides in a solution; and
f) subjecting the solution comprising the at least 800,000 non-identical polynucleotides to a polymerase chain assembly reaction to assemble the at least 6,000 preselected nucleic acids, wherein the assembled at least 6,000 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences without correcting errors in the assembled at least 6,000 preselected nucleic acids, and wherein the patterned surface provides for the aggregate error rate of less than 1 in 2000 bases.

18. The method of claim 17 , wherein the at least 800,000-non-identical polynucleotides attached to the patterned surface are clustered, wherein each cluster is separated from another cluster by a structural barrier of the patterned surface, and wherein each cluster includes a subset of the at least 800,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

19. The method of claim 18 , wherein each cluster comprises 100 to 150 loci.

20. The method of claim 19 , wherein each locus is separated from another locus by a structural barrier of the patterned surface.

21. The method of claim 18 , wherein each subset of the at least 800,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 1.0 to 1.5 mm in diameter.

22. The method of claim 17 , wherein the at least 800,000 non-identical polynucleotides released from the patterned surface are clustered, wherein each cluster includes a subset of the at least 800,000 non-identical polynucleotides, and wherein the subset of the at least 800,000-non-identical nucleotides collectively encodes for a single nucleic acid.

23. The method of claim 17 , wherein the first molecule is 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, or (3-aminopropyl)triethoxysilane.

24. The method of claim 17 , wherein the second molecule is a fluorosilane.

25. The method of claim 24 , wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

Assignees

• Twist Bioscience Corp

Inventors

• Banyai William
• Peck Bill James
• Fernandez Andres
• Chen Siyuan
• Indermuhle Pierre

Classifications

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• B01J2219/00612

  the surface being inorganic · CPC title

• C40B50/14 Primary

  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 title

• C40B50/00

  Methods of creating libraries, e.g. combinatorial synthesis · CPC title

• B01J2219/00585

  Parallel processes · CPC title