De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-9833761-B2
Application number	US-201514885962-A
Country	US
Kind code	B2
Filing date	Oct 16, 2015
Priority date	Aug 5, 2013
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

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 for synthesizing a library of oligonucleic acids encoding for predetermined sequences, the method comprising:
(a) providing predetermined sequences for a library of at least 100,000 non-identical oligonucleic acids, wherein the at least 100,000 non-identical oligonucleic acids collectively encode sequence for at least 750 genes;
(b) providing a structure having a surface, wherein the surface comprises a plurality of clusters, wherein each cluster is selected for synthesis of oligonucleic acids associated with a single gene of the at least 750 genes, and wherein each cluster comprises a plurality of loci for oligonucleic acid extension;
(c) adding a droplet of fluid comprising an extension reaction reagent specific to a locus, wherein the extension reaction reagent comprises a nucleoside phosphoramidite;
(d) allowing sufficient time for an extension reaction step to occur;
(e) repeating steps (c) and (d) to synthesize the at least 100,000 non-identical oligonucleic acids, wherein each non-identical oligonucleic acid is at least 30 bases in length and is attached to the surface; and
(f) releasing the at least 100,000 non-identical oligonucleic acids from the surface, wherein the method comprises no amplification step prior to releasing the at least 100,000 non-identical oligonucleic acids from the surface.

2. The method of claim 1 , wherein each locus comprises a first plurality of molecules, and wherein each molecule of the first plurality of molecules binds to the surface and comprises a reactive group capable of binding to a nucleoside phosphoramidite.

3. The method of claim 2 , wherein the first plurality of molecules comprises a silane, siloxane, or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.

4. The method of claim 2 , wherein the first plurality of molecules comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, or (3-aminopropyl)trimethoxysilane.

5. The method of claim 3 , wherein the silane is an aminosilane.

6. The method of claim 2 , wherein each locus is adjacent to a region that comprises a second plurality of molecules, and wherein each molecule of the second plurality of molecules binds to the surface and lacks the reactive group capable of binding to the nucleoside phosphoramidite.

7. The method of claim 6 , wherein the second plurality of molecules comprises a fluorosilane.

8. The method of claim 7 , wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane.

9. The method of claim 1 , wherein each locus of the plurality of loci is separated from another locus by a structural barrier of the surface.

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

11. The method of claim 1 , wherein steps (a) to (f) are completed in less than 14 days.

12. The method of claim 1 , wherein steps (a) to (f) are completed in less than 7 days.

13. The method of claim 1 , wherein steps (a) to (f) are completed in less than 2 days.

14. The method of claim 1 , wherein each of the at least 100,000 non-identical oligonucleic acids comprises a tether region comprising 2 to 20 bases.

15. The method of claim 14 , wherein the tether region comprises 2 to 20 bases having a homogeneous sequence.

16. The method of claim 15 , wherein the tether region comprises 10 bases.

17. The method of claim 1 , wherein at least 1,000,000 non-identical oligonucleic acids are synthesized.

18. The method of claim 1 , wherein each of the at least 100,000 non-identical oligonucleic acids comprises a cleavable region comprising at least one base chemically modified to detach from the oligonucleic acid in the presence of a cleaving reagent.

19. The method of claim 18 , wherein the cleaving reagent is gaseous ammonia or gaseous methylamine.

20. The method of claim 1 , wherein each of the at least 100,000 non-identical oligonucleic acids comprises a region which is complementary to another of the at 100,000 non-identical oligonucleic acids.

21. The method of claim 1 , wherein each non-identical oligonucleic acid is at least 50 bases in length.

22. The method of claim 1 , wherein 2, 3, or 4 of the loci within each cluster comprise oligonucleic acids encoding for identical sequence.

23. The method of claim 1 , further comprising amplifying the released at least 100,000 non-identical oligonucleic acids from step (f) to generate a plurality of amplification products.

24. The method of claim 23 , wherein the plurality of amplification products have an aggregate error rate of no more than 1:1000 compared to the predetermined sequences without correcting errors.

25. The method of claim 1 , wherein up to 5 of the loci within each cluster comprise oligonucleic acids encoding for identical sequence.

26. A method for synthesizing a library of oligonucleic acids encoding for predetermined sequences, the method comprising:
(a) providing predetermined sequences for a library of at least 20,000 non-identical oligonucleic acids, wherein the at least 20,000 non-identical oligonucleic acids collectively encode sequence for at least 200 genes;
(b) providing a structure having a surface, wherein the surface comprises a plurality of clusters, wherein each cluster is selected for synthesis of oligonucleic acids associated with a single gene of the at least 200 genes, and wherein each cluster comprises a plurality of loci for oligonucleic acid extension;
(c) adding a droplet of fluid comprising an extension reaction reagent specific to a locus, wherein the extension reaction reagent comprises a nucleoside phosphoramidite;
(d) allowing sufficient time for an extension reaction step to occur;
(e) repeating steps (c) and (d) to synthesize the at least 20,000 non-identical oligonucleic acids, wherein each non-identical oligonucleic acid is at least 30 bases in length and is attached to the surface; and
(f) releasing the at least 20,000 non-identical oligonucleic acids from the surface, wherein the method comprises no amplification step prior to releasing the at least 20,000 non-identical oligonucleic acids from the surface.

27. The method of claim 26 , further comprising amplifying the released at least 20,000 non-identical oligonucleic acids from step (f) to generate a plurality of amplification products.

28. The method of claim 27 , wherein the plurality of amplification products have an aggregate error rate of no more than 1:1000 compared to the predetermined sequences without correcting errors.

29. The method of claim 26 , wherein up to 5 of the loci within each cluster comprise oligonucleic acids encoding for identical sequence.

Assignees

• Twist Bioscience Corp

Inventors

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

Classifications

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• B01J2219/00623

  Immobilisation or binding · CPC title

• B01J2219/00497

  Features relating to the solid phase supports · CPC title

• B01J2219/00619

  using hydrophilic or hydrophobic regions · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title