De novo synthesized gene libraries

• US 9,833,761 B2
• Filed: 10/16/2015
• Issued: 12/05/2017
• 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
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/885,962
• Publication Number:
  US 20160090592A1
• Time in Patent Office: 781 Days
• Field of Search: None
• 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 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.