De novo synthesized gene libraries

• US 9,409,139 B2
• Filed: 08/05/2014
• Issued: 08/09/2016
• 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
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/452,429
• Publication Number:
  US 20150038373A1
• Time in Patent Office: 735 Days
• Field of Search: None
• 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 15/66 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 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.