DE NOVO SYNTHESIZED GENE LIBRARIES

• US 20160339409A1
• Filed: 06/20/2016
• Published: 11/24/2016
• 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
• Granted Patent: US 10,632,445 B2
• Time in Patent Office: Days
• 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 750 preselected nucleic acids;

b) providing a structure comprising a surface;

c) synthesizing at least 100,000 non-identical polynucleotides extending from the surface; and

d) assembling the at least 750 preselected nucleic acids, wherein each of the at least 750 preselected nucleic acids is at least 0.5 kb in length, wherein the at least 750 preselected nucleic acids comprise sequences encoded by the at least 100,000 non-identical polynucleotides, and wherein the assembled at least 750 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

2. The method of claim 1, wherein the at least 100,000 non-identical polynucleotides extending from the surface are clustered, and wherein each cluster includes a subset of the at least 100,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

3. The method of claim 2, wherein each cluster comprises 50 to 500 loci.

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

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

6. The method of claim 2, wherein each subset of the at least 100,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 0.5 to 1.5 mm in diameter.

7. The method of claim 2, wherein each subset of the at least 100,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 1.15 mm in diameter.

8. The method of claim 1, further comprising releasing the at least 100,000 non-identical polynucleotides from the surface, wherein the at least 100,000 non-identical polynucleotides are released from the surface and clustered, and wherein each cluster includes a subset of the at least 100,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

9. The method of claim 8, wherein the at least 100,000 non-identical polynucleotides are released by gas cleavage.

10. The method of claim 1, further comprising treating the assembled at least 750 preselected nucleic acids with an error correction enzyme.

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

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

13. The method of claim 1, wherein the assembled at least 750 preselected nucleic acids are at least 1 kb long on average.

16. The method of claim 1, wherein greater than 80% of the at least 750 preselected nucleic acids assembled are perfect sequences compared to the predetermined sequences without correcting errors.

17. The method of claim 1, wherein greater than 90% of the at least 750 preselected nucleic acids assembled are perfect sequences compared to the predetermined sequences without correcting errors.

18. The method of claim 1, wherein the surface comprises a plurality of loci, wherein each locus comprises a first plurality of molecules and is adjacent to a region that comprises a second plurality of molecules, 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, 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.

19. The method of claim 1, wherein at least about 90% of the at least 750 preselected nucleic acids assembled encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

14. (canceled)

15. (canceled)

20. A method of synthesizing nucleic acids, comprising:

a) providing predetermined sequences for at least 50 preselected nucleic acids;

b) providing a structure comprising a surface;

c) synthesizing at least 6,000 non-identical polynucleotides extending from the surface, wherein the at least 6,000 non-identical polynucleotides comprises a plurality of discrete subsets of non-identical polynucleotides, and wherein each discrete subset of non-identical polynucleotides comprises non-identical polynucleotides that collectively encode for a gene; and

d) assembling the at least 50 preselected nucleic acids, wherein each of the at least 50 preselected nucleic acids is at least 0.5 kb in length, and wherein the assembled at least 50 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

21. The method of claim 20, wherein the plurality of discrete subsets of non-identical polynucleotides comprises 50 to 500 non-identical polynucleotides.

22. The method of claim 20, wherein the assembled at least 50 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors.