DE NOVO SYNTHESIZED GENE LIBRARIES

• US 20160340672A1
• Filed: 08/10/2016
• 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 9,839,894 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. An oligonucleic acid library, comprising at least 100,000 non-identical oligonucleic acids that collectively encode sequences for at least 750 preselected genes, wherein each of the at least 100,000 non-identical oligonucleic acids is synthesized and comprises at least 30 bases in length, and wherein the at least 100,000 non-identical oligonucleic acids encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to predetermined sequences for the at least 100,000 non-identical oligonucleic acids.

2. The oligonucleic acid library of claim 1, wherein about 90% of the at least 100,000 non-identical oligonucleic acids encode sequences with no errors compared to the predetermined sequences for the at least 100,000 non-identical oligonucleic acids.

3. The oligonucleic acid library of claim 1, wherein at least about 90% of the at least 100,000 non-identical oligonucleic acids encode sequences with the aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences for the at least 100,000 non-identical oligonucleic acids.

4. The oligonucleic acid library of claim 1, wherein the at least 100,000 non-identical oligonucleic acids encode sequences with a deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences for the at least 100,000 non-identical oligonucleic acids.

5. The oligonucleic acid library 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 least 100,000 non-identical oligonucleic acids.

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

7. The oligonucleic acid library of claim 1, wherein each non-identical oligonucleic acid is at least 100 bases in length.

8. The oligonucleic acid library of claim 1, wherein each non-identical oligonucleic acid is 30 to 200 bases in length.

9. The oligonucleic acid library of claim 1, wherein the library comprises at least 700,000 non-identical oligonucleic acids.

10. The oligonucleic acid library of claim 9, wherein the at least 700,000 non-identical oligonucleic acids encode for at least 6000 genes.

11. The oligonucleic acid library of claim 1, wherein the at least 100,000 non-identical oligonucleic acids are attached to a structure.

12. The oligonucleic acid library of claim 1, wherein each of the at least 750 preselected genes is at least 0.5 kb in length.

13. An oligonucleic acid library, comprising at least 6,000 non-identical oligonucleic acids, wherein each of the at least 6,000 non-identical oligonucleic acids is synthesized, and wherein the at least 6,000 non-identical oligonucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to predetermined sequences for the at least 6,000 non-identical oligonucleic acids.

14. The oligonucleic acid library of claim 13, wherein about 90% of the at least 6,000 non-identical oligonucleic acids encode sequences with no errors compared to the predetermined sequences for the at least 6,000 non-identical oligonucleic acids.

15. The oligonucleic acid library of claim 13, wherein at least about 90% of the at least 6,000 non-identical oligonucleic acids encode sequences with the aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences for the at least 6,000 non-identical oligonucleic acids.

16. The oligonucleic acid library of claim 13, wherein the at least 6,000 non-identical oligonucleic acids encode sequences with a deletion error rate of less than 1 in 2000 bases compared to the predetermined sequences for the at least 6,000 non-identical oligonucleic acids.

17. The oligonucleic acid library of claim 13, wherein each of the at least 6,000 non-identical oligonucleic acids comprises a region which is complementary to another of the at least 6,000 non-identical oligonucleic acids.

18. The oligonucleic acid library of claim 13, wherein each non-identical oligonucleic acid is at least 50 bases in length.

19. The oligonucleic acid library of claim 13, wherein each non-identical oligonucleic acid is at least 100 bases in length.

20. The oligonucleic acid library of claim 13, wherein each non-identical oligonucleic acid is 30 to 200 bases in length.

21. The oligonucleic acid library of claim 13, wherein the at least 6,000 non-identical oligonucleic acids are attached to a structure.

22. The oligonucleic acid library of claim 13, wherein the at least 6,000 non-identical oligonucleic acids collectively encode sequences for at least 50 preselected genes each at least 0.5 kb in length.

23. A gene library, comprising at least 750 preselected genes, wherein each of the at least 750 preselected genes is synthesized and at least 0.5 kb length in length, and wherein the at least 750 preselected genes encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to predetermined sequences for the at least 750 preselected genes without correcting errors.

24. The gene library of claim 23, wherein about 90% of the at least 750 preselected genes encode sequences with no errors compared to the predetermined sequences for the at least 750 preselected genes without correcting errors.

25. The gene library of claim 23, wherein at least about 90% of the at least 750 preselected genes encode sequences with the aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences for the at least 750 preselected genes.

26. The gene library of claim 23, wherein the at least 750 preselected genes acids encode sequences with a deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences for the at least 750 preselected genes.

27. The gene library of claim 23, wherein each of the at least 750 preselected genes is synthesized and at least 1 kb length in length.

28. The gene library of claim 23, wherein each of the at least 750 preselected genes is synthesized and at least 2 kb length in length.

29. The gene library of claim 23, wherein the at least 750 preselected genes encode sequences with an aggregate error rate of less than 1 in 15,000 bases compared to predetermined sequences for the at least 750 preselected genes after correcting errors.