De novo synthesized gene libraries

• US 10,583,415 B2
• Filed: 05/10/2019
• Issued: 03/10/2020
• 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): Zara, Jane J
• Application Number:
  US16/409,608
• Publication Number:
  US 20190314783A1
• Time in Patent Office: 305 Days
• Field of Search: 435 61
• 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 oligonucleotides, comprising:

synthesizing a plurality of oligonucleotides at a rate of extending each of the oligonucleotides by at least 10 nucleotides per hour, wherein each of the oligonucleotides has a preselected sequence, and wherein the synthesizing comprises extending each oligonucleotide by a single base in an extension reaction.

2. The method of claim 1, wherein the rate of extending each of the oligonucleotides is at least 20 nucleotides per hour.

3. The method of claim 1, wherein the rate of extending each of the oligonucleotides is at least 25 nucleotides per hour.

4. The method of claim 1, wherein the plurality oligonucleotides are synthesized in parallel.

5. The method of claim 1, wherein at least 20,000 oligonucleotides are synthesized.

6. The method of claim 1, wherein at least 500,000 oligonucleotides are synthesized.

7. The method of claim 1, wherein each of the oligonucleotides is at least 25 nucleotides in length.

8. The method of claim 1, wherein the plurality of oligonucleotides are synthesized on a solid substrate.

9. The method of claim 8, wherein the solid substrate comprises silicon, silicon dioxide or silicon nitride.

10. The method of claim 1, wherein each of the oligonucleotides has a different preselected sequence.

11. The method of claim 1, wherein each of the oligonucleotides comprises 100 to 200 nucleotides in length and is synthesized in less than 5 hours.

12. The method of claim 1, wherein the rate of extending each of the oligonucleotides is at least 12 nucleotides per hour.

13. The method of claim 1, wherein the rate of extending each of the oligonucleotides is at least 15 nucleotides per hour.

14. The method of claim 1, wherein each of the oligonucleotides comprises 100 to 200 nucleotides in length.

15. The method of claim 1, wherein each of the oligonucleotides comprises 80 to 200 nucleotides in length.

16. The method of claim 1, wherein each of the oligonucleotides is at least 100 nucleotides in length.

17. The method of claim 1, wherein each of the oligonucleotides are the same length.

18. The method of claim 1, wherein the plurality of oligonucleotides comprise DNA.

19. The method of claim 1, wherein the plurality of oligonucleotides comprise RNA.

20. The method of claim 1, wherein each of the oligonucleotides comprises 100 to 300 nucleotides in length.

21. The method of claim 1, wherein each of the oligonucleotides comprises 10 to 500 nucleotides in length.