De novo synthesized gene libraries

• US 10,632,445 B2
• Filed: 06/20/2016
• Issued: 04/28/2020
• 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): Zhang, Kaijiang
• Application Number:
  US15/187,714
• Publication Number:
  US 20160339409A1
• Time in Patent Office: 1,408 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 15/66 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 computer-assisted nucleic acid synthesis, comprising:

receiving instructions in a computer readable non-transient medium for synthesis of cDNA sequences encoding for at least 750 genes;

processing the instructions in a computer and transmitting synthesis instructions to a material deposition device, wherein the synthesis instructions provide for synthesis of a plurality of polynucleotides that collectively encode for the cDNA sequences;

releasing synthesis reagents from the material deposition device to synthesize the plurality of polynucleotides each at least 100 bases in length, wherein the plurality of polynucleotides encode sequences with an aggregate error rate of less than 1 in 1000 bases without correcting errors compared to the cDNA sequences received in the instructions in the computer readable non-transient medium; and

assembling a plurality of nucleic acids from a subset of the plurality of polynucleotides, wherein the plurality of nucleic acids comprise sequences encoded by the cDNA sequences encoding for at least 750 genes.

2. The method of claim 1, wherein each nucleic acid of the plurality of nucleic acids is isolated.

3. The method of claim 1, wherein each nucleic acid of the plurality of nucleic acids is purified.

4. The method of claim 1, wherein each of the plurality of polynucleotides extends from a surface.

5. The method of claim 1, further comprising treating the plurality of nucleic acids with an error correction enzyme.

6. The method of claim 1, wherein each nucleic acid of the plurality of nucleic acids is at least 0.5 kb long.

7. The method of claim 1, wherein each nucleic acid of the plurality of nucleic acids is at least 3 kb long.

8. The method of claim 1, wherein the plurality of polynucleotides comprises at least 60,000 polynucleotides.

9. The method of claim 1, wherein the plurality of polynucleotides comprises at least 100,000 polynucleotides.

10. The method of claim 1, wherein the plurality of polynucleotides comprises at least 600,000 polynucleotides.

11. The method of claim 1, wherein the plurality of polynucleotides collectively encode and correspond to cDNA sequences for at least 1000 genes.

12. The method of claim 1, wherein each polynucleotide is 100 to 500 bases in length.

13. The method of claim 1, wherein each polynucleotide is 100 to 200 bases in length.

14. The method of claim 1, wherein the aggregate error rate is determined by sequencing following amplification of the plurality of polynucleotides.

15. The method of claim 14, wherein the sequencing is performed using Sanger sequencing method.

16. The method of claim 14, wherein the amplification is performed using a DNA polymerase.

17. The method of claim 1, wherein the plurality of polynucleotides are synthesized on a solid support.

18. The method of claim 17, wherein the solid support comprises microstructures functionalized with a coupling reagent for binding to a nucleoside.

19. The method of claim 17, wherein the plurality of polynucleotides are released from the solid support prior to assembling the plurality of nucleic acids.