De novo synthesized gene libraries

• US 10,618,024 B2
• Filed: 05/23/2017
• Issued: 04/14/2020
• 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:
  US15/602,991
• Publication Number:
  US 20170327819A1
• Time in Patent Office: 1,057 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 15/635 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 polynucleotide synthesis,

comprising:

a) providing instructions in a computer readable non-transient medium for synthesis of preselected cDNA sequences; and

b) synthesizing at least 20,000 polynucleotides encoding for the preselected cDNA sequences, wherein each of the at least 20,000 polynucleotides is at least 100 bases in length, wherein each of the at least 20,000 polynucleotides comprises a first overlap region which is complementary to a second overlap region of another polynucleotide of the at least 20,000 polynucleotides such that a plurality of genes are formed when a subset of the at least 20,000 polynucleotides are assembled, wherein the first overlap region comprises at least 10 bases in length, and wherein at least 89% of the at least 20,000 polynucleotides have no errors compared to the preselected cDNA sequences received in the instructions provided in the computer readable non-transient medium without error correction.

2. The method of claim 1, wherein at least 100,000 polynucleotides are synthesized.

3. The method of claim 1, wherein at least 750 genes are formed when a subset of the at least 20,000 polynucleotides are assembled.

4. The method of claim 1, wherein at least 10,000 genes are formed when a subset of the at least 20,000 polynucleotides are assembled.

5. The method of claim 1, wherein the first overlap region comprises a GC content of 35% to 65%.

6. The method of claim 1, wherein the first overlap region comprises 10 to 100 bases in length.

7. The method of claim 1, wherein each polynucleotide is 100 to 300 bases in length.

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

9. The method of claim 1, wherein each of the genes is at least 3 kb in length.

10. The method of claim 1, wherein the at least 20,000 polynucleotides are synthesized on a structure.

11. The method of claim 10, wherein the structure is a solid support.

12. The method of claim 1, further comprising hybridizing each polynucleotide in said subset to at least another polynucleotide in said subset to form the plurality of genes.