De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-11452980-B2
Application number	US-202016737401-A
Country	US
Kind code	B2
Filing date	Jan 8, 2020
Priority date	Aug 5, 2013
Publication date	Sep 27, 2022
Grant date	Sep 27, 2022

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.

First claim

The invention claimed is:

1. A method for synthesizing oligonucleotides, comprising:
synthesizing a plurality of oligonucleotides at least 50 nucleotides in length, wherein the synthesizing comprises coupling with nucleosides having a blocking group in an extension reaction, wherein the coupling has a coupling efficiency on average of at least 99.6%, and wherein the plurality of polynucleotides comprise a plurality of different nucleotide bases.

2. The method of claim 1 , wherein the coupling efficiency on average is at least 99.7%.

3. The method of claim 1 , wherein the coupling efficiency on average is at least 99.8%.

4. The method of claim 1 , wherein the coupling efficiency is measured by CE=Y{circumflex over ( )}(1/n−1), wherein CE is coupling efficiency per cycle, Y is yield of a full length product, and n is expected length of the full length product.

5. The method of claim 4 , wherein the yield of the full length product is measured by Sanger sequencing of amplified products of the synthesis method of claim 1 to determine an amount of actual full length product divided by an amount of maximum calculated full length product.

6. The method of claim 1 , wherein the nucleosides are nucleoside phosphoramidites.

7. The method of claim 1 , wherein the blocking group is an acid labile protecting group.

8. The method of claim 7 , wherein the acid labile protecting group is 4,4′-dimethoxytrityl.

9. The method of claim 1 , wherein the plurality of oligonucleotides comprises oligonucleotides at least 100 nucleotides in length.

10. The method of claim 9 , wherein the coupling efficiency on average is at least 99.7%.

11. The method of claim 9 , wherein the coupling efficiency on average is at least 99.8%.

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

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

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

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

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

17. The method of claim 16 , wherein at least 20,000 oligonucleotides are synthesized on the single solid substrate.

18. The method of claim 16 , wherein at least 500,000 oligonucleotides are synthesized on the single solid substrate.

19. The method of claim 16 , wherein the single solid substrate comprises silicon, silicon dioxide or silicon nitride.

20. The method of claim 16 , wherein the single solid substrate comprises resolved loci that are functionalized with a chemical moiety suitable for nucleotide coupling.

21. The method of claim 16 , wherein the single solid substrate comprises a plurality of clusters.

22. The method of claim 21 , wherein each cluster comprises a plurality of loci.

23. The method of claim 22 , wherein each cluster contains 50 to 500 loci.

24. The method of claim 22 , wherein the plurality of loci in each cluster comprise oligonucleotides having substantially the same sequence.

25. The method of claim 1 , wherein synthesizing further comprises contacting nucleosides with an activator.

26. The method of claim 25 , wherein the activator comprises an acidic azole catalyst.

27. The method of claim 26 , wherein the acidic azole catalyst comprises 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, 5-(benzylmercapto)-1H-tetrazole, or 4,5-dicyanoimidazole.

Assignees

• Twist Bioscience Corp

Inventors

• Banyai William
• Peck Bill James
• Fernandez Andres
• Chen Siyuan
• Indermuhle Pierre

Classifications

• C12N15/66

  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 title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• B01J19/0046 Primary

  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 title

• B01J2219/00585

  Parallel processes · CPC title

• B01J2219/00317

  Microwell devices, i.e. having large numbers of wells · CPC title