De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-10773232-B2
Application number	US-201916535779-A
Country	US
Kind code	B2
Filing date	Aug 8, 2019
Priority date	Aug 5, 2013
Publication date	Sep 15, 2020
Grant date	Sep 15, 2020

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 computer-assisted polynucleotide synthesis, comprising:
receiving instructions in a computer readable non-transient medium for synthesis of DNA sequences each having a full length sequence of at least 100 bases in length;
processing the instructions in a computer and transmitting synthesis instructions to a material deposition device, wherein the instructions provide for synthesis of a plurality of polynucleotides that encode for the DNA sequences; and
releasing synthesis reagents from the material deposition device to synthesize the plurality of polynucleotides, wherein 67% or more of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences compared to the DNA sequences received in the instructions in the computer readable non-transient medium without purification, and wherein the plurality of polynucleotides comprise a plurality of different nucleotide bases.

2. The method of claim 1 , wherein at least 70% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences compared to the DNA sequences received in the instructions in the computer readable non-transient medium without purification.

3. The method of claim 1 , wherein at least 81% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences compared to the DNA sequences received in the instructions in the computer readable non-transient medium without purification.

4. The method of claim 1 , wherein at least 89% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences compared to the DNA sequences received in the instructions in the computer readable non-transient medium without purification.

5. The method of claim 1 , further comprising performance of size selection to the plurality of polynucleotides, wherein the size selection is based on the DNA sequences received in the instructions in the computer readable non-transient medium.

6. The method of claim 1 , wherein the DNA sequences are cDNA sequences.

7. The method of claim 1 , wherein the plurality of polynucleotides comprises at least 5000 polynucleotides.

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

9. The method of claim 1 , wherein the plurality of polynucleotides collectively encode for at least 500 genes.

10. The method of claim 1 , wherein the DNA sequences have a length of 100 to 300 bases.

11. The method of claim 1 , further comprising purification of the plurality of polynucleotides that are synthesized.

12. A method for polynucleotide synthesis, comprising:
receiving DNA sequences each having a full length sequence of at least 100 bases in length; and
synthesizing a plurality of polynucleotides encoding for the DNA sequences, wherein 67% or more of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences without purification, and wherein the plurality of polynucleotides comprise a plurality of different nucleotide bases.

13. The method of claim 12 , wherein the DNA sequences are cDNA sequences.

14. The method of claim 12 , wherein the plurality of polynucleotides comprises at least 5,000 polynucleotides.

15. The method of claim 12 , wherein the plurality of polynucleotides comprises at least 100,000 polynucleotides.

16. The method of claim 12 , wherein the plurality of polynucleotides collectively encode for at least 500 genes.

17. The method of claim 12 , wherein the plurality of polynucleotides collectively encode for at least 10,000 genes.

18. The method of claim 12 , wherein the DNA sequences have a length of 100 to 300 bases.

19. The method of claim 12 , wherein at least 70% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences without purification.

20. The method of claim 12 , wherein at least 81% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences without purification.

21. The method of claim 12 , wherein at least 89% of the plurality of polynucleotides that are synthesized encode for the full length sequence of the DNA sequences without purification.

22. The method of claim 12 , further comprising performance of size selection to the plurality of polynucleotides, wherein the size selection is based on the DNA sequences received.

23. The method of claim 12 , further comprising purification of the plurality of polynucleotides that are synthesized.

Assignees

• Twist Bioscience Corp

Inventors

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

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01J2219/00623

  Immobilisation or binding · CPC title

• B01J2219/00596

  Solid-phase processes · CPC title

• C40B50/14 Primary

  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 title

• 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