De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-2016339409-A1
Application number	US-201615187714-A
Country	US
Kind code	A1
Filing date	Jun 20, 2016
Priority date	Aug 5, 2013
Publication date	Nov 24, 2016
Grant date	—

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

What is claimed is:

1 . A method of synthesizing nucleic acids, comprising:
a) providing predetermined sequences for at least 750 preselected nucleic acids; b) providing a structure comprising a surface; c) synthesizing at least 100,000 non-identical polynucleotides extending from the surface; and d) assembling the at least 750 preselected nucleic acids, wherein each of the at least 750 preselected nucleic acids is at least 0.5 kb in length, wherein the at least 750 preselected nucleic acids comprise sequences encoded by the at least 100,000 non-identical polynucleotides, and wherein the assembled at least 750 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

2 . The method of claim 1 , wherein the at least 100,000 non-identical polynucleotides extending from the surface are clustered, and wherein each cluster includes a subset of the at least 100,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

3 . The method of claim 2 , wherein each cluster comprises 50 to 500 loci.

4 . The method of claim 2 , wherein each cluster comprises 100 to 150 loci.

5 . The method of claim 3 , wherein each locus is separated from another locus by a structural barrier on the surface.

6 . The method of claim 2 , wherein each subset of the at least 100,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 0.5 to 1.5 mm in diameter.

7 . The method of claim 2 , wherein each subset of the at least 100,000 non-identical polynucleotides collectively encodes for a single nucleic acid and is located within a channel 1.15 mm in diameter.

8 . The method of claim 1 , further comprising releasing the at least 100,000 non-identical polynucleotides from the surface, wherein the at least 100,000 non-identical polynucleotides are released from the surface and clustered, and wherein each cluster includes a subset of the at least 100,000 non-identical polynucleotides that collectively encodes for a single nucleic acid.

9 . The method of claim 8 , wherein the at least 100,000 non-identical polynucleotides are released by gas cleavage.

10 . The method of claim 1 , further comprising treating the assembled at least 750 preselected nucleic acids with an error correction enzyme.

11 . The method of claim 1 , wherein steps a) to d) are completed in less than one week.

12 . The method of claim 1 , wherein steps a) to d) are completed in less than 48 hours.

13 . The method of claim 1 , wherein the assembled at least 750 preselected nucleic acids are at least 1 kb long on average.

14 . (canceled)

15 . (canceled)

16 . The method of claim 1 , wherein greater than 80% of the at least 750 preselected nucleic acids assembled are perfect sequences compared to the predetermined sequences without correcting errors.

17 . The method of claim 1 , wherein greater than 90% of the at least 750 preselected nucleic acids assembled are perfect sequences compared to the predetermined sequences without correcting errors.

18 . The method of claim 1 , wherein the surface comprises a plurality of loci, wherein each locus comprises a first plurality of molecules and is adjacent to a region that comprises a second plurality of molecules, wherein each molecule of the first plurality of molecules binds to the surface and comprises a reactive group capable of binding to a nucleoside phosphoramidite, and wherein each molecule of the second plurality of molecules binds to the surface and lacks the reactive group capable of binding to the nucleoside phosphoramidite.

19 . The method of claim 1 , wherein at least about 90% of the at least 750 preselected nucleic acids assembled encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

20 . A method of synthesizing nucleic acids, comprising:
a) providing predetermined sequences for at least 50 preselected nucleic acids; b) providing a structure comprising a surface; c) synthesizing at least 6,000 non-identical polynucleotides extending from the surface, wherein the at least 6,000 non-identical polynucleotides comprises a plurality of discrete subsets of non-identical polynucleotides, and wherein each discrete subset of non-identical polynucleotides comprises non-identical polynucleotides that collectively encode for a gene; and d) assembling the at least 50 preselected nucleic acids, wherein each of the at least 50 preselected nucleic acids is at least 0.5 kb in length, and wherein the assembled at least 50 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1000 bases compared to the predetermined sequences without correcting errors.

21 . The method of claim 20 , wherein the plurality of discrete subsets of non-identical polynucleotides comprises 50 to 500 non-identical polynucleotides.

22 . The method of claim 20 , wherein the assembled at least 50 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors.

Assignees

• Twist Bioscience Corp

Inventors

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

Classifications

• B01J2219/00709

  Type of synthesis · CPC title

• C40B50/00

  Methods of creating libraries, e.g. combinatorial synthesis · CPC title

• C12Q1/6806

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

• B01J2219/00378

  Piezoelectric or ink jet dispensers · CPC title

• B01J2219/00585

  Parallel processes · CPC title