De novo synthesized gene libraries

Patent metadata

Field	Value
Publication number	US-12569822-B2
Application number	US-202217818656-A
Country	US
Kind code	B2
Filing date	Aug 9, 2022
Priority date	Aug 5, 2013
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

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 of de-novo synthesis of n-mer oligonucleotides on a substrate, comprising:
a. providing a substrate with resolved loci in the form of microstructures that are functionalized with a chemical moiety suitable for nucleotide coupling; and b. coupling at least two nucleotide building blocks to a plurality of growing individual oligonucleotide chains, thereby synthesizing a plurality of oligonucleotides that are n bases long, wherein each oligonucleotide chain of the plurality of growing individual oligonucleotide chains resides on one resolved locus of the resolved loci, wherein the resolved loci comprise a pitch of no more than 0.1 mm, and wherein n is at least 50.

2 . The method of claim 1 , wherein the resolved loci are arrayed on a uniform pitch.

3 . The method of claim 1 , wherein the resolved loci have a density of at least 700 per 1 mm 2 .

4 . The method of claim 1 , wherein the resolved loci have a density of at least 5000 per 1 mm 2 .

5 . The method of claim 1 , wherein the building blocks comprise an adenine, guanine, thymine, cytosine, or uridine group, or a modified nucleotide.

6 . The method of claim 1 , wherein the method comprises chemical synthesis of oligonucleotides.

7 . The method of claim 5 , wherein the building blocks comprise phosphoramidite.

8 . The method of claim 1 , wherein the substrate comprises at least 100 resolved loci and wherein at least two of the plurality of growing oligonucleotides are different from each other.

9 . The method of claim 1 , wherein the substrate comprises at least 10,000 resolved loci and wherein at least two of the plurality of growing oligonucleotides are different from each other.

10 . The method of claim 1 , wherein n is at least 100.

11 . The method of claim 1 , wherein the building blocks comprise a blocking group.

12 . The method of claim 11 , wherein the blocking group comprises an acid-labile DMT.

13 . The method of claim 12 , wherein the acid-labile DMT comprises 4,4′-dimethoxytrityl.

14 . The method of claim 1 , wherein the microstructures are in two-dimension form or three-dimension form.

15 . The method of claim 1 , wherein the method further comprises cleaving the synthesized oligonucleotides from the substrate.

16 . The method of claim 1 , wherein the building blocks comprise a nucleotide, a dinucleotide, a trinucleotide, or any combination thereof.

17 . The method of claim 1 , wherein the at least two nucleotide building blocks are coupled to a resolved locus based on a predetermined sequence.

18 . The method of claim 1 , wherein each nucleotide building block is deposited via a droplet at each resolved locus of the resolved loci.

19 . The method of claim 18 , wherein the droplet has a volume of at least 2 pl.

Assignees

• Twist Bioscience Corp

Inventors

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

Classifications

• C12N15/74

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

• C12N15/09

  Recombinant DNA-technology · CPC title

• B01J2219/00605

  the compounds being directly bound or immobilised to solid supports · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• B01J2219/00709

  Type of synthesis · CPC title