High efficiency, small volume nucleic acid synthesis

Patent metadata

Field	Value
Publication number	US-10519439-B2
Application number	US-201715463363-A
Country	US
Kind code	B2
Filing date	Mar 20, 2017
Priority date	Sep 26, 2011
Publication date	Dec 31, 2019
Grant date	Dec 31, 2019

Abstract

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

First claim

What is claimed is:

1. A method for producing a product nucleic acid molecule, the method comprising:
(a) designing the product nucleic acid molecule;
(b) synthesizing a plurality of nucleic acid molecules on separate beads in wells of a plate, wherein each well is configured to accommodate a single bead, wherein a subset of the nucleic acid molecules synthesized on the separate beads are components of the product nucleic acid molecule, wherein the volume of each well is between 0.1 picoliters and 10 nanoliters, wherein the wells comprise an electrode for the production of electrochemically generated acid, and wherein the individual nucleic acid molecules that are the components of the product nucleic acid molecule are capable of hybridizing with one or more of the other individual nucleic acid molecules that are the components of the product nucleic acid molecule;
(c) selectively removing beads from the wells that comprise individual nucleic acid molecules that are the components of the product nucleic acid molecule, wherein beads comprising nucleic acid molecules that are not components of the product nucleic acid molecule are not removed from their respective wells;
(d) collecting the beads selectively removed from wells in step (c) in a vessel;
(e) releasing the nucleic acid molecules from the beads collected in the vessel in step (d) to form a pool of nucleic acid molecules in the vessel; and
(f) assembling the product nucleic acid molecule from the pool of nucleic acid molecules formed in step (e).

2. The method of claim 1 , wherein the beads in the wells are porous beads.

3. The method of claim 1 , wherein the beads in the wells of the plate are monodispersed beads.

4. The method of claim 3 , wherein the monodispersed beads are between 10 μm and 40 μm in diameter.

5. The method of claim 1 , wherein synthesis of the nucleic acid molecules on the separate beads involves deblocking in step (b) by an acid generated by in a redox reaction mixture in the wells of the plate.

6. The method of claim 5 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone.

7. The method of claim 6 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone and anthraquinone.

8. The method of claim 6 , wherein the electrochemically generated acid is generated in the well by passing an electrical current through the wells.

9. The method of claim 5 , wherein the plate contains between 1,000 and 50,000 wells.

10. The method of claim 5 , wherein the plate contains a 10 mm 2 area which contains 10,000 wells.

11. The method of claim 1 , wherein the product nucleic acid molecule is between 0.5 kilobase and 100 kilobases in size.

12. The method of claim 1 , wherein the volume of each well is between 0.1 picoliters and 1 nanoliter.

13. The method of claim 1 , wherein the volume of each well is between 0.1 picoliters and 100 picoliters.

14. The method of claim 1 , wherein the average amount of nucleic acid synthesized on each bead is between 1 femtomole and 0.001 nanomole.

15. The method of claim 9 , wherein the number of beads selectively removed from wells of the plate is between 100 and 1,000.

Assignees

• Life Technologies Corp
• Thermo Fisher Scient Geneart Gmbh

Inventors

• Peterson Todd
• Trefzer Axel
• Poehmerer Thomas

Classifications

• B01J2219/00351

  Means for dispensing and evacuation of reagents · CPC title

• C12N15/64

  General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host · 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

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · 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