High efficiency, small volume nucleic acid synthesis

• US 10,519,439 B2
• Filed: 03/20/2017
• Issued: 12/31/2019
• Est. Priority Date: 09/26/2011
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific GENEART GmbH (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Peterson, Todd, Trefzer, Axel, Poehmerer, Thomas
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/463,363
• Publication Number:
  US 20170260521A1
• Time in Patent Office: 1,016 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 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 Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00655 the compounds being bound to magnets embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 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 Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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).

Claims

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 μ

m and 40 μ

m 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 mm² 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.