High efficiency, small volume nucleic acid synthesis

Patent metadata

Field	Value
Publication number	US-12209239-B2
Application number	US-202117320572-A
Country	US
Kind code	B2
Filing date	May 14, 2021
Priority date	Sep 26, 2011
Publication date	Jan 28, 2025
Grant date	Jan 28, 2025

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 multiwell plate for non-template directed synthesis of nucleic acid molecules, the plate comprising individual beads located in a plurality of wells of the plate,
wherein the beads are between 1.0 μm and 100 μm in diameter,
wherein single-stranded nucleic acid molecules are attached to the beads and
wherein at least some of the single-stranded nucleic acid molecules attached to the beads comprise a terminal protecting group at the 5′ hydroxyl group location,
wherein each well of the multiwell plate is independently and operably connected to a pair of electrodes,
wherein the pair of electrodes are positioned to allow for the flow of current between them, and
wherein each electrode is operably connected to a current controller capable of regulating current to between the pair of electrodes to apply current to selected wells of the multiwell plate.

2. The multiwell plate of claim 1 , wherein the terminal protecting group is a dimethoxytrityl group.

3. The multiwell plate of claim 1 , wherein a plurality of wells of the plate comprise an acidic solution and a terminal protecting group that is not covalently linked to the single-stranded nucleic acid molecules.

4. The multiwell plate of claim 1 , wherein a plurality of wells of the plate comprise an electrochemically generated acid that is capable of cleaving the terminal protecting group at the 5′ hydroxyl group location of the single-stranded nucleic acid molecules.

5. The multiwell plate of claim 1 , wherein the single-stranded nucleic acid molecules are from 40 to 100 nucleotides in length.

6. The multiwell plate of claim 1 , wherein the single-stranded nucleic acid molecules attached to each bead are designed to have the same nucleotide sequence and capable of hybridizing with one or more of the nucleic acid molecules attached to other beads.

7. The multiwell plate of claim 1 , wherein the number of wells in the plate is between 10,000 and 500,000.

8. The multiwell plate of claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.

9. The multiwell plate of claim 1 , wherein the wells of the multiwell plate are cylindrical, barrel or conical in shape.

10. The multiwell plate of claim 1 , wherein the wells are formed in semiconductor or polymer substrate.

11. The multiwell plate of claim 1 , wherein each well of the multiwell plate is operably connected to a pair of electrodes comprising a first electrode located beneath each well and a second electrode.

12. The multiwell plate of claim 1 , wherein the electrodes are composed of platinum.

13. The multiwell plate of claim 11 , further comprising a cover containing the second electrode.

14. The multiwell plate of claim 11 , further comprising a reference electrode.

15. The multiwell plate of claim 1 , wherein the bead is composed of a polymer, optionally wherein the polymer is polystyrene.

16. The multiwell plate of claim 1 , wherein a non-nucleosidic linker is attached to the beads.

17. A multiwell plate for non-template directed synthesis of nucleic acid molecules, wherein each well of the multiwell plate is operably connected to a pair of electrodes comprising:
a first electrode beneath each well, and
a second electrode, and
wherein a bead is located in each of a plurality of wells of the multiwell plate and an electrochemically generated acid is present in one or more wells,
wherein the wells of the multiwell plate are sized to allow only a single bead to occupy a well and wherein the bead is between 10 μm and 60 μm in diameter,
wherein each well of the multiwell plate is independently and operably connected to a pair of electrodes, and
wherein the pair of electrodes are positioned to allow for the flow of current between them, and
wherein each electrode is operably connected to a current controller capable of regulating current to between the pair of electrodes to apply current to selected wells of the multiwell plate.

18. The multiwell plate of claim 17 , wherein the second electrode is placed at the bottom or along one or more sidewall of each well.

19. The multiwell plate of claim 17 , wherein the multiwell plate further comprises a cover and the cover contains the second electrode.

20. The multiwell plate of claim 17 , wherein the electrodes are composed of platinum.

Assignees

• Life Technologies Corp
• Thermo Fisher Scient Geneart Gmbh

Inventors

• Peterson Todd
• Trefzer Axel
• Poehmerer Thomas

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · 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

• C12N15/1068

  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 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

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title