High efficiency, small volume nucleic acid synthesis
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).
1. A method for producing a plurality of nucleic acid molecules, the method comprising:
(a) synthesizing the plurality of nucleic acid molecules on separate beads in wells of a plate, wherein each well is configured to accommodate a single bead, 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 nucleic acid molecules synthesized on each bead are designed to have the same nucleotide sequence;
(b) selectively removing a plurality of beads from the wells, wherein the beads removed from the wells comprise a first set of individual nucleic acid molecules, wherein beads comprising nucleic acid molecules that are not part of the first set of individual nucleic acid molecules are not removed from their respective wells; and
(c) collecting the beads selectively removed from wells in step (b) in a vessel.
2. The method of claim 1 , further comprising:
(d) selectively removing a plurality of beads from the wells, wherein the beads removed from the wells comprise a second set of individual nucleic acid molecules, wherein beads comprising nucleic acid molecules that are not part of the second set of individual nucleic acid molecules are not removed from their respective wells; and
(e) collecting the beads selectively removed from wells in step (d) in a vessel.
3. The method of claim 1 , wherein the number of beads collected in step (c) is between 100 to 1,000.
4. The method of claim 1 , further comprising releasing the nucleic acid molecules from the beads collected in the vessel in step (c).
5. The method of claim 4 , wherein the first set of individual nucleic acid molecules are primers.
6. The method of claim 5 , wherein the primers are 5 to 100 nucleotides in length.
7. The method of claim 5 , wherein the primers are lyophilized.
8. The method of claim 5 , wherein the vessel is a well of a multiwell plate.
9. The method of claim 1 , wherein the members of the first set of nucleic acid molecules are between 40 to 200 nucleotides in length.
10. The method of claim 1 , wherein the beads in the wells are porous beads.
11. The method of claim 1 , wherein the beads in the wells of the plate are monodispersed beads.
12. The method of claim 11 , wherein the monodispersed beads are between 10 μm and 40 μm in diameter.
13. The method of claim 1 , wherein synthesis of the nucleic acid molecules on the separate beads involves deblocking in step (a) by an acid generated in a redox reaction mixture in the wells of the plate.
14. The method of claim 13 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone.
15. The method of claim 14 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone and anthraquinone.
16. The method of claim 13 , wherein the electrochemically generated acid is generated in the well by passing an electrical current through the wells.
17. The method of claim 1 , further comprising:
(d) releasing the first set of individual nucleic acid molecules from the beads collected in step (c) to form a pool of nucleic acid molecules; and
(e) joining some or all of the nucleic acid molecules present in the pool formed in (d) to form a plurality of larger nucleic acid molecules.
18. The method of claim 17 , further comprising:
(f) eliminating nucleic acid molecules containing sequence errors from the plurality of larger nucleic acid molecules formed in (e) to produce an error corrected nucleic acid molecule pool; and
(g) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form an assembled nucleic acid molecule.