High efficiency, small volume nucleic acid synthesis
AssigneesLife Technologies Corporation, Thermo Fisher Scientific Geneart GmbH
InventorsTodd PETERSON, Axel TREFZER, Thomas POEHMERER
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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).
Claims14
-7. (canceled)
A multiwell plate for non-template directed synthesis of nucleic acid molecules, the plate comprising:
- (a) a magnetic bead located in each of a plurality of wells of the plate, and
- (b) an electrochemically generated acid being present in one or more well,
- wherein the bead is between 1.0 μm and 100 μm in diameter.
The multiwell plate of claim 1, wherein the number of wells in the plate is between 10 and 50,000.
The multiwell plate of claim 1, wherein the total volume of each well is between 0.1 μl and 50 μl.
The multiwell plate of claim 1, wherein each well is operably connected to a pair of electrodes.
The multiwell plate of claim 1, wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.
A system for generating assembled nucleic acid molecule, the system comprising:
- a processor;
- and a memory encoded with processor-executable instructions for:
- (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity in the well of a plate;
- (b) combining the nucleic acid molecules generated in (a) to produce a pool;
- (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;
- (d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool;
- and
- assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.
A method for the generation of an assembled nucleic acid molecule, the method comprising:
- (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from about 0.001 nanomoles to about 1,000 nanomoles;
- (b) combining the nucleic acid molecules generated in (a) to produce a pool of single-stranded nucleic acid molecules;
- (c) generating double-stranded nucleic acid molecules from nucleic acid molecules present in the pool of single-stranded nucleic acid molecules formed in step (b) to generate a pool of double-stranded nucleic acid molecules;
- (d) eliminating nucleic acid molecules which contain sequence errors from the pool of double-stranded nucleic acid molecules formed in step (c) to produce an error corrected pool of double-stranded nucleic acid molecules;
- and
- (e) simultaneously assembling the nucleic acid molecules in the error corrected pool of double-stranded nucleic acid molecules and an acceptor nucleic acid molecule to form the assembled nucleic acid molecule.
Dependencies are inferred from the claim text.