High efficiency, small volume nucleic acid synthesis

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).

1 . 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.   
     
     
         2 . The multiwell plate of  claim 1 , wherein the number of wells in the plate is between 10 and 50,000. 
     
     
         3 . The multiwell plate of  claim 1 , wherein the total volume of each well is between 0.1 μl and 50 μl. 
     
     
         4 . The multiwell plate of  claim 1 , wherein each well is operably connected to a pair of electrodes. 
     
     
         5 . The multiwell plate of  claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents. 
     
     
         6 . 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;   (c) oining 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   (e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.   
     
     
         7 . The method of  claim 6 , wherein the joining in (c) is mediated by polymerase chain reaction and/or ligases. 
     
     
         8 . The method of  claim 6 , wherein the assembled nucleic acid molecule is composed of at least five nucleic acid molecules. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 6 , wherein the assembled nucleic acid molecule is at least 20 kilobases. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 6 , wherein the assembled nucleic acid molecule is closed, circular. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The method of  claim 6 , wherein assembly of the nucleic acid molecules in the error corrected nucleic acid molecule pool occurs in a fungal cell. 
     
     
         16 . (canceled) 
     
     
         17 . A method for producing a product nucleic acid molecule, the method comprising:
 (a) designing the product nucleic acid molecule of between 10 kilobases and 500 kilobases in size, wherein the product nucleic acid molecule is defined by nucleotide sequence;   (b) synthesizing a plurality of individual nucleic acid molecules which differ in nucleotide sequence, wherein each individual nucleic acid molecule is synthesized to prepare a quantity of between 1.0×10 7  and 1.0×10 9  copies and wherein the individual nucleic acid molecules are capable of hybridizing with one or more of the other individual nucleic acid molecules;   (c) combining the individual nucleic acid molecules synthesized in (b) under conditions which allow for hybridization of the individual nucleic acid molecules under conditions which allow for the formation of at least one larger nucleic acid molecule; and   (d) combining the at least one larger nucleic acid molecule formed in (c) with one or more additional nucleic acid molecules to form the product nucleic acid molecule, wherein the product nucleic acid molecule contains less than one sequence error per kilobase.   
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 17 , wherein an error correction process is employed after step (b). 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 17 , wherein polymerase chain reactions are used to amplify the at least one larger nucleic acid molecule formed in step (c). 
     
     
         22 . The method of  claim 17 , wherein the product nucleic acid molecule is self replicable. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 17 , wherein the individual nucleic acid molecules are synthesized on beads, wherein each bead is containing in a well. 
     
     
         25 . (canceled) 
     
     
         26 . A method for the generation of a self replicating nucleic acid molecule, the method comprising:
 (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) joining some or all of the nucleic acid molecules present in the pool formed in (a) to form a plurality of larger nucleic acid molecules; and   (c) assembling the plurality of larger nucleic acid molecules to form the self replicating nucleic acid molecule.   
     
     
         27 . The method of  claim 26 , wherein the self replicating nucleic acid molecule is a chromosome or a plasmid. 
     
     
         28 . The method of  claim 26 , wherein the self replicating nucleic acid molecule is a genome. 
     
     
         29 . The method of  claim 28 , wherein the genome is a viral genome, a nuclear genome, an organelle genome, or a genome of a prokaryotic cell. 
     
     
         30 . A method for synthesizing and assembling a nucleic acid molecule which encodes more than one expression product, the method comprising:
 (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity;   (b) joining some or all of the nucleic acid molecules present in the pool formed in (a) to form a plurality of larger nucleic acid molecules; and   (c) assembling the plurality of larger nucleic acid molecules to form the nucleic acid molecule which encodes more than one expression product.   
     
     
         31 . The method of  claim 30 , wherein the more than one expression products are proteins that are involved in the same biological pathway. 
     
     
         32 . The method of  claim 31 , wherein the more than one expression products are proteins which are involved in the same biological pathway are enzymes that catalyze a series of chemical reactions in a biological pathway. 
     
     
         33 - 41 . (canceled)