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 method for assembling nucleic acid molecules, the method comprising:
 (a) forming a reaction mixture of:
 (1) one or more insert nucleic acid molecule, one or more acceptor nucleic acid molecule, a plurality of oligonucleotides, wherein each oligonucleotide shares sequence complementarity with (i) one terminus of the insert nucleic acid molecule and the insertion site of the acceptor nucleic acid molecule or (ii) one terminus of two different insert nucleic acid molecules and wherein the number of oligonucleotides is represented by the formula O=2+2I, where O is the number of oligonucleotides and I is the number of insert nucleic acid molecules, 
 (2) a cell extract, and 
 (3) a protein composition comprising an exonuclease and, optionally, a single-stranded binding protein, and 
   (b) incubating the reaction formed in (a) under conditions which allow for the introduction of the insert nucleic acid molecule into the acceptor nucleic acid molecule.   
     
     
         2 . The method of  claim 1 , wherein the cell extract is obtained from a single cellular organism selected from the group consisting of:
 (a)  Escherichia coli;      (b)  Bacillus subtilis;      (c)  Schizosaccharomyces pombe ; and   (d)  Saccharomyces cerevisiae.      
     
     
         3 . The method of  claim 2 , wherein the  Escherichia coli  cells do not express redET genes. 
     
     
         4 . The method of  claim 3 , wherein the  Escherichia coli  cells are strain DH10B. 
     
     
         5 . The method of  claim 1 , wherein the exonuclease activity is provided by a DNA polymerase. 
     
     
         6 . The method of  claim 1 , wherein the single-stranded binding protein is encoded by T4 gene 32. 
     
     
         7 . The method of  claim 1 , wherein the number of inserts is three and the number of oligonucleotides is six. 
     
     
         8 . 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.   
     
     
         9 . The method of  claim 8 , wherein synthesis of the nucleic acid molecules on the separate beads involves deblocking in step (a) by an acid generated by in a redox reaction mixture in the wells of the plate. 
     
     
         10 . The method of  claim 9 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone. 
     
     
         11 . The method of  claim 8 , wherein the joining in step (c) is mediated by polymerase chain reaction. 
     
     
         12 . The method of  claim 8 , wherein the accept nucleic acid molecule is a linearized vector. 
     
     
         13 . The method of  claim 8 , wherein the pool of double-stranded nucleic acid molecules have blunt termini. 
     
     
         14 . The method of  claim 13 , wherein at least two nucleic acid molecules in the pool of double-stranded nucleic acid molecules comprise a terminus comprising a region of sequence complementarity with a terminus of the acceptor nucleic acid molecule. 
     
     
         15 . The method of  claim 14 , wherein the region of sequence complementarity is from twenty to sixty nucleotides in length. 
     
     
         16 . 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, wherein the nucleic acid molecules synthesized on each bead are designed to have the same nucleotide sequence, and wherein less than 1% of the purine bases in the synthesizing a plurality of nucleic acid molecules are depurinated;   (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.   
     
     
         17 . The method of  claim 16 , further comprising:
 (e) 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   (f) collecting the beads selectively removed from wells in step (e) in a vessel.