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 removing a bead from a fluid-filled well of a microchip for synthesizing nucleic acid molecules, wherein a nucleic acid molecule is attached to the bead, the method comprising:
 providing a voltage between a first electrode that is arranged at a bottom of the fluid-filled well and a second electrode, wherein the voltage is sufficient to cause fluid in the fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the fluid-filled well along with the bead or to lift the bead to the top of the fluid-filled well.   
     
     
         2 . The method of  claim 1 , further comprising collecting the bead that has risen to the top of the fluid-filled well with a bead-collection device. 
     
     
         3 . The method of  claim 2 , further comprising transferring the bead that was collected to a well of a first multiwell collection plate. 
     
     
         4 . The method of  claim 1 , wherein the fluid comprises an aqueous or a non-aqueous buffer solution. 
     
     
         5 . The method of  claim 1 , wherein the fluid comprises water, methanol, acetonitrile, and Net4pTsO. 
     
     
         6 . The method of  claim 1 , wherein the first electrode is composed of platinum and the voltage is about 0.1 to about 100 volts. 
     
     
         7 . The method of  claim 1 , wherein each well of the microchip is individually addressable by a controller. 
     
     
         8 . The method of  claim 1 , wherein the bead is composed of: a synthetic polymer, a modified naturally occurring polymer, glass, controlled pore glass, magnetic controlled pore glass, magnetic beads, ceramics, or one or more metals. 
     
     
         9 .- 17 . (canceled) 
     
     
         18 . A method for selectively removing one or more beads from a microchip for synthesizing nucleic acid molecules having a plurality of fluid-filled wells, wherein each of the plurality of wells comprises an electrode formed at the bottom of the well and each bead of the one or more beads occupies a single well on the microchip, the method comprising:
 identifying one or more wells that contain one or more beads to be removed from the microchip;   providing a voltage between a first electrode in the one or more wells that have been identified and a second electrode, wherein the voltage is sufficient to cause fluid in the one or more fluid-filled wells to undergo electrolysis and produce one or more bubbles in the fluid to rise to a top of the one or more fluid-filled wells along with the one or more beads contained within the one or more wells or to lift the one or more beads to the top of the one or more fluid-filled wells;   collecting the one or more beads that have risen to the top of the one or more fluid-filled well with a bead-collection device; and   transferring the one or more beads that were collected to one or more wells of a multiwell collection plate.   
     
     
         19 . A system for synthesis of a nucleic acid molecule, the system comprising:
 a microchip comprising a plurality of well structures formed thereon, each well of the plurality of well structures sized to accommodate a monodisperse bead for synthesis of the nucleic acid molecule, wherein each well has formed therein a first electrode at a bottom of the well that is individually controllable by a controller, and wherein the diameter of the monodisperse bead is smaller than the diameter of each well by about 5% to about 20%; and   a lid member arranged on top of the microchip and comprising a fluidic channel formed therein to provide fluid path for the bead, wherein the lid member comprises a second electrode,   wherein the controller is operable to provide a voltage between the first electrode and the second electrode that is sufficient to cause fluid in the well to undergo electrolysis producing one or more bubbles in the fluid to to rise to a top of the well along with the bead or to lift the bead to the top of the fluid-filled well.   
     
     
         20 . The system of  claim 19 , wherein the diameter of the monodisperse bead varies less than 10%. 
     
     
         21 . (canceled) 
     
     
         22 . The system of  claim 19 , wherein the diameter of the monodisperse bead is between about 29 to 35 μm, the diameter of each well is between about 40 μm to about 45 μm, and the depth of each well is between about 45 μm to about 55 μm. 
     
     
         23 . The system of  claim 19 , wherein the monodisperse bead has a linker loading capacity of the oligonucleotide synthesis substrate within a range of 30 to 100 μmol/g. 
     
     
         24 . The system of  claim 19 , further comprising a bead-collection device operable to collect the monodisperse bead that is removed from the well of the microchip. 
     
     
         25 . The system of  claim 19 , further comprising a first multiwell collection plate operable to receive the monodisperse bead that is collected from the bead-collection device. 
     
     
         26 .- 40 . (canceled) 
     
     
         41 . 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 50 femtomoles to about 15,000 femtomoles, wherein the well is operably connected to a light source for the production of a photogenerated acid;   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   e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule,   wherein the number of sequence errors present in the assembled nucleic acid molecule is less than one base per 10,000 bases.   
     
     
         42 . The method of  claim 41 , wherein the joining in (c) is mediated by polymerase chain reaction and/or ligases. 
     
     
         43 . (canceled) 
     
     
         44 . The method of  claim 41 , wherein the assembled nucleic acid molecule is composed of between five and five thousand nucleic acid molecules. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 41 , wherein the assembled nucleic acid molecule is between 10 kilobases and 1 megabase.