Microfluidic-based Gene Synthesis

Abstract

A method for synthesizing long DNA constructs from oligonucleotide precursors directly within a microfluidic device uses several oligonucleotides at once. A precursor mix containing at least two oligonucleotide precursors with at least partial base complementarity is introduced into an input of a microfluidic chip and at least one cycle of at least one gene synthesis protocol are applied to fabricate a DNA construct containing the sequence of at least two oligonucleotide precursors. A method for the synthesis of a modified DNA construct includes electroporating at least one oligonucleotide encoding for at least one point mutation and having homology with at least one DNA region of a target cell into the target cell and incorporating the oligonucleotide into the target cell DNA through the action of recombination protein beta or a recombination protein beta functional homolog.

1 . A method for the synthesis of at least one DNA construct from oligonucleotide precursors, comprising the steps of: 
 introducing a precursor mix into an input of at least one microfluidic device having at least one input, the precursor mix containing at least two oligonucleotide precursors with at least partial base complementarity; and    applying at least one cycle of at least one gene synthesis protocol to fabricate at least one DNA construct containing the sequence of at least two of the oligonucleotide precursors.    
   
   
       2 . The method of  claim 1 , wherein the gene synthesis protocol comprises at least the step of applying a time varying thermal field to the precursor mix and at least one enzyme.  
   
   
       3 . The method of  claim 1 , wherein the gene synthesis protocol comprises at least the step of applying a force to move the precursor mix relative to a spatial thermal gradient.  
   
   
       4 . The method of  claim 1 , wherein the precursor mix comprises at least polymerase and nucleosides suitable for polynucleotide synthesis.  
   
   
       5 . The method of  claim 1  wherein the precursor mix comprises at least ligase and the gene synthesis protocol comprises at least the step of containing the mix for an effective incubation time.  
   
   
       6 . The method of  claim 1 , wherein the oligonucleotide precursors comprise a DNA tile set and the DNA construct is a DNA tile or a set of DNA tiles.  
   
   
       7 . The method of  claim 1 , wherein some portion of the oligonucleotide precursors are derived from an oligonucleotide array.  
   
   
       8 . The method of  claim 7 , wherein the oligonucletide precursors are cleaved from the oligonucleotide array.  
   
   
       9 . The method of  claim 8 , wherein the means of oligonucleotide cleavage is at least one enzyme.  
   
   
       10 . The method of  claim 8 , wherein the means of oligonucleotide cleavage is at least one chemical.  
   
   
       11 . The method of  claim 1 , wherein the microfluidic device further comprises means for hierarchical DNA assembly.  
   
   
       12 . The method of  claim 1 , further comprising the steps of: 
 combining fabricated DNA constructs from multiple microfluidic devices into at least an additional microfluidic device; and    applying at least one cycle of at least one gene synthesis protocol to combine at least two of the DNA constructs to fabricate at least one larger DNA construct.    
   
   
       13 . The method of  claim 1 , wherein the DNA construct is a gene and further comprising the step of expressing the gene as a protein gene product.  
   
   
       14 . The method of  claim 1 , further comprising the step of applying at least one form of error correction.  
   
   
       15 . The method of  claim 14 , wherein the error correction comprises at least one cycle of hybridization by selection.  
   
   
       16 . A method for the synthesis of a modified DNA construct comprising the steps of: 
 electroporating, into at least one target cell, at least one oligonucleotide having homology with at least one DNA region of the target cell and encoding for at least one point mutation; and    incorporating the oligonucleotide into the target cell DNA through the action of recombination protein beta or a recombination protein beta functional homolog.    
   
   
       17 . The method of  claim 16 , wherein a plurality of oligonucleotides is employed and the step of electroporating is repeated a plurality of times.  
   
   
       18 . The method of  claim 16 , wherein the step of electroporating is carried out using an electroporation means comprised within a microfluidic system.  
   
   
       19 . A method for the synthesis of a modified DNA construct, comprising the steps of: 
 introducing, into at least one target cell, at least one polynucleotide construct having homology with at least one DNA region of the target cell and encoding for at least one point mutation;    generating, using the polynucleotide construct, at least one oligonucleotide within the target cell having homology with at least one DNA region of the target cell and encoding for at least one point mutation; and    incorporating the oligonucleotide into the target cell DNA through the action of recombination protein beta or a recombination protein beta functional homolog.