De novo synthesized gene libraries

Abstract

De novo synthesized large libraries of nucleic acids are provided herein with low error rates. Further, devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Longer nucleic acids can be synthesized in parallel using microfluidic assemblies. Further, methods herein allow for the fast construction of large libraries of long, high-quality genes. Devices for the manufacturing of large libraries of long and high-quality nucleic acids are further described herein.

1 . A method for synthesizing oligonucleotides, comprising:
 synthesizing a plurality of oligonucleotides at a rate of extending of at least 15 nucleotides per hour, wherein each of the oligonucleotides has a preselected sequence, and wherein the synthesizing comprises extending each oligonucleotide by a single base in an extension reaction.   
     
     
         2 . The method of  claim 1 , wherein the rate of extending is at least 20 nucleotides per hour. 
     
     
         3 . The method of  claim 1 , wherein the rate of extending is at least 25 nucleotides per hour. 
     
     
         4 . The method of  claim 1 , wherein the plurality oligonucleotides are synthesized in parallel. 
     
     
         5 . The method of  claim 1 , wherein at least 20,000 oligonucleotides are synthesized. 
     
     
         6 . The method of  claim 1 , wherein at least 500,000 oligonucleotides are synthesized. 
     
     
         7 . The method of  claim 1 , wherein each of the oligonucleotides is at least 25 bases in length. 
     
     
         8 . The method of  claim 1 , wherein the plurality of oligonucleotides are synthesized on a solid substrate. 
     
     
         9 . The method of  claim 8 , wherein the solid substrate comprises silicon, silicon dioxide or silicon nitride. 
     
     
         10 . The method of  claim 1 , wherein each of the oligonucleotides has a different preselected sequence. 
     
     
         11 . The method of  claim 1 , wherein each of the oligonucleotides comprises 100 to 200 nucleotides in length and is synthesized in less than 5 hours. 
     
     
         12 . A method of synthesizing oligonucleotides, comprising:
 a. providing a solid substrate;   b. depositing droplets at a velocity of 5 m/s to 30 m/s on the substrate, wherein the droplets comprise nucleosides for oligonucleotide synthesis; and   c. synthesizing a plurality of oligonucleotides, wherein each of the oligonucleotides has a preselected sequence.   
     
     
         13 . The method of  claim 12 , wherein the velocity is about 15 m/s, about 20 m/s, or about 25 m/s. 
     
     
         14 . The method of  claim 12 , wherein the plurality of oligonucleotides are synthesized in parallel. 
     
     
         15 . The method of  claim 12 , wherein each droplet has a volume of 1 to 100 picoliters. 
     
     
         16 . The method of  claim 12 , wherein the nucleosides comprise adenine, guanine, thymine, cytosine, uridine group, or a modified nucleotide. 
     
     
         17 . The method of  claim 16 , wherein each of the oligonucleotides is at least 25 bases in length. 
     
     
         18 . The method of  claim 12 , wherein at least 20,000 oligonucleotides are synthesized. 
     
     
         19 . The method of  claim 12 , wherein at least 500,000 oligonucleotides are synthesized. 
     
     
         20 . The method of  claim 12 , wherein the plurality of oligonucleotides is synthesized on a solid substrate. 
     
     
         21 . The method of  claim 20 , wherein the solid substrate comprises silicon, silicon dioxide or silicon nitride. 
     
     
         22 . The method of  claim 12 , wherein each of the oligonucleotides has a different preselected sequence. 
     
     
         23 . A device for oligonucleotide synthesis, comprising:
 a. a solid support;   b. a plurality of locations on a top surface of the solid support, wherein each of the locations has a pitch of 0.5 mm or higher in length; and   c. a plurality of microwells within each of the locations, wherein each of the microwells has a width of up to 500 μm.   
     
     
         24 . The device of  claim 23 , wherein each of the locations has a pitch of 1, 1.1, 1.2, 1.3, 1.4, or 1.5 mm. 
     
     
         25 . The device of  claim 23 , wherein the solid support comprises nylon, nitrocellulose, glass, silica, silicon, polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, gold, platinum, agarose, dextran, polyacrylamides, or polydimethylsiloxane (PDMS). 
     
     
         26 . The device of  claim 23 , wherein the solid support is a plate or a film.