De novo synthesized gene libraries
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 computer-assisted nucleic acid synthesis, comprising:
receiving instructions in a computer readable non-transient medium for synthesis of cDNA sequences encoding for at least 750 genes;
processing the instructions in a computer and transmitting synthesis instructions to a material deposition device, wherein the synthesis instructions provide for synthesis of a plurality of polynucleotides that collectively encode for the cDNA sequences;
releasing synthesis reagents from the material deposition device to synthesize the plurality of polynucleotides each at least 100 bases in length, wherein the plurality of polynucleotides encode sequences with an aggregate error rate of less than 1 in 1000 bases without correcting errors compared to the cDNA sequences received in the instructions in the computer readable non-transient medium; and
assembling a plurality of nucleic acids from a subset of the plurality of polynucleotides, wherein the plurality of nucleic acids comprise sequences encoded by the cDNA sequences encoding for at least 750 genes.
2. The method of claim 1 , wherein each nucleic acid of the plurality of nucleic acids is isolated.
3. The method of claim 1 , wherein each nucleic acid of the plurality of nucleic acids is purified.
4. The method of claim 1 , wherein each of the plurality of polynucleotides extends from a surface.
5. The method of claim 1 , further comprising treating the plurality of nucleic acids with an error correction enzyme.
6. The method of claim 1 , wherein each nucleic acid of the plurality of nucleic acids is at least 0.5 kb long.
7. The method of claim 1 , wherein each nucleic acid of the plurality of nucleic acids is at least 3 kb long.
8. The method of claim 1 , wherein the plurality of polynucleotides comprises at least 60,000 polynucleotides.
9. The method of claim 1 , wherein the plurality of polynucleotides comprises at least 100,000 polynucleotides.
10. The method of claim 1 , wherein the plurality of polynucleotides comprises at least 600,000 polynucleotides.
11. The method of claim 1 , wherein the plurality of polynucleotides collectively encode and correspond to cDNA sequences for at least 1000 genes.
12. The method of claim 1 , wherein each polynucleotide is 100 to 500 bases in length.
13. The method of claim 1 , wherein each polynucleotide is 100 to 200 bases in length.
14. The method of claim 1 , wherein the aggregate error rate is determined by sequencing following amplification of the plurality of polynucleotides.
15. The method of claim 14 , wherein the sequencing is performed using Sanger sequencing method.
16. The method of claim 14 , wherein the amplification is performed using a DNA polymerase.
17. The method of claim 1 , wherein the plurality of polynucleotides are synthesized on a solid support.
18. The method of claim 17 , wherein the solid support comprises microstructures functionalized with a coupling reagent for binding to a nucleoside.
19. The method of claim 17 , wherein the plurality of polynucleotides are released from the solid support prior to assembling the plurality of nucleic acids.