De novo synthesized gene libraries
First Claim
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1. A method for synthesizing a library of oligonucleic acids encoding for predetermined sequences, the method comprising:
- (a) providing predetermined sequences for a library of at least 100,000 non-identical oligonucleic acids, wherein the at least 100,000 non-identical oligonucleic acids collectively encode sequence for at least 750 genes;
(b) providing a structure having a surface, wherein the surface comprises a plurality of clusters, wherein each cluster is selected for synthesis of oligonucleic acids associated with a single gene of the at least 750 genes, and wherein each cluster comprises a plurality of loci for oligonucleic acid extension;
(c) adding a droplet of fluid comprising an extension reaction reagent specific to a locus, wherein the extension reaction reagent comprises a nucleoside phosphoramidite;
(d) allowing sufficient time for an extension reaction step to occur;
(e) repeating steps (c) and (d) to synthesize the at least 100,000 non-identical oligonucleic acids, wherein each non-identical oligonucleic acid is at least 30 bases in length and is attached to the surface; and
(f) releasing the at least 100,000 non-identical oligonucleic acids from the surface, wherein the method comprises no amplification step prior to releasing the at least 100,000 non-identical oligonucleic acids from the surface.
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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.
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Citations
29 Claims
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1. A method for synthesizing a library of oligonucleic acids encoding for predetermined sequences, the method comprising:
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(a) providing predetermined sequences for a library of at least 100,000 non-identical oligonucleic acids, wherein the at least 100,000 non-identical oligonucleic acids collectively encode sequence for at least 750 genes; (b) providing a structure having a surface, wherein the surface comprises a plurality of clusters, wherein each cluster is selected for synthesis of oligonucleic acids associated with a single gene of the at least 750 genes, and wherein each cluster comprises a plurality of loci for oligonucleic acid extension; (c) adding a droplet of fluid comprising an extension reaction reagent specific to a locus, wherein the extension reaction reagent comprises a nucleoside phosphoramidite; (d) allowing sufficient time for an extension reaction step to occur; (e) repeating steps (c) and (d) to synthesize the at least 100,000 non-identical oligonucleic acids, wherein each non-identical oligonucleic acid is at least 30 bases in length and is attached to the surface; and (f) releasing the at least 100,000 non-identical oligonucleic acids from the surface, wherein the method comprises no amplification step prior to releasing the at least 100,000 non-identical oligonucleic acids from the surface. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for synthesizing a library of oligonucleic acids encoding for predetermined sequences, the method comprising:
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(a) providing predetermined sequences for a library of at least 20,000 non-identical oligonucleic acids, wherein the at least 20,000 non-identical oligonucleic acids collectively encode sequence for at least 200 genes; (b) providing a structure having a surface, wherein the surface comprises a plurality of clusters, wherein each cluster is selected for synthesis of oligonucleic acids associated with a single gene of the at least 200 genes, and wherein each cluster comprises a plurality of loci for oligonucleic acid extension; (c) adding a droplet of fluid comprising an extension reaction reagent specific to a locus, wherein the extension reaction reagent comprises a nucleoside phosphoramidite; (d) allowing sufficient time for an extension reaction step to occur; (e) repeating steps (c) and (d) to synthesize the at least 20,000 non-identical oligonucleic acids, wherein each non-identical oligonucleic acid is at least 30 bases in length and is attached to the surface; and (f) releasing the at least 20,000 non-identical oligonucleic acids from the surface, wherein the method comprises no amplification step prior to releasing the at least 20,000 non-identical oligonucleic acids from the surface. - View Dependent Claims (27, 28, 29)
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Specification