De novo synthesized gene libraries

摘要

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.

主权项

1. A method of synthesizing nucleic acids, comprising:
a) providing predetermined sequences for at least 200 preselected nucleic acids; b) providing a structure comprising a patterned surface, wherein the patterned surface comprises predetermined regions coated with a first molecule, wherein the first molecule comprises a silane, a siloxane, or is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, and wherein each of the predetermined regions coated with the first molecule is surrounded by a region coated with a second molecule, wherein the second molecule binds to the patterned surface and lacks an available reactive group that binds to a nucleoside phosphoramidite; c) synthesizing at least 20,000 non-identical polynucleotides attached to the patterned surface, wherein the at least 20,000 non-identical polynucleotides are each at least 80 bases long; d) releasing the at least 20,000 non-identical polynucleotides from the patterned surface; e) suspending the at least 20,000 non-identical polynucleotides in a solution; and f) subjecting the solution comprising the at least 20,000 non-identical polynucleotides to a polymerase chain assembly reaction to assemble the at least 200 preselected nucleic acids, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences without correcting errors in the at least 200 assembled preselected nucleic acids, and wherein the patterned surface provides for the aggregate error rate of less than 1 in 2000 bases.