Methods and apparatus for synthesizing nucleic acids
First Claim
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1. A method for synthesizing an oligonucleotide, comprising:
- exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,wherein the nucleotide analog comprises a nucleotide having an unmodified 3′
hydroxyl and an inhibitor coupled to the nucleotide by a cleavable linker, wherein the inhibitor prevents the nucleotidyl transferase from catalyzing incorporation of either a natural nucleotide or nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.
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Abstract
The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.
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22 Claims
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1. A method for synthesizing an oligonucleotide, comprising:
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exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide, wherein the nucleotide analog comprises a nucleotide having an unmodified 3′
hydroxyl and an inhibitor coupled to the nucleotide by a cleavable linker, wherein the inhibitor prevents the nucleotidyl transferase from catalyzing incorporation of either a natural nucleotide or nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for synthesizing an oligonucleotide, comprising:
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exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide, wherein the nucleotide analog comprises a nucleotide having an unmodified 3′
hydroxyl and an inhibitor coupled to the nucleotide by a cleavable linker, the inhibitor configured to prevent the nucleotidyl transferase from catalyzing incorporation of either a natural nucleotide or nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, andwherein the synthesized oligonucleotide remains attached to the solid support for subsequent use in an application. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22)
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Specification