Methods and apparatus for synthesizing nucleic acids
AssigneeMolecular Assemblies, Inc.
InventorsJ. William Efcavitch, Suhaib Siddiqi
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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.
Claims47
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 coupled, by a cleavable linker, to an inhibitor comprising a negatively-charged moiety and an amino acid with a negative charge that prevents the nucleotidyl transferase from catalyzing incorporation of a nucleotide or an additional nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.
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 coupled, by a cleavable linker, to an inhibitor comprising a positively-charged moiety and an amino acid with a positive charge that prevents the nucleotidyl transferase from catalyzing incorporation of a nucleotide or an additional nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker.
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 has the following inhibitor structure:
- wherein NTP=nucleotide triphosphate, n=1, 2, 3, or 4, and —X— is —O—, —S—, —NH—, or —CH2—.
Dependencies are inferred from the claim text.