A universal template strand built with universal base analogs is used as a template for polynucleotide synthesis. The universal template strand can hybridize to any sequence of nucleotides. A new polynucleotide is synthesized by using a polymerase to extend a primer hybridized to the universal template strand. Unlike primer extension in polymerase chain reactions, base pairing with nucleotides in the template strand does not specify the sequence of the new polynucleotide. Instead, the sequence of the new polynucleotide is specified by the order of addition of protected nucleotides. After addition of a single species of protected nucleotide, the blocking group is removed and another protected nucleotide is added. The order of nucleotide addition can be varied to create any sequence. After synthesis, the polynucleotide can be dehybridized from the universal template strand. The universal template strand may then be reused to synthesize a different polynucleotide.

Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.

Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. 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.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. 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.

Cleavable nucleotide analogs are provided. The nucleotide analog includes a nucleotide molecule attached to a cleavable moiety wherein the cleavable moiety comprises a protective group and/or a linker attached to a fluorophore. The cleavable moiety is linked to the oxygen atom of the 3′-OH of the pentose of the nucleotide molecule. The nucleotide analogs can be used in making polynucleotide molecules using template independent polymerases. The nucleotide analogs can act as reversible terminators during DNA sequencing by synthesis. The cleavage of the cleavable moiety restores a free 3′-OH functional group allowing growth of the polynucleotide molecule. The general structures as well as proposed synthetic schemes for the nucleotide analogs are also provided.

Cleavable nucleotide analogs are provided. The nucleotide analog includes a nucleotide molecule attached to a cleavable moiety wherein the cleavable moiety comprises a protective group and/or a linker attached to a fluorophore. The cleavable moiety is linked to the oxygen atom of the 3′-OH of the pentose of the nucleotide molecule. The nucleotide analogs can be used in making polynucleotide molecules using template independent polymerases. The nucleotide analogs can act as reversible terminators during DNA sequencing by synthesis. The cleavage of the cleavable moiety restores a free 3′-OH functional group allowing growth of the polynucleotide molecule. The general structures as well as proposed synthetic schemes for the nucleotide analogs are also provided.

Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.

Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.

Disclosed herein include methods and compositions for nucleic acid synthesis using a terminal deoxynucleotidyl transferase with deoxyribonucleotide trisphosphates each comprising a modified base with a photocleavable carbon chain moiety that enables single incorporations when present.