Abstract: The present disclosure provides methods of polynucleotide synthesis and compounds, compositions useful for synthesis of polynucleotides. The chemical compounds include nucleotides and their analogs that are attached to polymerase though a cleavable linker comprising an enzymatically cleavable amino acid ester.

Abstract: Methods are disclosed for high throughput identification of optimized template-independent polymerase variants. The methods comprise generation of polymerase gene-encoding variants, isolation of variants, expression of variants, exposing expressed polymerase variants to desired conditions for activity, and identification of active variants, such as by hybridization to synthesized polynucleotides or by amplification. Encoding genes of active variants can be sequenced. The method can be performed iteratively to enhance generation and selection of preferred variants.

Abstract: The present disclosure provides modified terminal deoxynucleotidyl (TdT) polymerases and compositions (e.g., conjugates) comprising modified TdT polymerases. The present disclosure also provides circularly permuted terminal deoxynucleotidyl (TdT) polymerases as well as modified TdT polymerases and compositions (e.g., conjugates) comprising such cpTdT or modified TdT polymerases. Also provided are nucleic acids, vectors, and host cells encoding modified TdT polymerases, as well as methods including methods of producing modified TdT polymerases, and methods of using modified TdT polymerases (e.g., methods of nucleic acid synthesis).

Abstract: Methods are disclosed for high throughput identification of optimized template-independent polymerase variants. The methods comprise generation of polymerase gene-encoding variants, isolation of variants, expression of variants, exposing expressed polymerase variants to desired conditions for activity, and identification of active variants, such as by hybridization to synthesized polynucleotides or by amplification. Encoding genes of active variants can be sequenced. The method can be performed iteratively to enhance generation and selection of preferred variants.

Abstract: Disclosed herein are compositions and methods for de novo enzymatic synthesis of nucleic acid molecules. In particular, and among other things, compositions and methods of the present disclosure provide for reduction of non-terminations in a nucleic acid molecule product generated by de novo enzymatic synthesis. Compositions and methods of the present disclosure also include the use of a phosphatase enzyme to facilitate the reduction of non-terminations.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: The present application is directed to, in part, nucleotide analogs with a novel design of a 3?-OH reversible terminator that comprises an enzyme-cleavable linkage.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Abstract: Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.