Abstract: The invention is directed to methods for synthesizing oligonucleotides directly on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3?-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3? hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Abstract: The present invention is directed to methods and kits for enzymatic synthesis of labeled polynucleotide probes using template-free DNA polymerases.

Abstract: The present invention relates to novel polypeptides having an endonuclease V activity, and uses thereof.

Abstract: The invention is directed to methods and compositions for inkjet assisted synthesis of a plurality of polynucleotides at reaction sites on a substrate using template-free polymerases, such as, terminal deoxynucleotidyl transferases (TdTs). Compositions of the invention include formulations of synthesis reagents for inkjet delivery including, but not limited to, TdT coupling reaction buffers and 3?-O-protected dNTP monomers.

Abstract: The invention is directed to systems, apparatus and kits for automated synthesis of a plurality of polynucleotides in an array of reaction chambers using a template-free polymerase. In some embodiments, adaptive elements and processes are provided to monitor and control disruption of the synthesis process and fluid movement by enzyme aggregation.

Abstract: The invention is directed to methods and apparatus for parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a tem-plate-free polymerase that has sequence-dependent coupling efficiencies. Whenever sequences causing low efficiency coupling occur at a 3? end of a growing chain of a polynucleotide being synthesized, one or more additional coupling cy-cies without de-protection steps are inserted into synthesis plans to provide additional time for completing the coupling reaction at that position of the polynucleotide.

Abstract: The invention is directed to compositions and methods for enzymatic template-free synthesis of polynucleotides wherein different terminal deoxynucleotidyl transferase (TdT) variants are used to incorporate different 3?-O-reversibly blocked deoxynucleoside triphosphates (dNTPs) into a growing chain. In part, the invention is a recognition and appreciation that different TdT variants can be engineered to preferentially incorporate specific dNTPs with higher efficiency than a single “general purpose” TdT variant used to incorporate all four 3-O-reversibly blocked dNTPs.

Abstract: The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides using chain elongation conditions that suppress the formation of DNA secondary structures including, but not limited to, intra-strand and between-strand duplexes, G-quadruplexes, and the like. In some embodiments, such chain elongation conditions include using 3?-O-blocked dNTP monomers that base protection groups or base analogs that suppress the formation of hydrogen bonding in the polynucleotide being synthesized.

Abstract: The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3?-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

Abstract: The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3?-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3? hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Abstract: The invention is directed to methods for synthesizing oligonucleotides directly on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3?-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3? hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Abstract: The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

Abstract: The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3?-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3? hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Abstract: The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3?-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3? hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.