Compositions for enzymatic polynucleotide synthesis and methods of use

The method of 16, further comprising cleaving the linker.

The method of any one of the preceding claims, wherein the polymerase is a template-independent polymerase.

A nucleic acid synthesis reaction buffer comprising at least one divalent metal ion, wherein when used in a synthesis reaction, the concentration of the divalent metal ion present when the synthesis reaction is being performed is no greater than about 2500 μM.

The method of 47, wherein the nucleotide is a modified nucleotide.

A method of performing a nucleic acid synthesis reaction comprising providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein said conjugates comprise a nucleotide covalently linked to a polymerase via a linker, and contacting a sample comprising a polynucleotide with said conjugate reagent, wherein said polymerase of the conjugate catalyzes an extension reaction comprising the covalent addition of the nucleotide of the conjugate onto the 3′ hydroxyl of said polynucleotide in the presence of a concentration of cobalt (Co2+), and, optionally, in the absence of Mg2+, wherein the concentration of Co2+ is selected from about 0.250 mM Co2+, about 0.125 mM Co2+, or about 0.050 mM Co2+, and wherein the extension reaction in the presence of the about 0.250 mM Co2+, about 0.125 mM Co2+, or about 0.050 mM Co2+ is faster than a reaction performed in the presence of a concentration of greater than 0.500 mM Co2+.

A method of performing a nucleic acid synthesis reaction comprising providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein said conjugates comprise a nucleotide covalently linked to a polymerase via a linker, and contacting a sample comprising a polynucleotide with said conjugate reagent, wherein said polymerase of the conjugate catalyzes an extension reaction comprising the covalent addition of the nucleotide of the conjugate onto the 3′ hydroxyl of said polynucleotide in the presence of a concentration of zinc (Zn2+), and, optionally, in the absence of Mg2+, wherein the concentration of zinc is selected from about 0.250 mM Zn2+, about 0.125 mM Zn2+, or about 0.050 mM Zn2+, and wherein the extension reaction in the presence of the about 0.250 mM Zn2+, about 0.125 mM Zn2+, or about 0.050 mM Zn2+ is faster than a reaction performed in the presence of a concentration of greater than 0.500 mM Zn2+.

A method of reducing free nucleotide incorporation in a polynucleotide generated using a nucleic acid synthesis reaction comprising providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein said conjugates comprise a nucleotide covalently linked to a polymerase via a linker, and contacting a sample comprising a polynucleotide with said conjugate reagent, wherein said polymerase of the conjugate catalyzes an extension reaction comprising the covalent addition of the nucleotide of the conjugate onto the 3′ hydroxyl of said polynucleotide in the presence of a concentration of cobalt (Co2+), and, optionally, in the absence of Mg2+, wherein the concentration of Co2+ is about 0.050 mM Co2+, and wherein the incorporation of free nucleotides into a polynucleotide using a nucleotide extension reaction in the presence of the about 0.050 mM Co2+ is reduced than in a reaction performed in the presence of a concentration of greater than 0.125 mM Co2+.

A method of improving a nucleotide synthesis reaction, the improvement comprising performing the nucleic acid synthesis in the presence of a divalent cation at a concentration of about 0.250 mM, about 0.125 mM, or about 0.050 mM of the divalent cation, wherein the speed of the extension reaction is greater than the speed of an extension reaction performed in the presence of a divalent cation at a concentration greater than about 0.500 mM.