A Process for the Preparation of Nucleic Acid by Means of 3'-O-Azidomethyl Nucleotide Triphosphate

The invention relates to a method of nucleic acid synthesis comprising the use of 3′-O-azidomethyl blocked nucleotide triphosphates which comprises the step of adding a capping group to any uncleaved 3′-O-azidomethyl groups and to the use of kits comprising said capping groups in a method of nucleic acid synthesis. The invention also relates to capped nucleotide triphosphates and 3′-O-azidomethyl capping groups.

NOVEL USE

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis. 1. A method of nucleic acid synthesis comprising a step of providing a terminal deoxynucleotidyl transferase (TdT) enzyme comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 to 5 and 8 , such as any of SEQ ID NOs: 1 , 2 , or 8.2. (canceled)3. A method of nucleic acid synthesis , comprising steps of:(a) providing an initiator sequence;{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, '(b) adding a 3′-blocked nucleotide triphosphate to said initiator sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) as defined in ;'}(c) removal of all reagents from the initiator sequence;(d) cleaving the blocking group from the 3′-blocked nucleotide triphosphate in the presence of a cleaving agent; and(e) removal of the cleaving agent.4. The method as defined in claim 3 , wherein greater than 1 nucleotide is added by repeating steps (b) to (e).5. The method as defined in claim 3 , wherein the 3′-blocked nucleotide triphosphate is blocked by either a 3′-O-azidomethyl claim 3 , 3′-aminoxy or 3′-O-allyl group.6Saccharomyces cerevisiae.. The method as defined in claim 3 , wherein the terminal deoxynucleotidyl transferase (TdT) is added in the presence of an extension solution comprising one or more buffers claim 3 , such as Tris or cacodylate claim 3 , one or more salts; and/or inorganic pyrophosphatase claim 3 , such as purified claim 3 , recombinant inorganic pyrophosphatase from7. The method as defined in claim 3 , wherein step (b) is performed at a pH range of between 5 and 10 ...

Azidomethyl Ether Deprotection Method

The invention relates to a method of converting an azidomethyl ether substituent to a free hydroxyl group. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of nucleotide triphosphates having a 3′-O-azidomethyl substituent, to kits comprising nucleotide triphosphates having a 3′-O-azidomethyl substituent and photoactivatable transition metal complex and to the use of said kits in methods of nucleic acid synthesis and sequencing. 1. A method of converting an azidomethyl ether substituent to a free hydroxyl group wherein said method comprises the step of exposing a compound having said azidomethyl ether substituent to a photoactivated transition metal complex.2. The method as defined in claim 1 , wherein the photoactivated transition metal complex comprises a transition metal selected from ruthenium claim 1 , platinum claim 1 , palladium claim 1 , rhodium and osmium.3. The method as defined in claim 2 , wherein the transition metal is ruthenium.4. The method as defined in any one of to claim 2 , wherein the photoactivated transition metal complex comprises a ligand which is a mono-dentate or bidentate ligand selected from phosphine claim 2 , thiocynate claim 2 , nitrogen claim 2 , pyridine claim 2 , phenanthroline claim 2 , cyclopentadienyl and N-heterocyclic carbine based ligands.5. The method as defined in claim 4 , wherein the photoactivated transition metal complex comprises a pyridine ligand claim 4 , such as a bipyridine ligand.6. The method as defined in claim 5 , wherein the photoactivated transition metal complex is tris(2 claim 5 ,2′-bipyridyl)ruthenium(II)).7. The method as defined in any one of to claim 5 , wherein the azidomethyl ether is present on a ribose or deoxyribose sugar moiety.8. The method as defined in claim 7 , wherein the azidomethyl ether is a 2′ or 3′-O-azidomethyl.9. The method as defined in any one of to claim 7 , which comprises the step of exposing a compound having said azidomethyl ether ...

COMPOSITIONS AND METHODS RELATED TO NUCLEIC ACID PREPARATION

The invention relates to a method of nucleic acid synthesis comprising the use of 3′-O-azidomethyl blocked nucleotide triphosphates which comprises the step of adding a capping group to any uncleaved 3′-O-azidomethyl groups and to the use of kits comprising said capping groups in a method of nucleic acid synthesis. The invention also relates to capped nucleotide triphosphates and 3′-O-azidomethyl capping groups. 1. A method of treating an oligonucleotide , which comprises the steps of:(a) providing an oligonucleotide with a 3′-O-azidomethyl group an initiator sequence; and(b) treating the 3′-O-azidomethyl group via a 1,3-dipolar cycloaddition reaction to prevent subsequent cleavage of the 3′-O-azidomethyl group.2. The method as defined in claim 1 , wherein the 3′-O-azidomethyl group is treated with an irreversible capping group.3. The method as defined in claim 2 , wherein the capping group is a dipolarophile.4. The method as defined in claim 3 , wherein the dipolarophile is an alkyne claim 3 , such as a strained alkyne.5. The method as defined in claim 3 , wherein the dipolarophile is dibenzocyclooctyne-amine.6. The method as defined in claim 1 , wherein the 1 claim 1 ,3-dipolar cycloaddition reaction of step (b) comprises an uncatalysed cycloaddition reaction.7. The method as defined in claim 1 , wherein the 1 claim 1 ,3-dipolar cycloaddition reaction of step (b) comprises a cycloaddition reaction catalysed by a copper or ruthenium-based catalyst.8. The method as defined in claim 2 , wherein the capping group comprises biotin.9. The method as defined in claim 2 , wherein the capping group comprises a fluorine containing moiety.10. The method as defined in claim 2 , wherein the capping group comprises a fluorescent moiety.1216-. (canceled)17. The method as defined in claim 1 , wherein the capping group is an alkyne containing reagent.20. (canceled) This application is a continuation of U.S. patent application Ser. No. 15/555,232 filed Sep. 1, 2017, which is a ...

Compositions and Methods Related to Nucleic Acid Synthesis

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis. 138-. (canceled)39. A method of nucleic acid synthesis , which comprises:(a) providing an initiator oligonucleotide immobilised on a solid support; and(b) adding a 3′-blocked nucleoside triphosphate to said initiator in the presence of a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 1 to 5 and 8 or a fragment of at least 300 amino acids thereof.40. The method as defined in claim 39 , further comprising the steps of:(c) removal of all reagents from the initiator oligonucleotide;(d) cleaving the blocking group from the 3′-blocked nucleoside in the presence of a cleaving agent; and(e) removal of the cleaving agent.41. The method as defined in claim 39 , wherein the terminal deoxynucleotidyl transferase (TdT) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a fragment of at least 300 amino acids thereof.42. The method as defined in claim 40 , wherein greater than 1 nucleotide is added by repeating steps (b) to (e).43. The method as defined in claim 39 , wherein the 3′-blocked nucleoside triphosphate is blocked by either a 3′-O-azidomethyl claim 39 , 3′-aminoxy or 3′-O-allyl group.44Saccharomyces cerevisiae.. The method as defined in claim 39 , wherein the terminal deoxynucleotidyl transferase (TdT) is added in the presence of an extension solution comprising one or more buffers claim 39 , such as Tris or cacodylate claim 39 , one or more salts claim 39 , and inorganic pyrophosphatase claim 39 , such ...

Method of nucleic acid synthesis

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3'-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis.

Compositions and methods related to nucleic acid synthesis

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis.

A process for the preparation of nucleic acid by means of 3'-o-azidomethyl nucleotide triphosphate

A process for the preparation of nucleic acid by means of 3'-o-azidomethyl nucleotide triphosphate

The invention relates to a method of nucleic acid synthesis comprising the use of 3'-O- azidomethyl blocked nucleotide triphosphates which comprises the step of adding a capping group to any uncleaved 3'-O-azidomethyl groups and to the use of kits comprising said capping groups in a method of nucleic acid synthesis. The invention also relates to capped nucleotide triphosphates and 3'-O-azidomethyl capping groups.

Compositions and Methods Related to Nucleic Acid Synthesis

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis.