Modification of 3′ terminal ends of nucleic acids by DNA polymerase theta
AssigneeTemple University of the Commonwealth System of Higher Education
InventorsRichard T. Pomerantz, Tatiana Kent
Life of this patent
Estimating the term…
Abstract
The invention provides compositions and methods for modifying the 3′-terminal ends of nucleic acids using DNA polymerase θ terminal transferase activity.
Claims27
A method of modifying a 3′ terminal end of a nucleic acid with a substrate, the method comprising:
- forming an in vitro mixture comprising an A family polymerase consisting essentially of SEQ ID NO 1, a substrate, a nucleic acid comprising a single-stranded 3′ end or overhang, and a reaction solution, wherein the reaction solution comprises at least one divalent metal;
- incubating the mixture, wherein the A family polymerase attaches the substrate to the single-stranded 3′ end or overhang of the nucleic acid in vitro by a template-independent terminal transferase activity thereby forming a 3′-terminal end modified nucleic acid;
- and
- isolating a 3′-terminal end modified nucleic acid.
A kit for modifying a 3′ terminal end of a nucleic acid with a substrate, the kit comprising an A-family polymerase consisting essentially of SEQ ID NO 1 operable to attach a substrate to a single-stranded 3′ end or overhang of nucleic acid in vitro by template-independent terminal transferase activity in the presence of a divalent metal and a reaction solution comprising the divalent metal.
A method of de novo synthesis of nucleic acids, the method comprising:
- forming an in vitro mixture comprising an A family polymerase consisting essentially of SEQ ID NO 1, at least one nucleobase, a nucleic acid comprising a single-stranded 3′ end or overhang, and a reaction solution, wherein the reaction solution comprises at least one divalent metal;
- incubating the mixture, wherein the A family polymerase attaches the nucleobase to the single-stranded 3′ end or overhang of the nucleic acid in vitro by a template-independent terminal transferase activity thereby forming an extended nucleic acid;
- and
- isolating the extended nucleic acid.
Dependencies are inferred from the claim text.