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US 10,865,396 B2Granted
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
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Abstract
The invention provides compositions and methods for modifying the 3′-terminal ends of nucleic acids using DNA polymerase θ terminal transferase activity.
Claims29
A method of modifying a 3′ terminal end of a nucleic acid with a substrate, the method comprising:
- forming a mixture comprising an A family polymerase, a substrate, a nucleic acid, and a reaction solution,
- wherein the A family polymerase is Polθ or an active fragment or variant thereof,
- wherein the nucleic acid is selected from the group consisting of a single stranded nucleic acid, double stranded nucleic acid possessing a 3′ single stranded overhang, double stranded nucleic acid lacking a 5′ single stranded overhang, and nucleic acid lacking a 5′ single stranded overhang;
- and
- wherein the reaction solution comprises at least one divalent metal,
- wherein the divalent metal comprises manganese (Mn2+);
- incubating the mixture;
- and
- isolating a 3′-terminal end modified nucleic acid.
6 more elements
A kit for modifying a 3′ terminal end of a nucleic acid with a substrate, the kit comprising an A-family polymerase and a reaction solution,
- wherein the nucleic acid is selected from the group consisting of a single stranded DNA (ssDNA), double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA;
- wherein the A-family polymerase is Polθ, or an active fragment or variant thereof;
- and
- wherein the reaction solution comprises manganese (Mn2+).
1 more element
A method of synthesizing sequence-specific nucleic acids, the method comprising:
- forming a mixture comprising an A family polymerase, at least one nucleotide, and a reaction solution, wherein the reaction solution comprises a nucleic acid and at least one divalent metal,
- wherein the nucleic acid is selected from the group consisting of a single stranded DNA (ssDNA), double stranded DNA possessing a 3′ ssDNA overhang, single stranded RNA, double stranded RNA possessing a 3′ single stranded RNA overhang, double stranded DNA/RNA hybrid possessing a 3′ ssDNA overhang, double stranded DNA/RNA hybrid possessing a 3′ single strand RNA overhang, nucleic acid lacking a 5′ ssDNA overhang, and telomeric ssDNA,
- wherein the A family polymerase is Polθ, or an active fragment or variant thereof;
- and
- wherein the divalent metal comprises manganese (Mn2+);
- incubating the mixture;
- and
- isolating a nucleic acid with a defined sequence.
5 more elements
Dependencies are inferred from the claim text.