Method for analyzing activity of nucleic acid polymerase using self-dimer primer
AssigneeSeegene, Inc.
InventorsHyobeen LEE, Byungjun AHN, Myung-Il KIM, Jaemin LEE
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Abstract
A method for analyzing the activity of a nucleic acid polymerase of interest using a self-dimer primer is disclosed. The method requires fewer factors to be considered in design and thus are easy to design, compared to conventional methods using both primers and templates. In addition, the method is simple, fast, cost-effective, safe, and user-friendly compared to conventional methods using radioisotopes.
Claims21
A method for analyzing the activity of a nucleic acid polymerase of interest, comprising the steps of:
- (a) incubating the nucleic acid polymerase of interest with a composition for analyzing activity comprising:
- (i) a self-dimer primer, (ii) an intercalating dye, and (iii) dNTPs or NTPs, wherein the self-dimer primer comprises, in the 5′ to 3′ direction, a 5′-hairpin-non-forming portion and a 3′-hairpin-forming portion, wherein during said incubating, the self-dimer primer hybridizes with another self-dimer primer between 3′-hairpin-forming portions to form a self-dimer, and both strands of the resulting self-dimer are extended by the activity of the nucleic acid polymerase of interest to form an extended dimer, wherein the formation of the extended dimer provides a detectable signal from the intercalating dye;
- and
- (b) measuring the detectable signal over time, wherein the change in signal over time is indicative of the activity of the nucleic acid polymerase of interest.
A composition for analyzing the activity of a nucleic acid polymerase of interest, comprising a self-dimer primer,
- wherein the self-dimer primer comprises, in the 5′ to 3′ direction, a 5′-hairpin-non-forming portion and a 3′-hairpin-forming portion,
- wherein during said incubating, the self-dimer primer hybridizes with another self-dimer primer between 3′-hairpin-forming portions to form a self-dimer, and both strands of the resulting self-dimer are extended by the activity of the nucleic acid polymerase of interest to form an extended dimer, wherein the formation of the extended dimer provides a detectable signal from the intercalating dye,
- wherein the change in signal over time is indicative of the activity of the nucleic acid polymerase.
A self-dimer primer comprising, in the 5′ to 3′ direction, a 5′-hairpin-non-forming portion and a 3′-hairpin-forming portion,
- wherein the 5′-hairpin-non-forming portion has a nucleotide sequence substantially non-complementary to the 5′-hairpin-non-forming portion and the 3′-hairpin-forming portion of another self-dimer primer, and the 3′-hairpin-forming portion has a nucleotide sequence substantially complementary to the 3′-hairpin-forming portion of another self-dimer primer,
- wherein the self-dimer primer is capable of hybridizing with another self-dimer primer between 3′-hairpin-forming portions to form a self-dimer.
Dependencies are inferred from the claim text.