METHOD FOR DIRECT QUANTIFICATION OF NUCLEIC ACIDS IN REAL TIME qPCR
AssigneeIndian Institute of Technology Madras (Iit Madras)
InventorsGuhan JAYARAMAN, Sudeshna SENGUPTA, Shivansh GOYAL
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Abstract
A method for direct quantification of nucleic acids in real time qPCR. The invention discloses a method for specific quantification of nucleic acids in real time qPCR. The disclosed invention can be achieved in three ways; 1) using a modified primer for qPCR quantification; 2) using strand displacement based probes for qPCR quantification; 3) using label-free endonuclease probe for qPCR quantification. The mechanism of quantification is based on the fact that, DNA, RNA or modified oligonucleotide based light-up dye-aptamer system, where dye is not fluorescent in free state but its fluorescence increases multi-fold when it binds to its specific aptamer.
Claims10
A method for direct quantification of nucleic acids in real time qPCR, said method comprising:
- using a simple and shorter Aptamer-based qPCR (Apt-qPCR) probe for quantification in real-time PCR wherein the probe uses a light-up dye-aptamer system in which the dye shows negligible fluorescence in a free state and its fluorescence increases manifold when it binds to its specific aptamer;
- placing an aptamer 5′ upstream of one of primers, wherein a primer initially in pre-annealed form shows fluorescence as aptamer is free and single-stranded and can bind to the dye;
- and
- performing annealing and extension step to make the aptamer double stranded and thereby loose its 3D structure to form a double helix wherein the double helix is not specific for the dye and do not bind, therefore reducing fluorescence of solution corresponding to each cycle of qPCR reaction.
Dependencies are inferred from the claim text.