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US 12,668,832 B2Granted
Method for detecting polynucleotide using RISC
AssigneesimfliBIO Inc.
InventorsSungchul Hohng, Soochul Shin
Life of this patent
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Abstract
Disclosed herein is a method for detecting small RNAs and discriminating single nucleotide polymorphism at a single molecule level, and specifically to a method for detection of small RNAs, whereby the detection and quantitation of small RNAs and the identification of single nucleotide polymorphism (SNP) can be achieved.
Claims13
A method for detecting a polynucleotide in a biological sample, the method comprising:
- (a) contacting a composition for detecting the polynucleotide with the biological sample,
- wherein the composition for detecting the polynucleotide comprises a first protein-nucleic acid complex comprising an Argonaute protein associated with a first nucleic acid molecule and a second protein-nucleic acid complex comprising an Argonaute protein associated with a second nucleic acid molecule,
- wherein the first nucleic acid molecule comprises a nucleic acid sequence complementary to a first target nucleic acid region in the polynucleotide to be detected, and the second nucleic acid molecule comprises a nucleic acid sequence complementary to a second target nucleic acid region in the polynucleotide to be detected, wherein the first target nucleic acid region and the second target nucleic acid region differ from each other, and wherein the first nucleic acid molecule is conjugated with a first fluorophore and the second nucleic acid molecule is conjugated with a second fluorophore, wherein the first fluorophore differs from the second fluorophore,
- wherein the first fluorophore generates a first fluorescent signal, and the second fluorophore generates a second fluorescent signal;
- (b) detecting the first and the second fluorescent signals resulting from the contacting of (a), wherein the first fluorescent signal is a fluorescent signal generated by the first fluorophore conjugated with the first nucleic acid sequence bound to the first target nucleic acid region and the second fluorescent signal is a fluorescent signal generated by the second fluorophore conjugated with the second nucleic acid sequence bound to the second target nucleic acid region;
- (c) calculating time fractions of bound state for the first fluorescent signal and for the second fluorescent signal according to formula:
- Time fraction of bound state=T/P.
- wherein T is a total time of sensing the first or the second fluorescent signal during a detection time, and P is the detection time;
- and
- (d) determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than a first threshold value set for the first target nucleic acid region and the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than a second threshold value set for the second target nucleic acid region,
- wherein each of the first and the second threshold values is a value in a range of from 0.03 to 0.12.
8 more elements
A method for detecting a single nucleotide polymorphism (SNP) in a polynucleotide, of the method comprising:
- (a) contacting a composition for detecting the polynucleotide with a biological sample comprising the polynucleotide to be detected,
- wherein the composition for detecting the polynucleotide comprises a first protein-nucleic acid complex comprising an Argonaute protein associated with a first nucleic acid molecule and a second protein-nucleic acid complex comprising an Argonaute protein associated with a second nucleic acid molecule,
- wherein the first nucleic acid molecule comprises a nucleic acid sequence complementary to a first target nucleic acid region in the polynucleotide to be detected, and the second nucleic acid molecule comprises a nucleic acid sequence complementary to a second target nucleic acid region in the polynucleotide to be detected, wherein the first target nucleic acid region and the second target nucleic acid region differ from each other, and wherein the first nucleic acid molecule is conjugated with a first fluorophore, and the second nucleic acid molecule is conjugated with a second fluorophore, wherein the first fluorophore differs from the second fluorophore,
- wherein the first fluorophore generates a first fluorescent signal, and the second fluorophore generates a second fluorescent signal;
- (b) detecting the first and the second fluorescent signals resulting from the contacting of (a), wherein the first fluorescent signal is a fluorescent signal generated by the first fluorophore conjugated with the first nucleic acid sequence bound to the first target nucleic acid region and the second fluorescent signal is a fluorescent signal generated by the second fluorophore conjugated with the second nucleic acid sequence bound to the second target nucleic acid region;
- (c) calculating time fractions of bound state for the first fluorescent signal and for the second fluorescent signal according to formula:
- Time fraction of bound state=T/P.
- wherein T is a total time of sensing the first or the second fluorescent signal during a detection time, and P is the detection time;
- and
- (d) determining that the biological sample comprises the polynucleotide to be detected when the calculated time fraction of bound state for the first fluorescent signal is equal to or higher than a first threshold value set for the first target nucleic acid region and the calculated time fraction of bound state for the second fluorescent signal is equal to or higher than a second threshold value set for the second target nucleic acid region,
- wherein each of the first and the second threshold values is a value in a range of from 0.03 to 0.12.
8 more elements
Dependencies are inferred from the claim text.