Multiplexed Real-Time PCR Using Temperature-Dependent Quenching
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Solution Overview
Problem
Current real-time PCR methods are limited in their ability to simultaneously detect and quantify multiple nucleic acid targets due to the constraints of spectrally-resolvable fluorophores, allowing for detection of only four to seven separate targets in a single reaction vessel, which is insufficient for the increasing number of loci of interest in medical and diagnostic applications.
Innovation Solution
The use of novel oligonucleotide probes with a non-complementary tag portion and a quenching molecule, where the tag portion is reversibly bound to a quenching oligonucleotide capable of quenching a reporter moiety, allowing for temperature-dependent signal measurement and calculation to detect multiple targets in a single reaction vessel.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorescently labeled probes are used to detect multiple targets, then the number of detectable targets increases, but the spectral overlap between fluorophores limits detection to only 4-7 targets simultaneously
Solution Approach 1:
The patent changes the detection parameter from spectral wavelength to temperature. By using a single fluorophore with temperature-dependent quenching, the system can distinguish multiple targets based on their different melting temperatures rather than requiring multiple spectrally distinct fluorophores. This allows detection of 12-21 targets using only one or two fluorophores.
Solution Approach 2:
The patent introduces a quenching oligonucleotide as an intermediary that mediates the quenching of the fluorophore. This quenching oligonucleotide binds to the probe in a temperature-dependent manner, allowing the system to modulate fluorescence signal based on temperature rather than relying on spectral properties of multiple fluorophores.
2Quantity of substance
If fluorescent dyes are used for probe labeling, then target detection is enabled, but only about 6-7 dyes can be fit within the visible spectrum without significant overlap interference
Solution Approach 1:
The patent changes the discrimination parameter from spectral wavelength to temperature. By using a single fluorophore whose signal is modulated by temperature-dependent quenching, the system can distinguish multiple targets based on their different melting temperatures rather than requiring multiple spectrally distinct fluorophores.
Solution Approach 2:
The patent adds the temperature dimension to the detection space. Instead of using multiple fluorophores differentiated by wavelength (one dimension), the system uses a single fluorophore differentiated by temperature-dependent quenching (another dimension), effectively expanding the multiplexing capacity.
3Quantity of substance
If a single fluorophore is used with temperature-dependent quenching, then multiplexing capacity increases to 12-21 targets, but the system requires precise temperature control and measurement
Solution Approach 1:
The patent employs periodic temperature cycling characteristic of PCR protocols. By cycling through specific temperature ranges, the system exploits the temperature-dependent binding and quenching behavior to generate distinguishable melting profiles for multiple targets, with each target producing a characteristic curve based on its melting temperature.
Solution Approach 2:
The system uses real-time monitoring of fluorescence signal during temperature cycling to generate melting profiles. The feedback from the fluorescence intensity at different temperature points allows differentiation of multiple targets based on their distinct melting characteristics, with each target producing a characteristic curve.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the detection and quantification of multiple nucleic acid targets in a single reaction vessel, overcoming the limitations of existing methods by allowing for the analysis of up to 12-21 or 16-28 targets using a single reporter moiety, enhancing the throughput and accuracy of nucleic acid sequence detection.
Implementation Method 1
a quenching oligonucleotide that comprises or is associated with one or more quencher moieties capable of quenching the reporter moiety when the quenching oligonucleotide is bound to the tag portion
Implementation Method 2
amplifying the target nucleic acid by PCR
Implementation Method 3
contacting the sample containing the target nucleic acid in a single reaction vessel with (i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid
Implementation Method 4
amplifying the target nucleic acid by PCR using a nucleic acid polymerase
Implementation Method 5
A melting assay involves determining the melting temperature (melting point) of a double-stranded target, or a duplex between the labeled probe and the target. As described in U.S. Patent No. 5,871,908, to determine melting temperature using a fluorescently labeled probe, a duplex between the target nucleic acid and the probe is gradually heated (or cooled) in a controlled temperature program.
Data Source
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AI summary
The present invention describes methods for performing higher multiplexed real-time PCR for detection and quantitation of target nucleic acids using tagged hydrolysis probes.