Multiplex Nucleic Acid Assay Using Distinct Melting Temperatures
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Solution Overview
Problem
Current real-time PCR methods are limited in their ability to simultaneously amplify, detect, and quantify multiple nucleic acid targets in a single tube due to the constraints of optical detection instruments, which can only distinguish a few separate fluorescent labels, restricting the number of targets that can be analyzed to four or less.
Innovation Solution
The method involves using multiple oligonucleotide probes labeled with the same reporter moiety but having distinct melting temperatures, allowing for the simultaneous multiplex real-time PCR amplification, detection, and quantification of multiple targets by generating unique melting profiles, which can be distinguished without the need for multiple fluorescent labels, and further multiplexing is possible by using several sets of probes labeled with different reporter moieties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorescent labels are used to detect multiple targets, then the number of detectable targets increases, but the device complexity and cost increase due to requiring multiple optical detection channels
Solution Approach 1:
The patent uses melting temperature differences to create distinct melting profiles for different probes, analogous to using color changes to differentiate targets. Each probe is designed with a unique melting temperature that produces a characteristic melting curve, allowing differentiation without requiring multiple fluorescent labels. This resolves the contradiction by enabling multiplex detection while maintaining a single optical detection channel.
Solution Approach 2:
The invention changes the detection parameter from fluorescent wavelength discrimination to melting temperature discrimination. By monitoring the melting temperature of probe-target hybrids, the system can distinguish between multiple targets using a single fluorescent label. This parameter change eliminates the need for multiple optical detection channels while maintaining the ability to detect multiple targets simultaneously.
2Quantity of substance
If multiple probes with distinct fluorescent labels are used, then multiple targets can be detected simultaneously, but the measurement precision decreases due to limitations in discriminating light signals at different wavelengths
Solution Approach 1:
The patent employs melting temperature differences to generate distinct melting profiles for each probe-target combination. These melting profiles serve as unique identifiers for each target, providing high measurement precision through thermal discrimination rather than optical wavelength discrimination. This approach overcomes the limitations of fluorescent label discrimination and enables accurate detection of multiple targets.
3Quantity of substance
If the number of optical detection channels is increased, then more targets can be detected, but the loss of substance increases due to higher reagent costs and reaction complexity
Solution Approach 1:
The patent makes a single fluorescent reporter moiety universal for detecting multiple different targets by combining it with probes of different melting temperatures. This multi-functional approach allows one reporter to serve multiple detection purposes, eliminating the need for multiple reporters and reducing reagent complexity and cost while maintaining the ability to detect multiple targets.
Solution Approach 2:
By changing from wavelength-based detection to melting temperature-based detection, the system reduces reagent requirements. A single fluorescent reporter can be used for all targets, and the differentiation is achieved through probe design parameters (melting temperature) rather than through multiple expensive fluorescent labels, thereby reducing substance loss and reaction complexity.
4Quantity of substance
If traditional real-time PCR with multiple fluorescent labels is used, then up to four targets can be detected, but the productivity decreases due to the need for separate analysis of each target
Solution Approach 1:
The patent incorporates a periodic melting step within the PCR cycle where all probe-target hybrids are simultaneously subjected to controlled heating. During this periodic melting phase, the system captures melting profiles for all targets in sequence based on their distinct melting temperatures. This periodic action enables simultaneous analysis of multiple targets within the existing PCR workflow, increasing productivity without requiring separate reactions for each target.
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
This approach enables the detection and quantification of multiple targets in a single tube, expanding the multiplexing capability beyond traditional real-time PCR, allowing for the analysis of up to sixteen or more targets, and providing independent quantitative data for each target sequence, while using a single wavelength channel.
Implementation Method 1
each said labeled oligonucleotide probe is capable of binding to the corresponding target nucleic acid with a melting temperature distinct from the melting temperatures of the other labeled oligonucleotide probes within the same set; detecting light emission from said reporter moiety
Implementation Method 2
each of said labeled oligonucleotide probes is capable of binding to the corresponding target nucleic acid with a melting temperature distinct from the melting temperatures of the other labeled oligonucleotide probes within the same set
Data Source
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AI summary
The invention is a single-tube multiplex assay, capable of simultaneously amplifying, detecting and quantifying multiple nucleic acid targets, using multiple hybridization probes, labeled with the same fluorescent reporter label, but each having a distinct melting temperature. The assay can be further multiplexed with the use of multiple sets of hybridization probes, each set labeled with a separate fluorescent reporter label.