Self-Quenched Probe for Nucleic Acid Sequence Variation Detection
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Solution Overview
Problem
Current methods for detecting nucleic acid sequence variations using melting curve analysis are limited by the need for multiple probes and channels, high cost, and complexity in synthesizing probes, especially for simultaneous detection of multiple mutations in a single tube.
Innovation Solution
A self-quenched probe method that uses oligonucleotide probes labeled with both a fluorescent group and a quenching group, allowing for simultaneous detection of multiple sequence variations in a single tube without the need for multiple probes or channels, and is cost-effective and easy to synthesize.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent probes and channels are used for simultaneous detection of multiple mutations, then detection capability is improved, but device complexity and cost increase
Solution Approach 1:
A single fluorescent probe is designed to perform multiple detection functions by detecting different melting temperature ranges. The probe can simultaneously identify wild-type sequences, heterozygous mutations, and homozygous mutations through a single fluorescence channel by analyzing the melting curve characteristics at different temperature ranges, eliminating the need for multiple probes and channels.
Solution Approach 2:
The detection method utilizes changes in melting temperature (Tm) as the key parameter to differentiate between wild-type and mutant sequences. By monitoring fluorescence intensity changes across a temperature gradient and identifying characteristic Tm values, the system can distinguish multiple mutation types using only one probe, thereby simplifying the detection system while maintaining high adaptability.
2Adaptability or versatility
If multiple fluorescent probes are used for simultaneous detection of multiple mutations, then detection capability is improved, but manufacturing cost increases
Solution Approach 1:
A single fluorescent probe is designed to perform multiple detection functions by detecting different melting temperature ranges. The probe can simultaneously identify wild-type sequences, heterozygous mutations, and homozygous mutations through a single fluorescence channel by analyzing the melting curve characteristics at different temperature ranges, eliminating the need for multiple probes and channels.
Solution Approach 2:
The invention uses a single probe design that can be replicated and reused for detecting multiple mutation types. Instead of synthesizing multiple different probes, the same probe sequence is used across different detection scenarios, significantly reducing synthesis costs and simplifying manufacturing while maintaining the ability to detect multiple mutations through differential melting temperature analysis.
3Measurement precision
If complex probe structures are used for specific detection, then measurement precision is improved, but ease of manufacture deteriorates
Solution Approach 1:
The detection method utilizes changes in melting temperature (Tm) as the key parameter to differentiate between wild-type and mutant sequences. By monitoring fluorescence intensity changes across a temperature gradient and identifying characteristic Tm values, the system can distinguish multiple mutation types using only one probe, thereby simplifying the detection system while maintaining high adaptability.
Solution Approach 2:
The invention employs a simple, easily synthesizable fluorescent probe that can be manufactured using standard oligonucleotide synthesis techniques. The probe design avoids complex modifications while achieving high measurement precision through clever use of melting temperature analysis and fluorescence monitoring, making it cost-effective and easy to manufacture compared to more complex probe structures.
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 efficient and cost-effective detection of nucleic acid sequence variations by increasing fluorescence intensity upon hybridization, allowing for real-time monitoring of melting curves to differentiate between wild-type and variant sequences, and can be used in commonly available PCR machines.
Implementation Method 1
A self-quenched probe refers to an oligonucleotide probe which is labeled with a fluorescent group and a quenching group, so that fluorescence (or fluorescence intensity) increases upon hybridization of the probe with a target sequence
Implementation Method 2
An increase in temperature leads to denaturation of the double-stranded DNA, resulting in a decrease of fluorescence
Data Source
AI summary
The present invention relates to a method and a kit for detecting nucleic acid sequence variation using melting curve analysis, especially relates to a method and a kit for detecting nucleic acid sequence variation by melting curve analysis using self-quenched probe. Said method provides the characteristics of the self-quenched probe employed, as well as the corresponding nucleic acid amplification conditions, so that the probe can bind to the amplified target sequence, and variations of the target sequence can be detected by melting curve analysis. The present invention also encompasses a kit assembled according to the method described.