Multiplex PCR Probes Using Melting Temperature Differentiation
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Solution Overview
Problem
Current multiplex real-time PCR methods are limited by low multiplexity and sensitivity, as they can only detect and quantify up to four or five fluorescence dyes simultaneously, requiring large differences in amplicon sizes for differentiation, which restricts the ability to analyze multiple target sequences effectively.
Innovation Solution
The method employs probes with distinct melting properties and emission changes based on their internal double-stranded portions, allowing for the simultaneous amplification and detection of multiple target nucleic acid sequences by measuring melting profiles and consumption of probes, even with similar labels, enabling the differentiation of probes with different melting temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorescence dyes are used for multiplex detection, then the number of detectable targets increases, but the system becomes limited to only four or five dyes due to spectral overlap and detection complexity
Solution Approach 1:
The invention changes the detection parameter from fluorescence emission spectra to DNA melting temperature characteristics. Probes are designed with distinct melting temperatures (Tm) that serve as unique identifiers for different target sequences. This allows multiplex detection without relying on multiple fluorescence dyes, as each probe can be detected by monitoring its specific Tm during a melting curve analysis, thereby increasing the number of detectable targets while simplifying the detection system.
Solution Approach 2:
The invention transitions from detecting targets in the fluorescence spectral dimension to detecting them in the thermal dimension through melting temperature analysis. By measuring the temperature at which probes dissociate from target sequences, the system can differentiate between multiple targets using a single fluorescence channel, effectively adding a thermal dimension to the detection process and overcoming the limitation of only four or five simultaneously detectable fluorescence dyes.
2Quantity of substance
If probes with similar labels are used to increase multiplexity, then more targets can be detected, but differentiation between probes becomes difficult
Solution Approach 1:
Instead of relying on different fluorescence labels for probe differentiation, the invention changes the distinguishing parameter to melting temperature (Tm). Each probe is designed with a unique Tm value based on its sequence composition and length. During detection, probes are differentiated by monitoring their specific Tm values during melting curve analysis, allowing multiple probes with similar or identical fluorescence labels to be clearly distinguished based on their thermal characteristics.
3Measurement precision
If large differences in amplicon sizes are required for differentiation, then target sequences can be distinguished, but the ability to analyze multiple targets effectively is restricted
Solution Approach 1:
The invention changes the differentiation parameter from amplicon size to probe melting temperature. By using probes with distinct Tm values that bind to different target sequences, the system can differentiate between multiple targets without requiring large differences in amplicon sizes. This allows for effective multiplex analysis of multiple targets in a single reaction, as each probe's unique Tm serves as a specific identifier regardless of the amplicon length.
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 enhances the multiplexity and sensitivity of multiplex real-time PCR, allowing for the analysis of a large number of different target sequences in a single reaction, improving the accuracy and reliability of nucleic acid detection and quantification.
Implementation Method 1
the distinct melting temperatures or melting profiles of probes
Implementation Method 2
emission of fluorescence from dyes directly or indirectly associated with the formation of newly-synthesized amplicons or the annealing of primers with DNA templates can be detected
Implementation Method 3
a pair of dyes (a reporter dye and an acceptor dye) that are involved in fluorescence resonance energy transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye
Data Source
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AI summary
The invention relates to the field of multiplex amplification. In particular, the invention relates to methods for assaying a sample for one or more nucleic acid targets in a single reaction based on the distinct melting temperatures or melting profiles of primers and/or probes. The invention also provides probes and kits for use in such methods.