Multiplex Nucleic Acid Detection via Probe Melting Profiles
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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 distinction, 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, even with probes having the same or similar labels, and consuming probes indicate target presence or quantity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence resonance energy transfer probes with distinguishable emission spectra are used for multiplex detection, then detection specificity is improved, but the number of simultaneously detectable targets is limited to four or five dyes
Solution Approach 1:
The probe is divided into two separate oligonucleotides (first and second oligonucleotides) that can be independently labeled. The first oligonucleotide is labeled with a first label and the second with a second label, allowing independent optimization of each label's properties and enabling more flexible multiplexing strategies beyond the limitations of single-probe FRET systems
Solution Approach 2:
Instead of relying solely on emission spectrum differentiation (one dimension), the invention introduces melting temperature differentiation as a second dimension for probe identification. Probes can be distinguished both by their label emission characteristics and by the thermal stability of their internal double-stranded portions, providing an additional degree of freedom for multiplex detection
2Measurement precision
If amplicon size differences are increased to distinguish multiple targets, then detection capability is improved, but the complexity of analyzing multiple target sequences increases
Solution Approach 1:
The invention changes the distinguishing parameter from amplicon size (physical dimension) to melting temperature (thermal property). By designing probes with internal double-stranded portions of different stability, each probe can be identified by its unique melting profile, allowing target distinction without requiring large amplicon size differences and simplifying the analysis of multiple targets
3Adaptability or versatility
If multiple probes with the same or similar labels are used, then multiplexity is improved, but detection sensitivity decreases due to overlapping emission spectra
Solution Approach 1:
By segmenting the probe into two independently labelable oligonucleotides, the system can use probes with identical or similar labels while maintaining distinguishability through melting temperature profiles. The first and second oligonucleotides can each carry the same label type, yet the probes remain distinguishable via their thermal characteristics
Solution Approach 2:
The invention adds melting temperature as a second differentiation dimension alongside label emission characteristics. This allows multiple probes to share the same or similar labels while being distinguished by their thermal profiles, thereby maintaining high multiplexity without sacrificing detection sensitivity
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 target sequences with enhanced multiplexity and sensitivity, allowing for the analysis of a large number of different nucleic acid sequences in a single reaction, overcoming the limitations of existing methods.
Implementation Method 1
a first oligonucleotide which comprises a first region which is substantially complementary to part of one target nucleic acid and a second region, and at least one second oligonucleotide which comprises a region which is substantially complementary to the second region of the first oligonucleotide, such that the first and second oligonucleotides are capable of forming a double-stranded portion
Implementation Method 2
measuring, at least once, the melting profile of the double-stranded portions between the first and second oligonucleotides of the unconsumed probes in the reaction mixture by detecting the signal(s) from the labels in those probes as a function of temperature
Implementation Method 3
each probe comprises a detectable label or detectable combination of labels which is/are capable of producing a changeable signal which is characteristic of the presence or absence of a double-stranded portion between the first and second oligonucleotides of that probe
Data Source
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.