Competing Oligonucleotide Probes for Multiplex Variant Detection
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Solution Overview
Problem
Existing nucleic acid detection technologies face limitations in multiplexing capacity, require complex and costly protocols, and struggle with detecting minor DNA variants in mixtures, especially in applications like cancer diagnostics and non-invasive prenatal testing.
Innovation Solution
A method utilizing two competing probes with adjusted melting temperatures (ΔTm) for real-time PCR, allowing simultaneous detection and quantification of multiple genetic variants without blocking amplification, using capillary electrophoresis for signal differentiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If standard qPCR technologies are used for detection, then detection capability is achieved, but multiplexing capacity is limited and device complexity increases
Solution Approach 1:
The patent combines multiple detection functions into a single qPCR assay by using probe sets with different Tm values that can simultaneously detect multiple analytes (wild-type and variant sequences) in one reaction, eliminating the need for separate reactions or complex multiplexing systems
Solution Approach 2:
The probe set design enables a single assay to perform multiple functions: detecting wild-type sequences, detecting variant sequences, and quantifying variant frequency, thereby reducing the need for multiple separate detection systems
2Measurement precision
If sequencing is used for variant detection, then detection precision is improved, but time consumption and cost increase
Solution Approach 1:
The patent replaces the complex sequencing process with a simplified qPCR-based detection method using Tm-differentiated probes, achieving accurate variant detection without the time-consuming and costly sequencing workflow
Solution Approach 2:
The invention utilizes melting temperature (Tm) as a discriminating parameter to distinguish between wild-type and variant sequences, enabling precise detection through thermal transitions rather than sequence reading, thereby reducing time and cost
3Reliability
If blocking amplification methods are used, then detection specificity is improved, but amplification efficiency decreases
Solution Approach 1:
The patent introduces Tm-differentiated probes as intermediaries that bind to specific sequences (wild-type or variant) and produce distinguishable signals, achieving detection specificity without blocking the amplification process itself
Solution Approach 2:
The invention changes the detection parameter from blocking amplification to measuring Tm differences of probe-target hybrids, maintaining amplification efficiency while achieving high detection specificity through thermal transition analysis
4Measurement precision
If multiple separate assays are used for variant detection, then detection precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent merges multiple detection assays into a single qPCR reaction by designing probe sets with distinct Tm values that can simultaneously detect multiple analytes, reducing assay complexity while maintaining detection precision
Solution Approach 2:
The probe set design creates a universal detection system that can identify wild-type sequences, variant sequences, and quantify variant frequency within a single assay, eliminating the need for multiple separate tests
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 fast, sensitive, and cost-effective detection of multiple genetic variants in a single PCR reaction, reducing the need for sequencing and providing reliable results within hours, suitable for applications like molecular tumor diagnostics.
Implementation Method 1
the downstream probe hybridizes with a decreasing hybridization rate in relation to the upstream probe to the target sequence
Implementation Method 2
the upstream probe has a sequence region (1) and the downstream probe has a sequence region (3), both have an overlapping region (2)
Implementation Method 3
hydrolysis probes (sometimes also referred to as TaqMan probes, a trademark of Roche Diagnostics International AG, Rotkreuz, CH) are hydrolysed after each PCR cycle by the intrinsic nuclease of Taq DNA polymerase
Implementation Method 4
using capillary electrophoresis for signal differentiation
Implementation Method 5
using a dsDNA selective fluorescent optical read out instruments (e.g. qPCR thermocycler) using a dsDNA selective fluorescent dye (like SYBRTM Green) or FRET (Foerster Resonance Energy Transfer) probes
Implementation Method 6
FRET (Foerster Resonance Energy Transfer) probes which bind specifically to the amplified target DNA
Data Source
AI summary
A method for detection of at least one molecular genetic analyte comprising a upstream and a competitive downstream oligonucleotide probe, a combination of robes and a kit for use in the method. According to the method the upstream probe has a sequence region (1) and the downstream probe has a sequence region (3), both have an overlapping region (2). The regions (1), (2) and (3) have similar melting temperatures (Tm) and wherein the downstream probe hybridizes with a decreasing hybridization rate in relation to the upstream probe to the target sequence with at least one analyte, and the upstream probe hybridizes with an increased hybridization rate in relation to the downstream probe to the target sequence with the at least one analyte. The Detection is based on the released hydrolysis product(s) from the respective probe and optionally in combination with the obtained amplified products.