Universal Probe Multiplex Genotyping via Mediator Strands
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Solution Overview
Problem
Current multiplex genotyping methods, such as TaqMan probe-based real-time PCR, are limited in testing capacity due to the need for multiple probes and high-cost modifications, leading to increased complexity and reduced sensitivity, especially when detecting multiple genetic variants or nucleic acid sequences.
Innovation Solution
The use of universal primers and probes, specifically designed for nucleic acid hybridization, which measure fluorescence changes in the presence or absence of quencher-labeled probes, allowing for the detection of multiple genetic variants using a single fluorescence channel and reducing the need for multiple probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple TaqMan probes with different fluorophores are used to increase testing capacity, then the number of detectable targets increases, but the system complexity and modification costs increase
Solution Approach 1:
The patent applies universality by designing a single universal probe that can detect multiple different targets through hybridization to various mediator strands. Instead of requiring separate probes for each target, one universal probe with quencher and fluorophore labels can interrogate multiple mediator strands (M1, M2, M3, etc.) that are complementary to different target sequences, thereby increasing testing capacity without proportionally increasing probe complexity
Solution Approach 2:
The patent introduces mediator strands as intermediary molecules between the universal probe and the target sequences. The mediator strands serve as intermediaries that bind to specific targets and provide binding sites for the universal probe, allowing the probe to indirectly detect multiple different targets through these intermediary mediators without needing multiple specialized probes
2Productivity
If multiple TaqMan probes with different fluorophores are used to increase testing capacity, then the number of detectable targets increases, but the modification costs increase
Solution Approach 1:
The universal probe design allows a single probe sequence to serve multiple detection functions across different targets. This eliminates the need to synthesize and modify multiple different probe sequences with various fluorophores, thereby reducing modification costs while maintaining the ability to detect multiple targets through the same universal probe structure
3Productivity
If multiple fluorogenic probes are used to increase testing capacity, then the number of detectable targets increases, but the fluorescence background increases and testing sensitivity decreases
Solution Approach 1:
The patent extracts the fluorophore and quencher labels from multiple separate probes and consolidates them into a single universal probe. This extraction approach reduces the total number of fluorescent molecules in the reaction, thereby lowering fluorescence background while maintaining the ability to detect multiple targets through the universal probe's interaction with multiple mediator strands
Solution Approach 2:
By using one universal probe instead of multiple target-specific probes, the system reduces the cumulative fluorescence background that would result from multiple probes. The single universal probe maintains adequate signal strength while detecting multiple targets, thereby preserving testing sensitivity across all targets rather than diluting it across multiple probes
4Reliability
If one TaqMan probe is used per target sequence, then detection specificity is maintained, but the design process becomes time-consuming and complexity increases
Solution Approach 1:
The universal probe design allows a single probe sequence to detect multiple different targets, eliminating the need to design and validate multiple separate probes. This dramatically reduces probe design time while maintaining detection specificity through the probe's specific hybridization to each mediator strand, which in turn is specific to its target sequence
Solution Approach 2:
The mediator strands serve as intermediaries that carry the target-specific recognition function. Instead of designing probes to directly bind to each target (which would require extensive design time for each), the system designs mediators to bind to targets and a universal probe to bind to mediators, thereby reducing overall design complexity and time while maintaining specificity through the mediator-target specificity
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 of multiple genetic variants with increased sensitivity and reduced complexity, expanding testing capacity while minimizing costs, as it utilizes a single set of fluorophore and quencher probes across multiple targets.
Implementation Method 1
measuring the fluorescence of fluorophore-labelled probes when bound to a target nucleotide sequence and in the presence of a quencher-labelled probe
Implementation Method 2
when unbound from a target nucleotide sequence and in the absence of a quencher-labelled probe
Implementation Method 3
methods of detecting genetic variants by nucleic acid hybridization
Data Source
AI summary
The subject invention pertains to the multiplex detection of nucleic acid molecules. This invention provides oligonucleotides labeled with a fluorophore and a quencher and oligonucleotides with target binding regions that are broadly compatible with the detection of multiple variant locus or multiple nucleic acid samples. Additionally, this disclosure also describes methods to label detection targets by asymmetric labeled amplification and differentiate the target sequences.