Multiplex Drop-Off dPCR for Multi-Locus Mutation Quantification
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Solution Overview
Problem
Existing digital polymerase chain reaction (dPCR) instruments have limited fluorescence detection channels, limiting the multiplex levels of dPCR assays for detecting multiple mutations at different genetic loci, especially in clinical applications with limited sample quantities, and there is a need for robust methods to quantify different genetic species.
Innovation Solution
A method using multiplex drop-off dPCR assays with probe sets comprising drop-off and reference probes, detectable via different channels, allowing for quantification of wildtype and mutant sequences at multiple target regions by hybridization detection, and calculating mutant and wildtype probabilities based on signal counts in partitions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple targets are detected using multiple separate dPCR assays, then each target can be accurately quantified, but the total reaction time increases due to sequential processing
Solution Approach 1:
The patent combines multiple dPCR assays into a single multiplex reaction by pooling target sequences, primers, probes, and polymerase into one reaction mixture. This allows simultaneous amplification and detection of multiple targets (e.g., wild-type and edited alleles) in parallel, reducing total reaction time while maintaining quantification accuracy through distinct probe fluorophores for each target
2Measurement precision
If multiple targets are detected using multiple separate dPCR assays, then each target can be accurately detected, but the number of required reagents and reactions increases
Solution Approach 1:
The invention merges multiple separate reactions into one multiplex reaction, reducing the total quantity of reagents required. By sharing common components (polymerase, buffer, nucleotides) across multiple targets and using distinct probes only for specific target differentiation, the method minimizes reagent consumption while maintaining detection accuracy for each target
Solution Approach 2:
The multiplex assay design allows a single reaction mixture to serve multiple detection functions simultaneously. The universal polymerase and reaction conditions support amplification of multiple target sequences, while specific probes provide targeted detection, making the system multi-functional and reagent-efficient
3Ease of manufacture
If conventional dPCR methods are used for detecting multiple targets, then simple assay design is maintained, but probe design becomes increasingly difficult with more targets
Solution Approach 1:
The patent applies local quality by designing probes that target specific local regions (unique sequences) within each target gene. Each probe is designed to bind to a distinct local region of its target, allowing multiple probes to coexist in the same reaction without cross-reactivity. This localized specificity simplifies the overall probe design process compared to requiring entirely distinct probe structures for each target
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 quantification of wildtype and mutant sequences at multiple genetic loci using fewer detection channels than the total number of probe sets, providing accurate concentration estimates and confidence intervals for clinical applications.
Implementation Method 1
digital polymerase chain reaction (dPCR) assays
Implementation Method 2
fluorescently-labeled probes
Data Source
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AI summary
The present application provides multiplex digital polymerase chain reaction (dPCR) assays such as multiplex drop-off dPCR assays, methods, systems, and kits. The methods described herein are useful in a variety of applications, such as detection of microsatellite instability and quantification of site-specific genome-edited products.