Generic Reporter Digital Assays for Multiplex PCR Quantification
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Solution Overview
Problem
Current digital assays face challenges in efficiently and cost-effectively detecting and quantifying multiple DNA targets in a single tube using a DNA intercalating dye without the need for sequence-specific probes, as existing methods are limited by instrument capability, resolution, and require time-consuming post-PCR processing.
Innovation Solution
A novel multiplexed PCR method using a DNA intercalating dye, such as EVAGREEN®, where primer concentrations are manipulated to generate distinct fluorescence signals for each target, allowing amplicon detection and quantification in emulsion droplets without sequence-specific probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple DNA targets are detected using sequence-specific probes, then detection precision is improved, but cost and complexity increase due to custom probe synthesis
Solution Approach 1:
A single generic fluorescent reporter molecule serves multiple functions by detecting amplification of different DNA targets through non-specific binding to double-stranded DNA, eliminating the need for multiple target-specific probes while maintaining detection capability across multiple targets
Solution Approach 2:
The invention changes the detection parameter from sequence-specific probe binding to generic fluorescent reporter binding, where detection is based on the physical state of DNA (double-stranded vs single-stranded) rather than sequence specificity, allowing one reporter to detect multiple targets
2Measurement precision
If gel electrophoresis is used for post-PCR processing, then amplicon separation is achieved, but time and operational complexity increase
Solution Approach 1:
The invention extracts the detection function from post-PCR processing steps like gel electrophoresis by implementing real-time fluorescent detection during the amplification process itself, eliminating the need for separate separation and analysis steps
Solution Approach 2:
The fluorescent reporter is incorporated into the reaction mixture before PCR amplification begins, enabling continuous monitoring of amplification in real-time without requiring subsequent processing steps to visualize or separate products
3Adaptability or versatility
If target-specific fluorescent probes are used for multiplexing, then multiple targets are detected, but instrument capability and resolution requirements increase
Solution Approach 1:
A single fluorescent detection channel with a generic reporter detects multiple targets by monitoring overall amplification signals, eliminating the need for multiple specialized detection channels while achieving multiplexing capability
Solution Approach 2:
The invention merges multiple target detection functions into a single detection system by using one generic fluorescent reporter that binds to double-stranded DNA from any target, combining multiple detection capabilities into one instrument channel
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 direct detection and quantification of multiple DNA targets of varying sizes in a single well, eliminating the need for gel electrophoresis and melt-curve analysis, and supporting higher multiplexing capabilities with a single DNA intercalating dye.
Implementation Method 1
A novel multiplexed PCR approach using a DNA intercalating dye, such as EVAGREEN®, where primer concentrations are manipulated to generate distinct fluorescence signals for each target
Implementation Method 2
Amplification of the target can be detected optically from a fluorescent probe included in the reaction. In particular, the probe can include a fluorophore that provides a fluorescence signal indicating whether or not the target has been amplified
Data Source
AI summary
Digital assay system, including methods, apparatus, and compositions, for assay of one or more targets in a set of partitions containing a generic reporter of target amplification.