Passive Reference Externalization in Multiplex PCR
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Solution Overview
Problem
Conventional real-time PCR methods face challenges due to reagent pipetting variations and non-optimal characteristics in amplification/detection, leading to systematically incorrect DNA concentration values and limitations in multiplex PCR due to the limited number of available dyes, which restricts the detection of multiple DNA regions simultaneously.
Innovation Solution
The method involves using a passive fluorescence dye in one set of wells and active fluorescence dyes in another set, with both emitting the same measurable spectrum, allowing for normalization and detection of regions of interest without dedicating a dye solely as a passive reference, thereby increasing multiplex level and reducing non-PCR-related imperfections.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a passive reference dye is used in multiplex PCR, then normalization accuracy is improved, but the number of detectable target regions decreases
Solution Approach 1:
The patent divides the plate into different well groups: some wells contain passive reference dye while others contain active fluorescence dyes for detecting different target regions. This segmentation allows the passive reference function to be distributed across multiple wells rather than requiring a dedicated dye in every well, thereby enabling higher multiplexing while maintaining normalization accuracy
Solution Approach 2:
The patent makes the passive reference dye configuration universal across multiple wells in the plate. Instead of each well needing its own passive reference dye (which would limit multiplexing), the system uses multiple wells with passive reference dye to collectively serve as reference for all active dye wells, allowing one dye to effectively serve multiple functions across the plate
2Reliability
If conventional normalization methods are used, then pipetting variations are compensated, but systematically incorrect DNA concentration values persist due to non-optimal amplification/detection characteristics
Solution Approach 1:
The patent implements a feedback mechanism where the passive reference dye signal is used to normalize the active dye signals. By continuously monitoring the passive reference signal across cycles and using it to adjust/normalize the active dye measurements, the system compensates for non-optimal amplification and detection characteristics, thereby improving DNA concentration measurement precision
Solution Approach 2:
The passive reference dye acts as an intermediary that mediates between the active fluorescence dyes and the detection system. It provides a reference signal that captures non-optimal characteristics of the amplification/detection process, which is then used to correct the measurements from active dyes, improving overall measurement precision
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 accurate normalization and detection of multiple DNA regions without sacrificing a dye for passive reference, enhancing the multiplex PCR capability and reducing the need for separate PCR runs, thus providing more reliable and efficient DNA analysis.
Implementation Method 1
providing a first mastermix including a passive fluorescence dye in at least a first well of a plate; providing a second mastermix including an active fluorescence dye in at least a second well of the plate; wherein the passive fluorescence dye and the active fluorescence dye emit a same measurable spectrum
Data Source
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AI summary
Methods for increasing multiplex level by externalization of a passive reference in polymerase chain reactions (PCR) are provided. An exemplary method comprises providing a plate including a plurality of wells; providing a first mastermix including a passive fluorescence dye in at least a first well of the plate; providing a second mastermix including an active fluorescence dye in at least a second well of the plate; wherein the passive fluorescence dye and the active fluorescence dye emit a same measurable spectrum; and wherein the first mastermix is devoid of the active fluorescence dye emitting the same measurable spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same measurable spectrum. Numerous other aspects are provided.