Digital PCR Droplet Analysis via Melting Temperature
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Solution Overview
Problem
Digital PCR measurement reproducibility and accuracy are compromised due to unequal PCR reaction efficiencies and the difficulty in distinguishing empty droplets lacking a target gene, which leads to low signal-to-noise ratios and inaccurate fluorescence intensity-based differentiation.
Innovation Solution
Measuring the ratio of fluorescence intensity at a lower temperature to that at a higher temperature during a melting curve analysis allows for the accurate identification and removal of empty droplets, improving measurement reproducibility and accuracy by distinguishing between droplets containing and lacking the target gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence intensity is measured to identify target gene presence in droplets, then target gene detection is enabled, but measurement reproducibility and accuracy decrease due to unequal PCR reaction efficiencies
Solution Approach 1:
The patent changes the measurement parameter from fluorescence intensity to melting temperature (Tm). By measuring Tm of the PCR amplicon instead of relying on fluorescence intensity, the method overcomes the problem of unequal PCR reaction efficiencies affecting measurement accuracy, since Tm is an intrinsic property of the DNA sequence and is independent of the amount of DNA present.
2Measurement precision
If empty droplets are identified based on fluorescence intensity, then target gene presence can be determined, but distinction accuracy decreases due to low signal-to-noise ratios
Solution Approach 1:
The patent changes the detection parameter from fluorescence intensity to melting temperature (Tm). Since Tm is determined by the DNA sequence composition rather than the amount of DNA, it provides a clear distinction between droplets containing target gene and empty droplets, significantly improving the signal-to-noise ratio and distinction accuracy.
3Adaptability or versatility
If molecular beacon probe is used for melting curve analysis, then genotype identification is enabled, but fluorescence intensity becomes weak and fluctuations increase
Solution Approach 1:
The patent changes the measurement parameter from fluorescence intensity to melting temperature (Tm). By focusing on Tm measurement rather than fluorescence intensity, the method enables genotype identification through Tm differences while avoiding the problem of weak and fluctuating fluorescence signals associated with molecular beacon probes.
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 method enhances the ability to differentiate between droplets with and without the target gene, leading to improved measurement accuracy and reproducibility in digital PCR by effectively distinguishing empty droplets and accounting for PCR reaction efficiency variations.
Implementation Method 1
measuring a fluorescence intensity in association with a temperature change
Implementation Method 2
calculating a melting temperature of a DNA double strand based on a change in the fluorescence intensity
Data Source
AI summary
The present invention addresses the problem of providing a novel digital PCR analysis method. One embodiment of the novel digital PCR analysis method is a DNA detection method including the steps of: partitioning a DNA solution containing a fluorescent-labeled probe or a DNA intercalator and a target DNA to be detected into a plurality of compartments; carrying out a nucleic acid amplification reaction in the compartments; measuring a fluorescence intensity in association with a temperature change; calculating a melting temperature of a DNA double strand based on a change in the fluorescence intensity in association with the temperature change; and calculating a ratio of a fluorescence intensity at a second temperature that is lower than a first temperature in association with the temperature change to that at the first temperature.