Base-Pairing Amplification Reporter for Digital PCR Signal Separation
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Solution Overview
Problem
Digital PCR assays face challenges in reliably distinguishing between negative and positive partitions due to varying fluorescence differences, which diminishes the ability to accurately detect the presence of nucleic acid targets.
Innovation Solution
The use of an amplification reporter system composed of a first and second oligomer that base-pair below a melting temperature, where the photoluminescence is affected by their pairing, allowing for enhanced signal differentiation between negative and positive partitions through a proximity-dependent energy transfer pair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a probe including an oligonucleotide conjugated to a fluorophore and a quencher is used for detection, then the ability to detect target amplification is improved, but the fluorescence difference between negative and positive partitions diminishes, reducing measurement precision
Solution Approach 1:
The invention divides the probe into two separate oligomers: a first oligomer containing the fluorophore and a second oligomer containing the quencher. These segmented oligomers can base-pair with each other below the melting temperature to form a reporter with reduced background fluorescence, while separating during amplification above the melting temperature to enable signal detection.
Solution Approach 2:
The invention utilizes temperature as a critical parameter to control the base-pairing state of the oligomers. By operating below the melting temperature for reporter formation and above it for amplification, the system dynamically changes the photoluminescence properties of the reporter to achieve both low background and high signal differentiation.
2Object-generated harmful factors
If the probe is designed to quench fluorophore emission in the absence of target amplification, then background noise is reduced, but the signal magnitude difference between negative and positive partitions decreases, worsening measurement precision
Solution Approach 1:
The reporter system dynamically transitions between quenched and unquenched states based on temperature. Below the melting temperature, the first and second oligomers base-pair to quench the fluorophore, minimizing background noise. Above the melting temperature during amplification, they separate, allowing strong fluorophore emission and creating a large signal magnitude difference for accurate detection.
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 reduces photoluminescent background in negative partitions, increasing the signal magnitude difference between negative and positive partitions, thereby improving the reliability of digital PCR assays.
Implementation Method 1
a first and second oligomer capable of base-pairing with one another below a melting temperature of the reporter
Implementation Method 2
The reporter has a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another
Implementation Method 3
through a proximity-dependent energy transfer pair
Data Source
Figure 1~2
Figure 3
Figure 4~6
AI summary
System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.