Fluorescent Dye Enhancing Agent for Reliable Microfluidic PCR
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Solution Overview
Problem
High-concentration nucleic acid dyes inhibit amplification reactions and cause false negative results in PCR, especially in microfluidic systems, limiting their use and reliability.
Innovation Solution
An enhancing agent with a domain that binds to fluorescent dye and a quenching group is used, having a lower affinity for the dye than the amplification products, temporarily storing excess dye and releasing it upon product formation to enhance signal intensity and reduce inhibition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If high-concentration nucleic acid dye is used to enhance fluorescence signal, then fluorescence intensity is improved, but amplification reaction is inhibited
Solution Approach 1:
The patent introduces an enhancing agent as an intermediary substance that mediates between the fluorescent dye and the amplification reaction. This enhancing agent binds to the fluorescent dye, preventing direct inhibition of the amplification reaction while still allowing fluorescence signal generation. The enhancing agent acts as a buffer that decouples the conflicting requirements of high fluorescence intensity and reliable amplification.
2Productivity
If nucleic acid dye is used in microfluidic systems, then miniaturization and reagent savings are achieved, but false negative results occur
Solution Approach 1:
The enhancing agent serves as a mediator that prevents false negative results in microfluidic PCR systems. It binds to fluorescent dye molecules, ensuring that sufficient free dye remains available to bind to amplification products even in the confined microfluidic environment, thereby maintaining reliable detection while preserving the advantages of miniaturization.
3Measurement precision
If fluorescent probe is used to specifically bind to PCR products, then specificity is improved, but synthesis cost and reaction condition complexity increase
Solution Approach 1:
The patent employs a generic enhancing agent that can be easily synthesized and reused, replacing the need for expensive, sequence-specific fluorescent probes. The enhancing agent is a simple molecular structure that can be mass-produced at low cost and does not require complex optimization for different target sequences, making it economically advantageous while maintaining sufficient binding specificity through its interaction with the fluorescent dye.
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
The enhancing agent effectively reduces dye inhibition and enhances fluorescence intensity, improving the reliability of nucleic acid amplification reactions, especially on microfluidic chips, preventing false negatives.
Implementation Method 1
the enhancing agent has a domain binding to the fluorescent dye and a quenching group for quenching fluorescence emitted by the fluorescent dye to which the domain binds
Implementation Method 2
a quenching group for quenching fluorescence emitted by the fluorescent dye to which the domain binds
Data Source
AI summary
The present disclosure discloses an enhancing agent and a kit for enhancing nucleic acid amplification reaction and a method for performing nucleic acid amplification reaction, relating to the field of biotechnology. The enhancing agent disclosed in the present disclosure has a domain capable of binding to fluorescent dye and a quenching group for quenching fluorescence emitted by the fluorescent dye bound to the domain binds. As the binding affinity between the above domain and the fluorescent dye is lower than the binding affinity between a target amplification product of nucleic acid amplification reaction and the fluorescent dye, the enhancing agent not only can reduce inhibition of high-concentration fluorescent dye to amplification reaction, but also can enhance signal intensity of the fluorescent dye in the nucleic acid amplification reaction based on microfluidic chip, improving reliability of application of the fluorescent dye as amplification indicator on the microfluidic chip.