Nucleic Acid Quantification via Fluorescence Thresholds
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for quantifying initial nucleic acid concentration from nucleic acid amplification data require differentiation or integration, which can be complex and inefficient.
Innovation Solution
A method that amplifies nucleic acid, produces a function correlating fluorescence intensity with amplification cycle or time, calculates characteristic amplification cycles or times based on maximum or minimum fluorescence values, and determines initial concentration without differentiation or integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods using differentiation or integration are used to quantify initial nucleic acid concentration, then quantification can be performed, but the calculation process becomes complex and computationally intensive
Solution Approach 1:
The patent transforms the quantification approach by changing from derivative-based parameters to direct fluorescence intensity parameters. Instead of calculating n-th order derivatives of fluorescence intensity functions, the method uses characteristic amplification cycle numbers (Ct values) where fluorescence intensity reaches specific thresholds, thereby simplifying the calculation while maintaining quantification accuracy
Solution Approach 2:
The patent inverts the conventional approach by not calculating derivatives to find concentration, but rather using the amplification cycle number at which fluorescence reaches a threshold to directly determine concentration. This inversion simplifies the mathematical operations from complex differentiation to simple threshold-based measurement
2Ease of operation
If conventional PCR with endpoint detection is used, then the method is simple, but quantitative analysis cannot be performed
Solution Approach 1:
The patent introduces real-time fluorescence detection as an intermediary between the PCR amplification process and the final quantification result. The fluorescence signal serves as a mediator that provides continuous information about amplification progress, enabling quantitative analysis while maintaining the simplicity of the PCR protocol
Solution Approach 2:
The patent replaces the mechanical endpoint detection method (agarose gel analysis) with an optical detection system that measures fluorescence intensity in real-time during amplification, thereby enabling quantitative analysis without complicating the overall process
3Measurement precision
If real-time PCR with derivative-based quantification is used, then quantitative analysis is possible, but the number of amplification cycles required increases
Solution Approach 1:
The patent performs preliminary determination of the characteristic amplification cycle number (Ct) during the amplification process itself, rather than requiring additional post-amplification analysis steps. This allows quantification to be obtained directly from the amplification data, reducing the total number of cycles needed
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 rapid and accurate quantification of initial nucleic acid concentration using characteristic amplification cycles or times, reducing the number of amplification cycles needed and improving quantification accuracy.
Implementation Method 1
A nucleic acid is amplified
Implementation Method 2
a function representing a correlation between fluorescence intensity which increases or decreases in proportion to the amount of the nucleic acid
Data Source
AI summary
Provided is a method for quantifying an initial concentration of a nucleic acid from a real-time nucleic acid amplification data. Nucleic acid (DNA or RNA) extracted from organism or virus is amplified using an enzyme. Then, the initial concentration of the nucleic acid is found by calculating the characteristic amplification cycle number or the characteristic amplification time at which the fluorescence intensity of the nucleic acid subtracted by the background fluorescence intensity of the nucleic acid has half of its maximum value, or the characteristic amplification cycle number or the characteristic amplification time at which the amplification efficiency has the maximum or the minimum value, or the prior-to-amplification fluorescence intensity of the nucleic acid subtracted by the background fluorescence intensity of the nucleic acid. Accordingly, the initial concentration of the nucleic acid can be calculated without differentiation or integration.