Nucleic Acid Amplification Determination via Signal Differentiation
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Solution Overview
Problem
Conventional melting curve analysis methods for detecting nucleic acid polymorphisms face challenges in accurately determining nucleic acid amplification, requiring specialized knowledge and varying criteria among individuals, which hinders expansion into general analysis and diagnosis, especially for multiple specimens.
Innovation Solution
A method involving signal value processing through first and second-order differentiation to determine nucleic acid amplification, calculating a threshold value (X) based on maximum and differential values, allowing for automated and reliable amplification assessment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If visual observation of melting curves is used to determine nucleic acid amplification, then specialized knowledge and manual judgment are required, but determination accuracy varies between individuals and the process cannot be easily automated
Solution Approach 1:
The patent replaces manual visual observation with automated image processing and pattern recognition algorithms. The system captures melting curve images, processes them through computer algorithms to extract features, and automatically determines amplification results, eliminating the need for specialized manual judgment while ensuring consistent accuracy across all determinations.
Solution Approach 2:
The patent introduces an intermediate image processing system that acts as a mediator between the melting curve data and the final determination. This intermediary layer captures the melting process visually, processes the images through algorithms, and translates them into objective determination results, bridging the gap between raw data and accurate interpretation.
2Reliability
If manual analysis of melting curves is performed, then specialized knowledge is required, but this limits the method's applicability to general analysis and diagnosis
Solution Approach 1:
The patent creates a universal automated determination system that can handle various nucleic acid analysis applications. The image processing and pattern recognition algorithms are designed to work across different sample types and experimental conditions, making the method adaptable to general analysis and diagnosis while maintaining reliable and consistent determination results.
3Productivity
If multiple specimens are analyzed manually, then the process becomes time-consuming and labor-intensive, but automated methods were previously unavailable
Solution Approach 1:
The patent combines multiple functions into an integrated automated system. The melting curve acquisition, image processing, feature extraction, and determination algorithms are merged into a single automated workflow that can handle multiple specimens sequentially or in parallel, dramatically increasing productivity while the system manages its own complexity through unified software control.
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 easy and reliable determination of nucleic acid amplification, reducing the need for specialized knowledge and enabling fully automated genotype determination, facilitating broader application in gene analysis and diagnosis for multiple specimens.
Implementation Method 1
The absorbance at 260 nm increases as a solution containing a double-stranded nucleic acid is heated. This increase is caused by the fact that the hydrogen bond between both the strands in a double-stranded nucleic acid is released by heating, and the double-stranded nucleic acid is dissociated into single-stranded nucleic acids (melting of a double-stranded nucleic acid).
Data Source
AI summary
The present invention provides an amplification determining method that can determine whether or not an objective nucleic acid has been amplified with respect to a sample treated so as to amplify the nucleic acid. Signal values showing molten states of the treated sample at respective temperatures are provided, and the maximum signal value (A) is searched for. Further, signal differential values at the respective temperatures are calculated by differentiation of the signal values at two successive points, and second-order differential values of the differential values are calculated by differentiation at four successive points. Among the second differential values, from those in a predetermined temperature range including a Tm value of the objective nucleic acid, the maximum second differential value (Dmax′) and the minimum second differential value (Dmin′) are selected. Then, the maximum difference (B) is calculated based on the formula (B)=(Dmax′)−(Dmin′). The calculation of the formula X=(B)/(A) is performed, and when X satisfies the condition [X>predetermined threshold value], the amplification is determined as normal amplification, and when X satisfies the condition [X≦predetermined threshold value], the amplification is determined as poor amplification.