Nucleic Acid Quantification Using Calibration Curves
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Solution Overview
Problem
Current methods for nucleic acid quantification, such as PCR and real-time PCR, face challenges like the plateau effect and require labor-intensive serial dilutions or preparation of competitors, making them inefficient and costly for accurate target nucleic acid quantification.
Innovation Solution
A method involving the collection and processing of initial signal data from experimental samples and standard control samples with known target copy numbers, using a threshold signal line to generate intersecting time values, which are then used to create a slope equation for quantifying target copy numbers, facilitating rapid and accurate nucleic acid quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR is used for nucleic acid quantification, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent uses standard control samples with known target copy numbers as copies of the quantification process. By plotting the relationship between Ct values and log10(target copy numbers) for these controls, a calibration curve is generated that can be used to quantify unknown samples without requiring complex real-time detection equipment. The calibration curve acts as a mathematical model that replicates the quantification function.
Solution Approach 2:
The patent replaces the mechanical/physical real-time detection system with a computational approach. Instead of continuously monitoring fluorescence during PCR cycles using expensive real-time PCR instruments, the method uses endpoint PCR data combined with mathematical modeling (calibration curves and slope calculations) to achieve quantification. This substitutes physical measurement complexity with computational analysis.
2Measurement precision
If real-time PCR with serial dilution is used, then measurement precision is improved, but loss of time and labor increase
Solution Approach 1:
The patent performs preliminary quantification using standard control samples with known concentrations before analyzing unknown samples. By pre-establishing calibration curves and slope values from controls, the method enables rapid quantification of experimental samples without requiring time-consuming serial dilutions. The preliminary work with controls creates reusable reference data that accelerates subsequent measurements.
Solution Approach 2:
The patent changes the approach from analyzing multiple dilution series to analyzing a single endpoint PCR product. Instead of performing serial dilutions and tracking growth rates over time, the method uses the final PCR product amount combined with mathematical transformations (log10 conversion and slope calculations) to achieve quantification. This parameter change dramatically reduces the time and labor required.
3Ease of operation
If quantitative competitive PCR is used, then ease of operation is improved, but device complexity increases due to competitor preparation
Solution Approach 1:
The patent uses standard control samples as copies of the target nucleic acid with known concentrations. These controls serve as reference standards that eliminate the need to prepare complex competitors for each primer. By using identical control samples across different primer sets, the method simplifies the overall procedure while maintaining quantification accuracy.
Solution Approach 2:
The patent creates universal standard control samples that can be used with multiple different primer sets and target nucleic acids. These controls serve multiple functions: they provide calibration data, establish slope values, and enable quantification across different experimental conditions. This universality eliminates the need to prepare separate competitors for each specific target, significantly reducing operational complexity.
Data Source
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AI summary
The present invention provides methods and software applications for quantifying a target in an experimental sample by collecting and processing initial signal data from the experimental sample and at least two standard control samples containing known target copy numbers. In this regards, the present invention allows the quantification of target copy number in the experimental sample.