Real-Time PCR Reference Table for Copy Number Analysis
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Solution Overview
Problem
Current nucleic acid quantification methods, such as absolute quantification using quantitative PCR (qPCR), face challenges including high reagent consumption, variability in Cycle Threshold (Ct) values due to external factors, and the need for complex operational procedures, which increase testing costs and require professionally trained operators.
Innovation Solution
A method that standardizes copy number by constructing a reference table through calibration of serial-diluted reference samples, allowing for real-time monitoring and analysis of amplification signals to determine copy number independently of Ct values, eliminating variations from amplification efficiency, polymerase activity, primer concentration, and instrument differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard curves are constructed for each qPCR using serial-diluted standard controls, then accurate copy number quantification is achieved, but reagent consumption increases significantly
Solution Approach 1:
The patent applies preliminary action by pre-establishing a reference table using serial dilutions of reference samples with known copy numbers before actual quantification. This reference table, constructed once and stored digitally, eliminates the need to physically prepare and run standard curves for each qPCR experiment, thereby significantly reducing reagent consumption while maintaining quantification accuracy through digital reference comparison
Solution Approach 2:
The patent uses copying by creating a digital reference table that stores the relationship between Ct values and copy numbers from initial calibration experiments. Instead of physically copying standard curves in each experiment, the system digitally stores and retrieves reference data, replacing physical reagent-based standard curves with virtual references that can be reused indefinitely without additional reagent consumption
2Reliability
If serial-diluted standard controls are amplified along with unknown samples, then Ct value variability is minimized, but operational complexity increases
Solution Approach 1:
The patent applies preliminary action by performing the calibration to establish the reference table once beforehand, capturing the relationship between Ct values and copy numbers under controlled conditions. This preliminary calibration eliminates the need to repeatedly run standard controls with each sample batch, simplifying operational procedures while maintaining reliability through the use of the pre-established reference data
Solution Approach 2:
The patent replaces the mechanical/physical system of running parallel standard curves with each qPCR reaction with a computational system that uses stored reference data and algorithmic comparison. This substitution eliminates the need for complex physical manipulation of standard controls during each experiment, reducing operational complexity while maintaining Ct value consistency through digital reference matching
3Measurement precision
If multiple standard controls are used for calibration, then quantification accuracy is improved, but testing costs increase
Solution Approach 1:
The patent applies preliminary action by performing the calibration experiments once to build a comprehensive reference table that captures the Ct-value to copy-number relationship across multiple concentrations. This one-time calibration investment replaces the need for continuous consumption of standard controls in each subsequent test, thereby improving quantification accuracy while significantly reducing ongoing testing costs
Solution Approach 2:
The patent uses copying by creating a digital replica of the calibration data in the form of a reference table that can be reused indefinitely. Instead of physically copying standard controls for each test, the system digitally stores and retrieves calibration information, eliminating the recurring cost of consumable standard controls while maintaining quantification accuracy through reference data comparison
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 reagent consumption, minimizes operator expertise requirements, and provides accurate copy number quantification by using a reference table to normalize amplification data, thereby reducing testing costs and variability.
Implementation Method 1
amplify several serial-diluted standard controls
Implementation Method 2
detecting the fluorescent signals
Data Source
AI summary
The present invention discloses a method of real-time quantification of a target nucleic acid in a sample by constructing a reference table of copy number vs. designated parameter from reference samples which sharing the same nucleic acid sequences with the target nucleic acid. The method includes (a) constructing a reference table of copy number vs. designated parameter from reference samples; (b) amplifying the target nucleic acid; (c) monitoring and detecting the amplification of the target nucleic acid in real-time; (d) analyzing the detected signals to get the designated parameter of the target nucleic acid; and (e) looking up and interpolating to the reference table to get the copy number of the target nucleic acid.