Automated Nucleic Acid Quantification via Randomness Analysis
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Solution Overview
Problem
Current quantitative PCR methods require human intervention for threshold setting, are not suitable for high-throughput automation, and have limited sensitivity, especially for low copy number targets, making them inefficient for large-scale sample analysis and comparative data standardization across laboratories.
Innovation Solution
A method for fully automated nucleic acid quantification using cycle-to-cycle amplification efficiency calculations and a measure of randomness to identify characteristic cycle numbers, allowing for relative and absolute quantification without human input, and enabling the use of signals from the early exponential phase for precise analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If conventional quantitative PCR methods are used, then nucleic acid quantification can be performed, but human intervention is required for threshold setting and automation is not possible
Solution Approach 1:
The system performs self-service by automatically determining threshold values and characteristic cycle numbers through algorithmic analysis of amplification curves. The software independently calculates Ct-values and quantifies nucleic acids without requiring manual threshold setting or human intervention in the quantification process.
Solution Approach 2:
The patent replaces the mechanical/manual system of human threshold setting with an automated computational system. Algorithms analyze fluorescence signals and automatically determine characteristic cycle numbers, substituting human operational decisions with computer-based calculations and decision-making.
2Productivity
If high-throughput analysis is performed, then large numbers of samples can be analyzed, but complete automation is required which conflicts with manual threshold setting methods
Solution Approach 1:
The automated system performs self-service by independently analyzing multiple samples through standardized algorithms. Each sample's amplification curve is automatically processed to determine Ct-values and quantify nucleic acids, enabling high-throughput analysis without requiring manual intervention for each sample.
Solution Approach 2:
The system achieves universality by applying a single standardized algorithm to analyze multiple different samples simultaneously. The same automated threshold determination and quantification process works across all samples, enabling consistent high-throughput processing of diverse nucleic acid targets.
3Measurement precision
If sensitivity is improved for low copy number targets, then detection capability increases, but measurement precision becomes more difficult to achieve
Solution Approach 1:
The system performs preliminary action by automatically determining optimal threshold values and characteristic cycle numbers before final quantification. This preliminary algorithmic analysis of the amplification curve morphology enables accurate detection even at low copy numbers, establishing reliable measurement parameters in advance.
Solution Approach 2:
The method incorporates feedback mechanisms where the system continuously monitors amplification curve characteristics and adjusts threshold determinations based on the actual signal patterns. This feedback loop ensures that measurement precision is maintained across different copy number ranges by adapting to the specific characteristics of each sample's amplification profile.
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 enables reliable, high-sensitivity, fully automated nucleic acid quantification, including low copy number targets, and facilitates multiplex analyses, improving comparability of analytical data and reducing the need for human interaction in high-throughput applications.
Implementation Method 1
The present invention is related to the field of nucleic acid quantification by means of the polymerase chain reaction (PCR)
Implementation Method 2
Detection of PCR product is achieved e.g. by means of fluorescently labeled hybridization probes emitting specific signals when bound to the target
Implementation Method 3
by means of DNA-intercalating fluorescence dyes that allow to detect double strand product
Data Source
AI summary
The invention relates to a method for quantification of amplified nucleic acids comprising the steps of: calculation of a measure of randomness M of the cycle-to-cycle amplification efficiency Ê(C) for target and comparative nucleic acids,—identification of the cycle numbers CM where M is minimal for target and comparative nucleic acids, calculation of the characteristic cycle numbers Cc from the values of CM-.