Nucleic Acid Quantification by Dilution-Series Fluorescence Fitting
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Solution Overview
Problem
Existing nucleic acid quantification methods using intercalating fluorescent dyes face challenges in accurately determining concentration when samples exceed the linear range of fluorescence intensity, leading to inaccurate measurements due to interference and non-linear relationships.
Innovation Solution
A method involving repeated thinning and fluorescence measurement of nucleic acid solutions with intercalating dyes, followed by plotting fluorescence signals against thinning steps and fitting a function to identify a local maximum, allowing for precise concentration determination within the linear range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a defined amount of fluorescent dye is added to the DNA solution for quantification, then fluorescence intensity increases significantly after intercalation, but measurement accuracy deteriorates when the sample concentration exceeds the linear range
Solution Approach 1:
The patent implements dynamic dilution where the system automatically performs multiple dilutions of the sample solution and measures fluorescence intensity at each dilution step. This dynamic approach allows the measurement system to adapt to samples of varying concentrations by finding the appropriate dilution level that falls within the linear measurement range, thereby resolving the contradiction between maintaining measurement precision and expanding adaptability to different concentration levels
Solution Approach 2:
The patent changes the concentration parameter of the sample solution through systematic dilution steps. By measuring fluorescence intensity at multiple concentration levels (original and diluted samples), the system can identify the linear range and accurately determine DNA concentration even when the original sample exceeds the linear measurement range, thus maintaining measurement accuracy while handling a broader range of sample concentrations
2Measurement precision
If repeated thinning and fluorescence measurement steps are performed to ensure linear range measurements, then measurement accuracy within the linear range is maintained, but measurement time and process complexity increase
Solution Approach 1:
The patent performs preliminary dilution steps before the actual fluorescence measurement to ensure that the sample concentration falls within the linear measurement range. By pre-diluting the sample and measuring at multiple dilution levels, the system avoids the need for complex post-measurement calculations and corrections, thereby maintaining high measurement accuracy while managing the time investment through structured preliminary preparation
Solution Approach 2:
The patent implements a feedback mechanism where fluorescence intensity measurements from multiple dilution steps are used to determine whether the original sample was within the linear range. The system analyzes the relationship between dilution factor and fluorescence intensity to identify the linear range, and uses this feedback information to accurately calculate the original sample concentration, optimizing the measurement process by adapting to the actual sample characteristics
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 accurate and automated quantification of nucleic acid concentrations by ensuring measurements remain within the linear range, avoiding inaccuracies caused by dye interference and non-linearity.
Implementation Method 1
a fluorescent dye whose emission increases after intercalation or binding of nucleic acids
Implementation Method 2
after intercalation or binding of nucleic acids
Data Source
AI summary
A method for quantifying a first solution containing at least one nucleic acid includes thinning the first solution with a second solution containing at least one intercalating fluorescent dye, carrying out a fluorescence measurement on the first solution in order to obtain a fluorescence signal, carrying out a specified number of repetitions of the two preceding steps, plotting the fluorescence signals against a thinning of the first solution, fitting a function to the fluorescence signals, determining a local maximum of the function or its first derivative, and determining a concentration of the nucleic acid from a fluorescence signal positioned before the local maximum.