UV-VIS Spectral Fitting for DNA Quantification
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Solution Overview
Problem
Current UV-VIS spectrophotometry techniques lack an accurate and efficient method for analyzing DNA and RNA samples, particularly in complex mixtures like PCR reactions, due to broad absorbance features and interference from contaminants.
Innovation Solution
A computer-implemented method and system that analyze UV-VIS spectrophotometer data by fitting the data with reference spectra representative of base pairs and nucleobases, allowing for the quantification of double-stranded DNA without the need for purification, and accounting for contaminants and turbidity components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional UV-VIS spectrophotometry is used for DNA/RNA analysis, then the measurement process is simple and fast, but the analysis accuracy is insufficient due to broad absorbance features and interference from contaminants
Solution Approach 1:
The patent segments the complex spectral analysis by decomposing the measured spectrum into contributions from individual components (DNA, RNA, contaminants) using reference spectra. Each component's absorbance profile is separately characterized and combined through linear combination, allowing accurate quantification despite broad overlapping features in the total spectrum.
Solution Approach 2:
The patent introduces reference spectra as intermediary data that mediate between the raw measured spectrum and the final component quantification. These reference spectra serve as templates that enable the decomposition of complex mixtures by providing known absorbance profiles for pure components, thus improving measurement precision without requiring complex instrumentation.
2Measurement precision
If sample purification is performed before analysis, then interference from contaminants is reduced, but the analysis time and process complexity increase
Solution Approach 1:
The patent extracts information about individual components (DNA, RNA, contaminants) directly from the complex mixture spectrum without physical separation. By using reference spectra to identify and quantify each component's contribution, the method extracts pure component signals mathematically, achieving the effect of purification without the time-consuming physical purification steps.
Solution Approach 2:
The patent uses reference spectra as copies of pure component absorbance profiles to represent and quantify components in the mixture. These spectral copies allow the system to identify and measure each component's concentration based on pattern matching, eliminating the need for physical isolation while maintaining measurement accuracy.
3Measurement precision
If complex spectral analysis methods are used, then the quantification accuracy improves, but the ease of operation decreases
Solution Approach 1:
The patent implements self-service through automated spectral decomposition algorithms that perform the complex analysis without user intervention. The system automatically fits the measured spectrum using reference spectra, calculates component concentrations, and handles the mathematical computations, making the sophisticated method as easy to operate as simple absorbance measurement while maintaining high quantification accuracy.
Solution Approach 2:
The patent transforms the complex spectral analysis problem into a parameter optimization problem by adjusting the linear combination coefficients of reference spectra to minimize the difference between the calculated and measured spectra. This parameter-based approach automates the complex analysis, improving quantification accuracy while maintaining operational simplicity through algorithmic optimization.
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 accurate and efficient characterization of DNA and RNA samples, including PCR reactions, by automatically accounting for AT and GC balance and compensating for contaminants, resulting in conclusive and relevant results without the need for sample purification.
Implementation Method 1
UV-VIS absorbance spectroscopy. During such experiments, samples are irradiated with UV-VIS radiation of different wavelengths, the radiation remaining after passage through the sample is detected and the absorbance at different wavelengths is determined
Data Source
AI summary
A method for analyzing UV-VIS spectrophotometer data of a sample comprising at DNA and/or RNA is described. The method comprises receiving UV-VIS spectrophotometer data, fitting the UV-VIS spectrometer data taking into account at least one spectrum representative for a base pair being any of more of adenine-thymine (AT) or guanine-cytosine (GC) or adenine-uracil, and deriving from the fitting a quantification of an amount of DNA and/or RNA.