DNA Quantification via Fluorescent Dye Elution
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Solution Overview
Problem
Current methods for determining DNA amounts in samples, especially when DNA is anchored on surfaces or in cells, face challenges such as the need for DNA isolation, metabolic state dependence, and non-linear signal responses, making them inefficient for precise cell counting and DNA quantification across a wide range of concentrations.
Innovation Solution
A method using fluorescent substances like DAPI and Hoechst, which bind to DNA, followed by washing and elution with specific detergents, allowing for the measurement of DNA in an elution solution, providing a linear signal response from 100 cells to full well capacity in 96-well plates, enabling accurate DNA quantification and cell counting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA isolation is performed before measurement, then measurement precision is improved, but device complexity and time consumption increase
Solution Approach 1:
The patent extracts only the essential step of DNA quantification from the complex isolation process. By using fluorescent dyes that bind directly to DNA in cells or on surfaces, the method eliminates the need for complete DNA isolation while maintaining measurement precision. This selective extraction of the quantification function resolves the contradiction by simplifying the overall process.
Solution Approach 2:
The patent replaces mechanical/chemical isolation procedures with a direct fluorescent binding method. Instead of physically isolating DNA through multiple steps, the method uses fluorescent dyes that directly interact with DNA in its natural state, substituting complex mechanical isolation with a simpler optical measurement approach.
2Device complexity
If direct cell counting is performed, then device complexity is reduced, but time consumption increases
Solution Approach 1:
The patent replaces manual mechanical cell counting with automated fluorescent measurement. By using fluorescent dyes that bind to DNA and can be detected optically, the method substitutes time-consuming manual microscopy with rapid automated fluorescence measurement, significantly reducing time loss while maintaining simplicity.
3Device complexity
If metabolic-based cell counting methods are used, then device complexity is reduced, but measurement precision deteriorates due to metabolic state dependence
Solution Approach 1:
The patent extracts the DNA quantification function from metabolic processes. By directly measuring DNA content through fluorescent binding, the method eliminates dependence on cellular metabolism, providing precise cell number measurements regardless of metabolic state while maintaining method simplicity.
4Measurement precision
If fluorescent DNA binding methods are used, then measurement precision is improved, but device complexity increases due to multiple processing steps
Solution Approach 1:
The patent makes the fluorescent measurement system universal by enabling it to work with DNA in multiple states (free, cell-bound, surface-anchored) using the same basic protocol. This multi-functionality reduces the need for different processing steps for different sample types, thereby reducing overall device complexity while maintaining precision.
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 method provides a fast, sensitive, and linear measurement of DNA across a broad concentration range, suitable for both anchored DNA and cells, with minimal material requirements and the ability to process multiple samples efficiently, while being independent of cellular metabolic state and density.
Implementation Method 1
fluorescent substances, which bind to DNA, and after their binding to DNA there is a significant increase of their fluorescence
Implementation Method 2
The fluorescent substances used in the invention are intercalating substances, which intercalate into the double helix of DNA
Implementation Method 3
the fluorescent substances bound to the DNA in the sample are eluted from the DNA with an aqueous elution solution of at least one substance selected from the group consisting of sodium dodecylsulphate, lithium dodecylsulphate and sodium [dodecanoyl(methyl)amino]acetate
Data Source
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AI summary
The present invention concerns a method of determining the amount of DNA in samples, comprising the following steps: (i) a sample is incubated in a solution of at least one fluorescent substance selected from the group consisted of 4',6-diamidino-2-phenylindole, 2-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole, 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole and N,N-dimethyl-4-[5-(4-methyl-1-piperazinyl)[2,5'-bi-1H-benzimidazole]-2'-yl]benzenamine, which, in the course of this incubation, binds to the DNA present in the sample; (ii) the sample is washed with a washing solution and/or water, which washed away the fluorescent substances not bound to the DNA in the sample; (iii) the fluorescent substances bound to the DNA in the sample are eluted from the DNA with an aqueous elution solution of at least one substance selected from the group consisted of sodium dodecylsulphate, lithium dodecylsulphate and sodium [dodecanoyl(methyl)amino] acetate, wherein the concentration of sodium dodecylsulphate, lithium dodecylsulphate and/or sodium [dodecanoyl(methyl)amino]acetate is at least 0.1 % (w/v); (iv) the amount of DNA is determined in the elution solution from the previous step by measuring the fluorescent signal of the fluorescent substance used in step (i). The present invention further relates to a method of determining the amount of 5-ethynyl-2'-deoxyuridine and/or 5-bromo-2'-deoxyuridine and/or 5-chloro-2'-deoxyuridine and/or 5-iodo-2'-deoxyuridine incorporated into DNA and/or the content of proteins and/or the amount of RNA and/or the amount of specific DNA sequences and/or the amount of sugars and/or the amount of lipids and/or the amount of fluorescently-labelled DNA and/or fluorescently-labelled RNA and/or fluorescently-labelled proteins in cells and tissues; to a method of determining the replication activity in cells and tissues; to a kit for determining the amount of DNA in a sample, and to the use thereof.