Acidic Detergent Lysis for DNA Quantification
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Solution Overview
Problem
Current methods for determining DNA or RNA content in cells are complex, time-consuming, and often result in cell loss and aggregation, making it difficult to accurately quantify nucleic acid in adherent cell lines and promoting noise in analysis.
Innovation Solution
A two-step method involving the use of a weakly acidic solution with a non-ionic detergent and a DNA-specific dye like DAPI to release and label cell nuclei, followed by neutralization, allowing for direct fluorescence quantification without fixation, which reduces aggregation and noise, and can be performed at low magnification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA staining methods are used with non-ionic detergents or alcohol fixation, then cells can be permeabilized for staining, but the methods require cells to be in suspension, require washing steps with centrifugation that results in cell loss, and promote cell aggregation
Solution Approach 1:
The invention changes the chemical parameters of the lysis solution by using a weakly acidic solution (pH 2.0-3.0) containing a non-ionic detergent instead of conventional neutral pH buffers or alcohol fixation methods. This parameter change enables direct lysis of adherent cells without requiring detachment or washing steps, thereby preventing cell loss while maintaining effective DNA staining capability
Solution Approach 2:
The invention extracts the cell lysis function into a single weakly acidic detergent solution that simultaneously performs lysis, permeabilization, and prevents aggregation. This eliminates the need for separate detachment, washing, and resuspension steps that conventionally cause cell loss
2Measurement precision
If conventional DNA staining methods are used with non-ionic detergents or alcohol fixation, then cells can be permeabilized for staining, but washing steps and centrifugation are required which result in cell loss
Solution Approach 1:
The invention merges multiple functions (cell lysis, membrane permeabilization, and aggregation prevention) into a single weakly acidic detergent solution treatment step. This consolidation eliminates the need for separate washing and centrifugation steps, significantly reducing analysis time while maintaining DNA staining quality
Solution Approach 2:
The invention enables continuous processing by using a weakly acidic solution that maintains cells in a stable, non-aggregated state throughout the staining process without requiring interruption for washing or centrifugation, thereby reducing total analysis time
3Measurement precision
If conventional DNA staining methods are used with non-ionic detergents or alcohol fixation, then cells can be permeabilized for staining, but they promote cell aggregation which hampers DNA content quantification of individual cells
Solution Approach 1:
The invention changes the pH parameter to weakly acidic (pH 2.0-3.0) and uses a non-ionic detergent to create a solution environment that prevents cell aggregation during lysis. This parameter change simplifies sample preparation by eliminating the need for additional dispersing steps while maintaining individual cell integrity for accurate DNA quantification
4Productivity
If acid lysis is performed to release nuclei from cells, then nuclei can be released efficiently, but the sample remains acidic which may interfere with subsequent nucleic acid labeling and analysis
Solution Approach 1:
The invention performs preliminary neutralization of the acidic lysis solution immediately after nuclei release. This preliminary action prepares the sample for subsequent nucleic acid labeling by adjusting the pH to a neutral range, ensuring optimal conditions for fluorescent dye binding while maintaining the benefits of efficient acid lysis
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 simple, rapid, and reliable means to quantify nucleic acid content with reduced variability and noise, enabling accurate cell cycle analysis and other cellular assessments without the need for cell detachment or centrifugation.
Implementation Method 1
Adding an acidic agent comprising a detergent causing lysis to the sample causing acidification of the sample to a pH level between 2.0 and 3.0
Implementation Method 2
Adding a fluorescent labelling agent to the sample, wherein the fluorescent labelling agent interacts with nucleic acid
Implementation Method 3
Adding a neutralizing agent for neutralising the sample after or simultaneous to adding the labelling agent
Data Source
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AI summary
Disclosed is a fast and simple method for quantification of nucleic acid of biological cells as 2 -step protocol. In the first step cells are treated with an acidic solution containing a non- ionic detergent and a fluorescent DNA specific label. In the second step the sample may be neutralised. Determining of the content of nucleic can be performed by fluorescence microscopy. The method may also be used for obtaining information of cell cycle analysis, ploidy determination, measurements of nucleotide incorporation and assays for proliferation, health, stress level, apoptosis, necrosis, or other state of conditions of cells. The invention also relates to a kit of parts comprising an acidic agent, a detergent, a labelling agent and optionally a neutralization agent.