Single-Cell Epigenetic Mark Visualization via ISH and Proximity Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current techniques lack the ability to visualize epigenetic marks, such as DNA methylation, at a single genomic locus in individual cells, which is crucial for diagnosing diseases like cancer.
Innovation Solution
A method combining in situ hybridization with a proximity ligation assay (PLA) to detect modified nucleotides in specific nucleic acid sequences within cells, using labeled nucleic acid probes and binding agents to visualize epigenetic marks like DNA methylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional techniques are used to study epigenetic marks, then molecular-level or genomic-scale analysis is achieved, but single-cell single-locus visualization capability is lost
Solution Approach 1:
The method divides the detection process into distinct sequential steps: in situ hybridization with labeled probes to target specific genomic loci, followed by proximity ligation assay steps that separately bind to the probe label and modified nucleotide. This segmentation allows each step to be optimized independently while achieving single-cell resolution visualization
Solution Approach 2:
The methodology nests multiple detection layers within a single cell: first nesting the labeled nucleic acid probe within the cell during hybridization, then nesting the first binding agent (anti-label antibody) around the probe, and finally nesting the second binding agent (anti-modified nucleotide antibody) at the target site. This nested arrangement enables simultaneous detection of multiple markers in the same cell without cross-interference
2Adaptability or versatility
If multiple binding agents are used to detect both probe label and modified nucleotide, then detection capability is improved, but procedure complexity increases
Solution Approach 1:
The proximal binding of the first binding agent (anti-label antibody) to the probe label serves as an intermediary step that bridges the probe and the target site. This intermediary positioning allows the second binding agent (anti-modified nucleotide antibody) to access and bind to the modified nucleotide while maintaining spatial proximity, enabling the proximity ligation assay to function effectively
Solution Approach 2:
The method employs different labels (such as biotin, digoxigenin, or fluorescent dyes) on the nucleic acid probes that can be detected by specific binding agents. These labels act as visual markers that change or become detectable through their interaction with binding agents, allowing the modified nucleotides to be visualized through the resulting signal (fluorescence, color change, or other detectable properties)
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 the visualization of epigenetic marks at a single genomic locus in individual cells, facilitating the detection of cancer cells and providing diagnostic tools for cancer biopsy samples.
Implementation Method 1
the nucleic acid probe hybridizes with the specific nucleic acid sequence to form a hybridized cell
Implementation Method 2
contacting the hybridized cell with a first binding agent that binds to the label of the nucleic acid probe and a second binding agent that binds to the modified nucleotide
Data Source
Figure 1
Figure 2
Figure 3A~3C
AI summary
Methods for visualizing modified nucleotides in a specific nucleic acid sequence or specific nucleic acid sequence interactions in single cells, wherein the methods comprise coupling an in situ hybridization (ISH) reaction with a proximity ligation assay (PLA) reaction.