In Situ Nucleic Acid Proximity Detection via Fragment Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods struggle to effectively identify and understand the complex three-dimensional interactions between nucleic acids in cells, particularly how long-range interactions regulate cellular processes, which is crucial for understanding gene activity and disease mechanisms.
Innovation Solution
An in situ method for detecting spatial proximity relationships between nucleic acid sequences involves fragmenting nucleic acids, marking ends with labeled nucleotides, joining fragments in close proximity, and determining sequences at junctions to map spatial relationships, allowing for the identification of disease-related junctions and environmental conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional linear DNA analysis methods are used, then the analysis process is simple, but the three-dimensional spatial proximity relationships between nucleic acids cannot be detected
Solution Approach 1:
The DNA is fragmented into smaller pieces through enzymatic digestion, creating multiple discrete segments that can be individually labeled and analyzed. This segmentation allows proximity ligation to occur between specific fragments that were spatially close in the intact chromosome, enabling three-dimensional spatial relationship detection while managing the complexity through systematic processing of fragments
Solution Approach 2:
A labeled nucleotide serves as an intermediary marker that is incorporated at the junctions where proximal DNA fragments are ligated. This labeled intermediate enables the detection and isolation of ligation products, providing a bridge between the spatial proximity information and the sequencing analysis, thus resolving the contradiction between detection capability and method complexity
2Measurement precision
If high-resolution mapping of nucleic acid interactions is achieved, then distal regulatory sequences can be identified, but the amount of data and analysis required increases
Solution Approach 1:
The method extracts and isolates only the ligation junctions that represent actual spatial proximity relationships by using labeled nucleotides as capture handles. This extraction approach pulls out the relevant interaction data from the complex genomic background, achieving high-resolution mapping while reducing the effective data volume that needs to be analyzed by focusing only on proximity-ligated fragments
Solution Approach 2:
The analysis focuses on local junction regions where ligation events occurred, rather than analyzing entire genomic sequences. By concentrating sequencing and analysis efforts on these localized junction points, the method achieves high mapping resolution for spatial relationships while minimizing the overall data volume and computational requirements
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 comprehensive mapping of nucleic acid interactions with high resolution, enabling the identification of distal regulatory sequences and disease diagnostics, as well as the assessment of genomic variations and drug effects on cellular circuits.
Implementation Method 1
fragmenting the nucleic acids present in the cells, wherein the fragmented nucleic acids are fragmented to create overhanging ends, such as by enzymatic digestion with a endonuclease that leaves overhanging or blunt ends
Implementation Method 2
joining, for example using a DNA ligase, the labeled end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having a junction
Implementation Method 3
the nucleic acids present in the cell, or cells, are fixed in position relative to one another by crosslinking, for example by treatment of the cells with a chemical cross-linker, for example an aldehyde, such as formaldehyde
Data Source
AI summary
Disclosed is an in situ method for detecting spatial proximity relationships between nucleic acid sequences, such as DNA, in a cell. The method includes: providing a sample of one or more cells comprising nucleic acids; fragmenting the nucleic acids present in the cells that leaves 5′ overhanging ends; filling in the overhanging ends with at least one labeled nucleotide; joining the filled in end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having a junction; isolating the one or more end joined nucleic acid fragments using the labeled nucleotide; and determining the sequence at the junction of the one or more end joined nucleic acid fragments.