Genome Architecture Mapping for Unbiased Spatial Proximity Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for analyzing the three-dimensional structure of the genome, such as chromosome conformation capture techniques and fluorescence in situ hybridization, face limitations including bias in detecting nucleic acid interactions, inability to identify binding sites genome-wide, and difficulty in measuring long-range chromatin contacts, which hinders the understanding of genome stability and function.
Innovation Solution
A method called Genome Architecture Mapping (GAM) that involves separating nucleic acids based on their localization in the cell nucleus, determining their presence in fractions, and analyzing co-segregation to infer spatial proximity, allowing for unbiased detection of nucleic acid interactions and simultaneous analysis of multiple interactions across the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chromosome conformation capture techniques are used to analyze three-dimensional genome structure, then spatial proximity information can be obtained, but bias in detecting nucleic acid interactions occurs and binding sites cannot be identified genome-wide
Solution Approach 1:
The genome is divided into many small bins (e.g., 5kb bins) across the entire genome, allowing comprehensive coverage. Each bin can be independently analyzed for spatial proximity, enabling both precise local measurements and genome-wide identification of binding sites without bias toward pre-selected regions.
Solution Approach 2:
The GAM method serves multiple functions simultaneously: it identifies spatial proximity, discovers binding sites genome-wide, measures chromatin contacts, and detects nucleic acid interactions without requiring prior knowledge of specific loci. This universal approach replaces the need for targeted, locus-specific methods.
2Measurement precision
If fluorescence in situ hybridization is used to measure spatial distances, then direct spatial measurement is achieved, but only a small number of genomic regions can be analyzed at a time and repeated re-probing causes structural artefacts
Solution Approach 1:
Instead of using a limited number of fluorescent probes for specific loci, the genome is segmented into many small bins that can all be analyzed simultaneously through sequencing. This allows thousands of genomic regions to be measured in parallel without the need for repeated experiments on the same cells.
Solution Approach 2:
The mechanical process of repeated probe hybridization and washing in FISH is replaced with a single-step crosslinking followed by fragmentation and sequencing. This substitution eliminates the need for repeated manual operations on the same samples, preventing structural artefacts while maintaining spatial measurement capability.
3Measurement precision
If FISH is used to analyze known loci, then specific locus interactions can be detected, but endogenous or exogenous DNA sequences cannot be detected unless they are known a priori
Solution Approach 1:
The method is designed to be universally applicable to any DNA sequence in the genome without requiring prior knowledge. By fragmenting crosslinked chromatin and sequencing all DNA fragments, both known and unknown loci can be detected, including endogenous sequences, exogenous viral integrations, and novel binding sites that were not previously characterized.
Solution Approach 2:
Instead of designing probes for known sequences and trying to find their interactions, the approach is inverted: all DNA fragments are sequenced first, and then spatial proximity is determined by analyzing which fragments co-occur in the same chromatin crosslinks. This allows discovery of unknown interactions and sequences without prior hypotheses.
4Measurement precision
If chromosome conformation capture techniques are used, then chromatin interactions can be analyzed, but ability to measure long-range chromatin contacts is limited and genome stability understanding is hindered
Solution Approach 1:
The genome is divided into small bins that can be combined in various ways to analyze interactions at different scales. This segmentation allows measurement of both short-range contacts (adjacent bins) and long-range contacts (distant bins) with the same method, overcoming the limited range detection of traditional techniques.
Solution Approach 2:
The analysis moves from one-dimensional linear genomic distance to three-dimensional spatial proximity by analyzing which bins co-occur in chromatin crosslinks. This dimensional transformation enables detection of long-range contacts that are far apart linearly but close in 3D space, providing comprehensive coverage of all interaction ranges.
Data Source
AI summary
The present invention relates to the field of analysis of the three-dimensional structure of the genome, i.e., for genome architecture mapping (GAM). The invention provides a method of determining spatial proximity of a plurality of nucleic acid loci in a compartment such as the cell nucleus, by exploiting their co-segregation amongst fractions of that compartment, identified upon separation of the nucleic acid loci from each other depending on their localization in the compartment to obtain a collection of fractions, e.g., by cryo-sectioning or cryo-milling the compartment; determining the presence or absence of the plurality of loci in the fractions; and determining the co-segregation of the plurality of loci. Co-segregation may then be analysed with statistical methods to determine spatial proximity. The method can be used e.g., for determining physical distance between a plurality of loci; and mapping loci and/or genome architecture, e.g., in the nucleus; identification of regulatory regions directing expression of a specific gene through spatial contacts; identifying the nuclear position of an exogenous nucleic acid in the nucleus and/or diagnosing a disease associated with a disturbed co-segregation of loci.