Promoter Capture Hi-C Method for Targeted Genomic Interaction Analysis
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Solution Overview
Problem
Current methods for identifying nucleic acid interactions, such as Chromosome Conformation Capture (3C) and Hi-C, are limited by the need for prior knowledge of interactions, high costs, and complexity of libraries, making it difficult to interrogate specific interactions on a genome-wide scale.
Innovation Solution
A method involving crosslinking, fragmentation, biotin marking, ligation, and PCR-based promoter capture to identify nucleic acid segments interacting with a sub-group of target segments, allowing for the isolation and sequencing of interacting sequences, even if they are far apart in the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If Hi-C method is used to isolate all ligated interacting sequences, then information on all interactions is obtained, but library complexity becomes excessive (800,000 ligatable fragments per cell) making specific region interrogation impossible
Solution Approach 1:
The patent divides the genome into specific regions of interest (e.g., promoters, enhancers, TADs) and uses targeted capture probes to isolate only the interacting sequences within these predefined segments. This segmentation approach reduces the library complexity from 800,000 fragments to a manageable subset of relevant interactions, while still capturing comprehensive information within the targeted regions.
Solution Approach 2:
The patent extracts only the specific interacting sequences of interest from the complex Hi-C library by using hybridization capture with region-specific probes. This extraction process removes irrelevant fragments and enriches for target sequences, enabling focused analysis of specific genomic regions without being overwhelmed by the full library complexity.
2Quantity of substance
If 4C method is used to identify interactions with one bait sequence, then resource cost is reduced, but only semi-quantitative data is obtained and only one locus is detected
Solution Approach 1:
The patent creates a methodology that can detect interactions with multiple bait sequences simultaneously by using a panel of capture probes targeting different genomic regions. This multi-functional approach allows the same experimental workflow to be applied to study interactions for many different loci in parallel, providing both cost efficiency and broad detection scope.
Solution Approach 2:
The patent changes the parameter of detection sensitivity by using hybridization capture with enriched target sequences, which provides quantitative measurement capability unlike the semi-quantitative 4C method. By increasing the abundance of specific interacting sequences through enrichment, the method achieves both quantitative accuracy and multi-locus detection capability.
3Measurement precision
If 3C method is used to identify interactions, then previous knowledge is required, but this limits detection to known interactions only
Solution Approach 1:
The patent performs preliminary action by defining regions of interest based on existing genomic annotations (promoters, enhancers, TADs) before conducting the interaction analysis. This preliminary segmentation allows the method to leverage known genomic structure while simultaneously discovering new interactions within and between these predefined regions, combining the precision of targeted analysis with the discovery potential of unbiased sampling.
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 capture of over 22,000 promoters and their interacting genomic loci in a single experiment, providing a more quantitative readout and identifying interacting nucleic acid segments that are indicative of disease states, thus potentially revealing new drug targets.
Implementation Method 1
crosslinking a nucleic acid composition comprising the sub-group of target nucleic acid segments
Implementation Method 2
marking the ends of the fragments with biotin; pulldown of fragments with streptavidin
Implementation Method 3
amplification using PCR
Data Source
Figure 1A
Figure 1B
Figure 2A~2C
AI summary
The invention relates to a method for identifying nucleic acid segments which interact with a target nucleic acid segment by use of an isolating nucleic acid molecule, and to kits for use in said method. The invention also relates to a method of identifying one or more interacting nucleic acid segments that are indicative of a particular disease state.