Exogenous DNA-Binding Molecule for Genomic Region Isolation
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Solution Overview
Problem
Conventional methods for isolating specific genomic regions while maintaining interaction with their interacting molecules are limited, leading to difficulties in biochemical and molecular biological analysis of chromatin domains, such as incomplete digestion, high background noise, and inability to detect unidentified interactions.
Innovation Solution
A method involving the insertion of a DNA fragment with a recognition sequence for an exogenous DNA-binding molecule into genomic DNA, followed by treatment to maintain interaction with the molecule, fragmentation of genomic DNA, and collection of a complex formed by a molecule capable of specifically binding to the exogenous molecule, allowing for the specific isolation of genomic regions while preserving their interacting molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Chromatin Immunoprecipitation (ChIP) is used to isolate a specific genomic region, then interaction with DNA-binding proteins is maintained, but the method cannot isolate a single specific genomic region and requires prior knowledge of DNA-binding proteins
Solution Approach 1:
The patent introduces an exogenous DNA-binding molecule as an intermediary that binds to a specific recognition sequence inserted into the target genomic region. This intermediary molecule serves as a bridge between the target DNA and the purification system, enabling specific isolation without requiring prior knowledge of endogenous DNA-binding proteins. The exogenous molecule can be tagged for easy detection and purification while maintaining the native chromatin interactions.
Solution Approach 2:
The method segments the genomic DNA by inserting a specific recognition sequence into the target region. This insertion creates a unique binding site that distinguishes the target region from other genomic regions. The exogenous DNA-binding molecule specifically binds to this inserted sequence, allowing selective isolation of the targeted segment while maintaining its interaction with associated molecules.
2Loss of information
If Chromosome Conformation Capture (3C) is used to identify interacting genomic regions, then interaction information can be obtained, but restriction enzyme digestion is incomplete under crosslinking conditions resulting in extremely high background
Solution Approach 1:
The patent uses an exogenous DNA-binding molecule as a mediator that binds to the inserted recognition sequence. This intermediary approach avoids the need for restriction enzyme digestion under crosslinking conditions, thereby eliminating the background noise problem while still enabling detection of genomic interactions through the specific binding of the exogenous molecule to the target region.
3Loss of information
If Fluorescence In Situ Hybridization (FISH) is used to detect genomic interactions, then interaction detection is possible, but the method has low resolution and cannot detect unidentified molecules
Solution Approach 1:
The exogenous DNA-binding molecule acts as a high-resolution intermediary that can be specifically tagged and detected. Unlike FISH probes that have limited resolution, the exogenous molecule binds precisely to the inserted recognition sequence, enabling high-resolution mapping of genomic interactions. Additionally, the tagged exogenous molecule allows detection of both known and unidentified interacting molecules through affinity purification and mass spectrometry.
4Measurement precision
If affinity purification with Cre-loxP system is used to isolate genomic regions, then specific genomic region isolation is achieved, but chromatin structures are altered and bound molecules may dissociate
Solution Approach 1:
The exogenous DNA-binding molecule serves as a stable intermediary that binds to the inserted recognition sequence without requiring chromatin structure alteration. Unlike the Cre-loxP system that requires enzymatic recombination, the exogenous molecule binds directly and stably, preserving the native chromatin structure and maintaining associations with bound molecules throughout the purification process.
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 specific isolation of any genomic region while keeping it interacting with its molecules, providing a valuable sample for analyzing molecular mechanisms of epigenetic regulation, with improved resolution and reduced background noise.
Implementation Method 1
a DNA fragment having a recognition sequence(s) for an exogenous DNA-binding molecule is inserted in genomic DNA
Implementation Method 2
performing a treatment for maintaining interaction of the genomic DNA of the cell with a molecule(s) interacting with the genomic DNA
Implementation Method 3
performing a treatment for fragmenting the genomic DNA
Implementation Method 4
allowing a complex to be formed by binding of a molecule capable of specifically binding to the exogenous molecule, and then collecting the complex
Data Source
AI summary
Provided is a method for specifically isolating any genomic region while maintaining interaction of the genomic region with its interacting molecule(s). According to the method comprising the following steps 1 to 5:Step 1:preparing a cell in which a DNA fragment having a recognition sequence(s) for an exogenous DNA-binding molecule is inserted in genomic DNA of a genomic region to be isolated;Step 2:bringing genomic DNA of the cell into contact with an exogenous molecule at least having a DNA-binding domain of the exogenous DNA-binding molecule;Step 3:performing a treatment for maintaining interaction of the genomic DNA of the cell with a molecule(s) interacting with the genomic DNA;Step 4:performing a treatment for fragmenting the genomic DNA; andStep 5:allowing a complex to be formed by binding of a molecule capable of specifically binding to the exogenous molecule, and then collecting the complex,specific genomic regions can be specifically isolated while kept interacting with their interacting molecules.