MAPit-patch Method for Targeted Epigenetic Analysis
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Solution Overview
Problem
Current genome-wide DNA footprinting techniques face limitations in determining intratumoral epigenetic heterogeneity due to requirements for large DNA amounts, high costs, and short sequencing reads that destroy structural integrity, making it difficult to analyze minority subpopulations and maintain continuity of epigenetic information.
Innovation Solution
The MAPit-patch method allows for high-resolution determination of chromatin structure and DNA methylation states using deep sequencing with long reads, enabling analysis of target loci with limiting DNA input and maintaining epigenetic information continuity, thereby identifying previously obscured subpopulations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If genome-wide DNA footprinting techniques are used, then chromatin structure and methylation state can be determined, but large amounts of DNA are required and costs increase
Solution Approach 1:
The invention extracts and focuses on specific target loci of interest from the entire genome, using targeted enrichment methods to isolate and analyze only the relevant regions. This extraction approach eliminates the need for whole-genome analysis, thereby reducing the DNA input requirement while maintaining measurement precision for the specific loci being studied.
Solution Approach 2:
The invention segments the genome into specific target loci for analysis, dividing the complex whole-genome problem into manageable, focused segments. By segmenting the analysis to target only specific regions of interest, the method reduces the total DNA quantity needed while preserving the ability to determine chromatin structure and methylation states with high precision.
2Quantity of substance
If short sequencing reads are used, then sequencing cost is reduced, but structural integrity and continuity of epigenetic information is destroyed
Solution Approach 1:
The invention introduces molecular barcodes as intermediary tags that link short sequencing reads to their original long DNA templates. These barcode intermediaries preserve the continuity information that would otherwise be lost, allowing reconstruction of the original structural context while enabling the use of cost-effective short-read sequencing technologies.
Solution Approach 2:
The invention creates copies of the original long DNA molecules with attached molecular barcodes, then fragments them into short reads for sequencing. The barcode copies serve as information carriers that maintain the structural integrity data throughout the sequencing process, allowing reconstruction of the original epigenetic landscape from inexpensive short-read data.
3Area of stationary object
If genome-wide analysis is performed, then comprehensive coverage is achieved, but ability to detect minority subpopulations is reduced
Solution Approach 1:
The invention applies local quality enhancement by concentrating sequencing depth and analytical resources on specific target loci rather than distributing them uniformly across the entire genome. This localized focus increases the measurement precision for detecting minority subpopulations at these critical regions, even though the overall genomic coverage area is reduced.
Solution Approach 2:
The invention introduces dynamic adaptability by allowing researchers to adjust and update the set of target loci based on emerging hypotheses and preliminary findings. This dynamic targeting strategy enables the method to adaptively focus on regions most likely to reveal subpopulation heterogeneity, improving detection sensitivity without requiring fixed comprehensive genome-wide coverage.
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
MAPit-patch provides scalable and multiplexed processing, enabling the identification of epigenetic configurations and comparing chromatin structures and methylation states between cells, facilitating the detection of cancerous cells and understanding drug tolerance mechanisms.
Implementation Method 1
exploits exogenous addition of DNA methyltransferases (DNMTs), such as the GC DNA methyltransferase (M.CviPI) to probe accessibility of GC sites in chromatin
Implementation Method 2
Following bisulfite conversion of isolated genomic DNA and sequencing of clonally amplified molecules
Data Source
AI summary
The subject invention pertains to a method of determining methylation state and chromatin structure of target loci. The method comprises treating the genetic material obtained from the cells with DNA methyltransferase, capturing target genetic loci using a set of oligonucleotides, ligating the target loci with oligonucleotide patches that flank the target loci, treating the target loci flanked by oligonucleotide patches with bisulfite, optionally amplifying the target loci by polymerase chain reaction, sequencing the PCR products, and analyzing the sequences to determine methylation state and chromatin structure of the target loci. The current invention also provides a method to identify genes associated with a disease. The invention also provides a method to detect cells suffering from a disease in a group of cells. The current invention also provides kits suitable for carrying out the method of determining methylation state and chromatin structure of the target loci.