Tissue-Specific Methylation Markers for Circulating DNA Analysis
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Solution Overview
Problem
Current methods for analyzing circulating DNA rely on genetic differences between tissues of interest and hosts, limiting their utility in diagnosing and monitoring conditions like myocardial infarction and sepsis, as they cannot distinguish between cell death from specific tissues and other sources.
Innovation Solution
The development of novel target sequences as tissue-specific methylation markers, allowing for the identification of cell types by analyzing the methylation status of specific DNA sequences in cell-free DNA, using bisulfite treatment and digital droplet PCR to differentiate between methylated and unmethylated cytosines, thereby determining the origin of circulating DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If genetic differences between tissues of interest and hosts are used for cfDNA analysis, then high specificity can be achieved for fetal DNA and tumor DNA detection, but the method cannot be applied to diagnose conditions like myocardial infarction and sepsis where no genetic differences exist between the tissue of interest and the host
Solution Approach 1:
The patent changes the detection parameter from genetic sequence (which varies between individuals) to DNA methylation pattern (which is tissue-specific and consistent within tissue types). By analyzing methylation status at specific CpG sites, the method can identify tissue of origin without requiring genetic differences between host and tissue of interest, thus resolving the contradiction between versatility and precision
Solution Approach 2:
The patent performs preliminary action by treating cfDNA with bisulfite before analysis, which converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged. This preliminary chemical modification creates detectable differences in methylation patterns that can be used to identify tissue-specific DNA, enabling the method to work across different diagnostic conditions
2Ease of operation
If total cfDNA levels are measured to assess tissue damage, then a simple assay can be performed, but the source of elevated cfDNA cannot be determined, compromising diagnostic utility
Solution Approach 1:
The patent segments the analysis by targeting specific genomic regions with characteristic methylation patterns for different tissues. Instead of measuring total cfDNA as a single value, the method divides the genome into specific loci (e.g., genes with tissue-specific methylation) and analyzes methylation status at each segment, thereby preserving tissue origin information while maintaining assay feasibility
Solution Approach 2:
The patent introduces methylation status as an intermediary property that links cfDNA to tissue of origin. By measuring methylation patterns at specific CpG sites within cfDNA molecules, the method creates an intermediate indicator that reveals tissue source information without requiring direct observation of the parent tissue, thus maintaining simplicity while preventing information loss
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 approach enables precise identification of cell death in specific tissues, improving diagnostic accuracy and monitoring capabilities, particularly in conditions like myocardial infarction and sepsis, by distinguishing between cardiac and non-cardiac DNA sources.
Implementation Method 1
using bisulfite treatment and digital droplet PCR to differentiate between methylated and unmethylated cytosines
Implementation Method 2
using bisulfite treatment and digital droplet PCR to differentiate between methylated and unmethylated cytosines
Data Source
AI summary
A method of ascertaining the methylation status of a double-stranded, cell-free DNA molecule in a specimen is disclosed. The method comprises ascertaining the methylation status of at least two methylation sites of the same double-stranded cell-free DNA molecule, wherein said double-stranded, cell-free DNA molecule comprises a nucleotide sequence which comprises no more than 300 base pairs and is comprised in a sequence as set forth in any one of SEQ ID NOs: 2-117 or 121-177.