cfDNA Methylation Detection via Antibody Immunoprecipitation
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Solution Overview
Problem
Conventional methods for detecting cell-free DNA methylation in cancer diagnosis suffer from limited sensitivity, high costs, and degradation loss due to bisulfite treatment, leading to unreliable results.
Innovation Solution
A method and kit for detecting cfDNA methylation using immunoprecipitation, which involves extracting cfDNA, constructing a sequencing library, co-immunoprecipitating anti-5-methylcytosine antibodies, and sequencing on an Illumina platform without bisulfite treatment, thereby capturing and enriching methylated DNA fragments for accurate analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used for DNA methylation detection, then methylation detection can be performed, but DNA degradation loss occurs (about 84% loss) and detection sensitivity is limited
Solution Approach 1:
The patent extracts and enriches methylated DNA fragments from the complex cfDNA mixture using immunoprecipitation with anti-5mC antibodies, separating the methylated fraction from the total DNA. This extraction approach avoids bisulfite treatment while concentrating the target methylated sequences for sensitive detection.
Solution Approach 2:
The patent introduces an intermediary substance - anti-5mC antibody - that specifically binds to methylated cytosine residues. This antibody acts as a mediator to selectively capture and enrich methylated DNA fragments without requiring chemical modification of the DNA itself, thereby avoiding bisulfite-induced degradation.
2Measurement precision
If conventional bisulfite treatment method is used, then methylation detection is achieved, but detection costs increase due to limited information recovery rate and high reagent requirements
Solution Approach 1:
The patent changes the fundamental parameter of the detection approach from chemical modification (bisulfite treatment) to immunological recognition (antibody-based immunoprecipitation). This parameter change enables direct enrichment of methylated DNA without the need for extensive bisulfite conversion steps, reducing reagent costs and improving information recovery.
3Reliability
If bisulfite treatment is applied to cfDNA, then methylation status can be determined, but reliability of results decreases due to limited amount of cfDNA and high degradation
Solution Approach 1:
The patent performs preliminary enrichment of methylated DNA fragments through immunoprecipitation before sequencing. This preliminary action concentrates the target methylated sequences and reduces the impact of cfDNA degradation, as the enrichment step occurs before any potential degradation can affect the final detection results.
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 significantly enhances detection sensitivity and specificity, reduces false positives, and lowers experimental costs by avoiding DNA degradation, resulting in more reliable and accurate methylation detection for early cancer screening.
Implementation Method 1
co-immunoprecipitating anti-5-methylcytosine (5mC) antibody with the mixture of the cfDNA library and the filler DNA obtained in step 2, conducting methylation capture on differentially methylated DNA fragments in the mixture
Data Source
AI summary
The present disclosure provides a method and a kit for detecting cell-free DNA (cfDNA) methylation, and belongs to the technical field of early cancer screening. The method includes the following steps: constructing and mixing a cfDNA library and a filter DNA to obtain a mixture of the cfDNA library and the filter DNA, co-immunoprecipitating the mixture with anti-5-methylcytosine (5mC) antibody, conducting methylation capture on methylated DNA fragments in the mixture, and purifying and eluting to obtain a captured product fragment; conducting amplification and enrichment, purification, recovery and screening to obtain a sequencing library; and sequencing on an Illumina sequencing platform, and bioinformatically analyzing acquired experimental data to know about the cfDNA methylation. The detection method and the kit provided by the present disclosure feature high detection sensitivity and low experimental cost, and substantially reduce the false positive rate of conventional detection to obtain more reliable results.