Cell-free DNA: A Non-Invasive Cancer Detection

Circulating DNA or cell-free DNA (cfDNA) is released into the bloodstream during apoptosis or necrosis. The size of cfDNA can range from less than 100 base pairs to 5000 base pairs (1^✔ ✔Trusted Source
Extracting regulatory active chromatin footprint from cell-free DNA

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Role of Nucleosomes in Cancer Detection

The size difference is because of different mechanisms involved in DNA release, protection of cfDNA by regulatory proteins, and the degradation process in the bloodstream and after blood sample collection.

As cfDNA was found in cancer patients, they are a potential non-invasive biomarker. Nucleosomes are the fundamental unit of chromatin as they maintain the structure of chromosomes. They also help regulate gene transcription by controlling the accessibility of regulatory proteins to DNA.

Regulatory proteins, such as transcription factors, bind to nucleosomes and can protect cfDNA from degradation, creating cell-free chromatin (cfDNAac). The cfDNAac provides information on the tissue of origin, molecular processes, and cellular states.

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Why cfDNA Fragment Size is Important?

Compared to nucleosome-associated cfDNA (cfDNAnuc), cfDNAac fragments can give information about gene regulation and its relevance in precision medicine and molecular diagnostics.

Extracting cfDNA from plasma is done using commercial kits. However, these kits are used for shorter cfDNAnuc fragments associated with mono-nucleosomes. This can lose the important information present in longer fragments.

The extraction process is also affected by the type of blood collection tubes, which can influence the size distribution of the cfDNA fragments. So it is vital to optimizing the extraction process to know the complete data of cfDNA fragments and when associated with active chromatin.

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New Method for Better cfDNA Analysis

A new study has introduced an optimized extraction method for cfDNA that can capture the full range of cfDNA fragment sizes in the blood by adjusting lysis, binding buffers, and altering salt and detergent concentrations.

The fragments of regulatory-active chromatin such as gene promoters, enhancers, transcription start sites, and gene bodies, provide information on gene expression and regulation.

Computational techniques were developed to analyze these cfDNA fragments and correlate them with known biological markers. With the new technique, they were able to obtain up to 3.9- to 5.5-fold increases in the recovery of longer (>200 bp and >400 bp) cfDNA fragments, compared to conventional cfDNA capture kits.

These enriched cfDNAac fragments can provide gene expression patterns and physiological changes like circadian rhythms. The cfDNAac fragments aligned with RNA polymerase II have a role in gene transcription regulation.

cfDNAac vs. Traditional Methods

Traditional methods like ChIP-seq and ATAC-seq were used in epigenome mapping and understanding the chromatin structure, but they have some limitations. ChIP-seq is time-consuming and resource-intensive as it requires antibodies to immunoprecipitate specific regulatory proteins bound to DNA. ATAC-seq requires intact cells to identify open chromatin regions. So it cannot be used to study cfDNA.

The innovative antibody-independent approach of cfDNAac contains a variety of epigenetic signals, including multiple methylation and acetylation marks. This enables a more comprehensive analysis of gene expression and regulation, overcoming the traditional regulatory regions such as promoters and enhancers.

cfDNA for Disease Detection

Recent advancements in cfDNA fragmentomics have given insights into gene regulation allowing us to infer gene regulation and identify tissue origins by analyzing the cfDNA motifs. However, this method faces challenges in finding the molecular complexity and heterogeneity of diseases. Capturing the complex biology of diseases remains challenging.

One of the approaches that identifies "fragmentation hotspots" in cfDNA aims to enrich gene-regulatory elements and open chromatin regions. This new method enhances the extraction process, improving data quality for more reliable analysis. By isolating and analyzing cfDNAac, we gain deeper insights into gene regulation in disease, allowing for earlier detection of subtle pathological changes that cannot be detected by traditional methods.

The cfDNAac is used in monitoring clinical conditions such as chronic inflammation, autoimmune diseases, and cancer. The ability to detect epigenetic modifications offers early disease detection and intervention.

The non-invasive nature of cfDNAac analysis, combined with its ability to reflect dynamic changes in gene expression, makes it an ideal tool for advancing molecular diagnostics and improving patient care across a wide range of diseases.

Reference:

1. Extracting regulatory active chromatin footprint from cell-free DNA - (https://www.nature.com/articles/s42003-024-06769-3)

Source-Medindia