Cell-free DNA and Unique Molecular Identifier Technology
What is Cell-free DNA (cfDNA)?
Cell-free DNA (cfDNA), also known as circulating DNA or cell-free nucleic acids, refers to small fragments of DNA that are present in bodily fluids, such as blood, urine, saliva, and other fluids, outside of cells. These fragments are released into the bloodstream through processes like cell death and active secretion. Collecting cfDNA from bodily fluids like blood or urine avoids the need for invasive procedures like tissue biopsies. This is particularly beneficial for monitoring patients over time, as it allows for more frequent sampling without discomfort. Based on this advantage, various clinical applications of cfDNA are being researched.
1. Clinical Applications of cfDNA
One of the most well-known applications of cfDNA is NIPT (Non-Invasive Prenatal Testing). Pregnant women’s blood contains fragments of DNA from the developing fetus, allowing for the detection of genetic abnormalities and conditions like Down syndrome without the need for invasive procedures like amniocentesis.
Tumors shed DNA into the bloodstream as they grow and undergo changes. Analyzing cfDNA from cancer patients can provide valuable information about the presence of cancer, its genetic mutations, and its response to treatment. This approach, known as liquid biopsy, has the potential to revolutionize cancer diagnosis and monitoring.
After organ transplantation, monitoring cfDNA levels in the recipient’s bloodstream can help detect signs of rejection. The donor’s DNA will be foreign to the recipient’s immune system, and an increase in donor-derived cfDNA can indicate an ongoing immune response against the transplanted organ.
2. Technical Challenges in cfDNA Analysis
In clinical diagnostics using Next-Generation Sequencing (NGS), cfDNA is highly useful, but several technical hurdles make its analysis challenging. First, cfDNA has a short half-life in the bloodstream, so the amount of obtainable DNA is very low.
It also exists as very short fragments, typically 100-200 bp in size, as it is degraded by enzymes during cell death. Furthermore, because DNA originating from blood cells and various other tissues is mixed together, the proportion of specific mutations of interest is diluted to extremely low levels. To detect these trace amounts of mutations, maximizing the sequencing depth of the NGS analysis is required.
However, general NGS analysis has a fundamental limitation. Due to the nature of cfDNA fragmentation, distinct original molecules can coincidentally have identical sequences. Without UMIs, these unique molecules cannot be distinguished from one another and are incorrectly regarded as PCR duplicates of a single molecule and removed during data analysis. This causes the sequencing depth to be artificially lower than the reality and, consequently, leads to a problem where the effective depth no longer increases even if the total sequencing amount is raised. The key technology to overcome this limitation is the Unique Molecular Identifier (UMI).
3. The Role of Unique Molecular Identifiers (UMIs)
UMI is a technology that attaches a unique molecular barcode to each DNA fragment during the library preparation stage. This barcode is attached early in the preparation and remains connected to the molecule throughout all experimental processes.
Therefore, all duplicates sharing the same UMI can be bundled into a single ‘group’ derived from a single original molecule. Through this process, the numerous duplicates generated during PCR amplification can be accurately identified, and the resulting amplification bias can be effectively removed. By precisely removing this PCR duplication bias, sequencing depth can be increased compared to conventional methods, and this becomes a key factor that greatly improves the sensitivity and accuracy of mutation analysis.
Conclusion
Celemics’ Unique Molecular Identifier (UMI) technology contribute to obtaining more reliable data in clinical settings. This enhances the accuracy and reproducibility of non-invasive testing, applicable in both research and diagnostic fields. Moving forward, Celemics will continue to focus on delivering customized solutions based on these technologies.
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