Mitochondrial DNA (mtDNA), the genetic material found inside mitochondria—often called the cell's "powerhouses"—and existing separately from the cell's nuclear genome, has unique biological features: it's inherited only from the mother, mutates at a high rate, and doesn't recombine. These traits make mtDNA an invaluable tool across many research areas.
From an evolutionary biology perspective, mtDNA acts as a key genetic marker for tracing species origins, migration paths, and genetic ties between populations. In medicine, mutations in mtDNA are closely tied to the start and progression of various human diseases, such as mitochondrial encephalomyopathies and Leber hereditary optic neuropathy. In forensic science, mtDNA's rich genetic variation and relative stability in degraded samples make it a critical resource for solving tough cases. In recent years, as sequencing technology has advanced rapidly, the cost of mtDNA sequencing has dropped sharply, while data quality has improved significantly. This has further driven its widespread use across different fields.
This article will explore practical applications of mtDNA sequencing through real-world case studies, aiming to provide thorough references and insights for researchers in related fields.
Case 1: Evolutionary Biology Research
In the field of evolutionary biology, mtDNA sequencing serves as a powerful tool for unveiling the evolutionary journey of species. Taking the study of human origins as an example, through the sequencing and analysis of mtDNA from people in different regions, scientists have discovered a variety of mtDNA haplogroups. The global distribution of these haplogroups shows certain geographical patterns. For instance, African populations have the richest mtDNA haplogroup diversity, which supports the "Out-of-Africa" hypothesis. This hypothesis posits that modern humans originated in Africa and gradually migrated and spread to various parts of the world.
Journal: Cell
Impact Factor: 66.85
Publication Date: May 15, 2025
DOI: 10.1016/j.cell.2025.03.023
Sample Selection: The study analyzed 9,251 publicly available human metagenomic datasets from 48 cohorts across 31 countries.
Research Techniques: Through BLASTn analysis, the assembled contigs in the samples were compared for homology with the human mitochondrial genome reference sequence (rCRS).
Background: Human DNA is inevitably present in metagenomic analyses of the human microbiome. Although current protocols remove human DNA before submission to public databases, mitochondrial DNA (mtDNA) is often overlooked and frequently remains.
Objective: To discuss the privacy risks and scientific research opportunities brought about by the presence of human mtDNA in public metagenomic data.
Research Approach and Results: The study found that human mtDNA was detected in 19.6% of the samples. These samples were distributed across 21 countries and mainly came from fecal, oral, and skin samples. The lengths of the mtDNA fragments detected in the samples ranged from 0.5 kb to the complete mitochondrial genome (about 16.5 kb). More than 500 cases successfully reconstructed nearly full-length mtDNA sequences. In addition, the study assigned mitochondrial haplogroups to 1,716 samples, accounting for 94.4% of all positive samples. The study also discovered that at least 26 single-nucleotide polymorphisms (SNPs) in the mtDNA sequences were consistent with known potentially pathogenic variants in the ClinVar database.
The study pointed out that the presence of mtDNA may pose privacy risks. Since mtDNA can trace maternal lineage, when combined with demographic data, it may indirectly identify individuals, especially in isolated populations with low genetic diversity. Moreover, genetic characteristics at the population level may lead to identity-related associations and stigmatization risks. However, mtDNA also offers new opportunities for studying maternal migration, geographical origins, and their associations with microbial communities.
Sequencing of mitochondrial DNA (mtDNA) elucidates the evolutionary trajectory of species (Sarhan et al., 2024)
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Case 2: Oncology Research
In the medical field, mtDNA sequencing holds significant importance for the diagnosis and treatment of diseases associated with mtDNA mutations. This technology can comprehensively and accurately detect mutations in mtDNA, providing a reliable basis for disease diagnosis and classification. The development of tumors is a complex process, and mtDNA variations play a crucial role in it.
Journal: Science Advances
Impact Factor: 14.957
Publication Date: November 4, 2024
DOI: 10.1126/sciadv.ade7297
Sample Selection: The study utilized a KRASG12D-driven lung cancer mouse model, as well as data from 107 human lung adenocarcinoma (LUAD) samples in The Cancer Genome Atlas (TCGA) database.
Research Techniques: Western blot analysis was used to assess TFAM protein levels, real-time quantitative PCR (qPCR) was employed to measure mtDNA levels, and laser-capture microdissection was applied to obtain tumor tissues.
Background: Lung adenocarcinoma is a common and aggressive type of cancer and one of the leading causes of global mortality. Previous studies have shown that mtDNA levels in lung adenocarcinoma patients are higher than those in normal lung tissues.
Objective: To investigate the role of mtDNA copy number in the progression of lung adenocarcinoma and explore its causal relationship in tumor development.
Research Approach and Results: The study found that mtDNA levels increased in KRASG12D-induced lung tumors in mice, and mitochondrial respiratory function was enhanced. In the mouse model, increasing mtDNA copy number led to a greater tumor burden, while depleting mtDNA in tumor cells reduced tumor growth. Additionally, the study revealed a positive correlation between mtDNA levels and the expression of genes related to mitochondrial respiration. This research provides experimental evidence for the intrinsic causal role of mtDNA in the progression of lung cancer, which may offer new targets for the future development of cancer therapies.
The application of mtDNA sequencing facilitates the investigation of tumorigenesis mechanisms (Mennuni et al., 2024)
Case 3: Forensic Science Research
In the field of forensic science, mtDNA sequencing is frequently employed to tackle challenging cases that can't be resolved through nuclear DNA analysis. These cases often involve highly degraded bone and tooth samples, as well as mixed samples. Since mtDNA has a high copy number in cells and remains relatively stable in degraded samples, it's possible to obtain sufficient genetic information from these samples where nuclear DNA extraction is difficult. This genetic data can provide crucial clues for solving cases.
Journal: PeerJ
Impact Factor: 2.4
Publication Date: July 2019
DOI: 10.7717/peerj.7314
Sample Selection: Specific sample selection details weren't explicitly mentioned.
Research Techniques: The research mainly focused on mitochondrial DNA sequencing technologies, including Sanger sequencing and whole-genome sequencing.
Background: Mitochondrial DNA holds significant value in forensic science due to its maternal inheritance, high copy number, and lack of recombination. This is especially true when dealing with samples containing low amounts of DNA.
Objective: To review the application of mitochondrial DNA in human identification, with a particular focus on its prospects and challenges in forensic science.
Research Approach and Results: The study examined the historical development of mtDNA analysis methods, which transitioned from early control-region sequence analysis to whole-genome sequence analysis. This technological advancement has significantly enhanced the utility of mtDNA in forensic science, particularly when dealing with samples containing low DNA content, such as ancient bones, teeth, and hair. The research also highlighted key biological issues that need to be considered when using mtDNA in forensic science. These include the standardization of nomenclature and the establishment of reference population databases. Moreover, it discussed the prospects of mtDNA in forensic science, including its potential applications in complex cases, such as the analysis of mixed samples and the identification of low-quality DNA.
Mitochondrial DNA analysis plays a crucial role in forensic science, especially when dealing with samples containing low amounts of DNA. The development of whole-genome sequencing technology has further improved the application value of mtDNA in human identification. However, some issues still need to be addressed, such as heteroplasmy and the possibility of paternal inheritance, to ensure the robustness of mtDNA as a forensic tool.
mtDNA sequencing finds its application in forensic research (Amorim et al., 2024)
Case 4: Pharmaceutical Research
In the field of pharmaceutical research, mtDNA sequencing plays a crucial role in gaining insights into how drugs affect mitochondrial function. It provides a crucial foundation for drug development and safety evaluation. Many drugs, while treating diseases, may have toxic effects on mitochondria, leading to mitochondrial dysfunction and subsequently triggering a series of adverse reactions. By using mtDNA sequencing technology, we can detect changes in mtDNA in cells or tissues before and after drug treatment, and assess the mitochondrial toxicity risk of drugs.
Journal: Frontiers in Genetics
Impact Factor: 2.8
Publication Date: April 17, 2021
DOI: 10.1186/s12885-021-08155-2
Sample Selection: The study selected four cancer cell lines and one non-cancer cell line to evaluate the relationship between mitochondrial DNA (mtDNA) variations and drug responses.
Research Techniques: DCFDA and MitoSOX probes were used to measure intracellular and mitochondria-specific reactive oxygen species (ROS) levels. Sanger sequencing and qPCR techniques were employed to screen for variations in the mitochondrial genome and relatively quantify mtDNA copy numbers.
Background: Mitochondrial DNA (mtDNA) variations are associated with elevated ROS levels in cancer cells, and excessively high ROS levels can trigger apoptosis. Therefore, the study aimed to explore how mtDNA variations can predict the responses of cancer cells to ROS-stimulating therapies.
Objective: Through a systematic review, investigate the role of mitochondrial DNA variations in drug responses, particularly their potential applications in cancer treatment.
Research Approach and Results: The study found a significant positive correlation between the number of non-synonymous variations in mitochondrial respiratory chain complex I and III genes, mtDNA copy numbers, intracellular ROS levels, and drug resistance. These variations may play a role in cancer cells by affecting ROS levels and drug resistance. The results suggest that mtDNA copy numbers and functional variations in complex I/III could serve as biomarkers for predicting the efficacy of ROS-ROS-stimulating therapies. This study offers new biomarkers for cancer treatment, which can help predict patients' responses to ROS-stimulating therapies and provide a theoretical basis for designing personalized treatment plans.
mtDNA sequencing is utilized in pharmaceutical research endeavors (Jones et al., 2024)
Conclusion
To sum up, mtDNA sequencing technology shows broad application prospects across multiple fields, including evolutionary biology, medicine, forensic science, and pharmaceutical research. In evolutionary biology, it provides crucial genetic evidence for uncovering the origin and evolutionary history of species. In the medical field, it aids in the accurate diagnosis and treatment of diseases associated with mtDNA mutations. Forensic identification can solve challenging cases, offering technical support for judicial fairness. In pharmaceutical research, it provides strong guarantees for drug development and safety evaluation. As sequencing technologies continue to advance and innovate, the cost of mtDNA sequencing will further decrease, and the quality of data will keep improving. Consequently, its application scope will expand.
In the future, mtDNA sequencing is expected to be combined with other omics technologies. This integration will provide more comprehensive and in-depth information for a deeper understanding of fundamental questions in life sciences and for solving practical problems.
For relevant researchers, mastering mtDNA sequencing technology and its application methods will help them achieve more research results and make innovative breakthroughs in their respective fields. At the same time, we also look forward to more interdisciplinary collaborations to jointly promote the development and application of mtDNA sequencing technology, thus making greater contributions to human health and social development.
References:
- Sarhan MS, Antonello G, Weissensteiner H, Mengoni C, Mascalzoni D, Waldron L, Segata N, Fuchsberger C. "Human mitochondrial DNA in public metagenomes: Opportunity or privacy threat?" Cell (2025) 188(10):2561-2566 https://doi.org/10.1016/j.cell.2025.03.023
- Mennuni M, Wilkie SE, Michon P, Alsina D, Filograna R, Lindberg M, Sanin DE, Rosenberger F, Schaaf A, Larsson E, Pearce EL, Larsson NG. "High mitochondrial DNA levels accelerate lung adenocarcinoma progression." Sci Adv (2024) 10(44):eadp3481 https://doi.org/10.1126/sciadv.adp3481
- Amorim A, Fernandes T, Taveira N. "Mitochondrial DNA in human identification: a review." PeerJ (2019) 7:e7314 https://doi.org/10.7717/peerj.7314
- Jones SW, Ball AL, Chadwick AE, Alfirevic A. "The Role of Mitochondrial DNA Variation in Drug Response: A Systematic Review." Front Genet (2021) 12:698825 https://doi.org/10.3389/fgene.2021.698825
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