Detection Of Mitochondrial DNA Research Articles

Robustness and reliability of single-cell regulatory multi-omics with deep mitochondrial mutation profiling.

The detection of mitochondrial DNA (mtDNA) mutations in single cells holds considerable potential to define clonal relationships coupled with information on cell state in humans. Previous methods focused on higher heteroplasmy mutations that are limited in number and can be influenced by functional selection, introducing biases for lineage tracing. Although more challenging to detect, intermediate to low heteroplasmy mtDNA mutations are valuable due to their high diversity, abundance, and lower propensity to selection. To enhance mtDNA mutation detection and facilitate fine-scale lineage tracing, we developed the single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM) approach, an integrated experimental and computational framework. Recently, some concerns have been raised about the analytical workflow in the ReDeeM framework. Specifically, it was noted that the mutations detected in a single molecule per cell are enriched on edges of mtDNA molecules, suggesting they resemble artifacts reported in other sequencing approaches. It was then proposed that all mutations found in one molecule per cell should be removed. We detail our error correction method, demonstrating that the observed edge mutations are distinct from previously reported sequencing artifacts. We further show that the proposed removal leads to massive elimination of bona fide and informative mutations. Indeed, mutations accumulating on edges impact a minority of all mutation calls (for example, in hematopoietic stem cells, the excess mutations on the edge account for only 4.3%-7.6% of the total). Recognizing the value of addressing edge mutations even after applying consensus correction, we provide an additional filtering option in the ReDeeM-R package. This approach effectively eliminates the position biases, leads to a mutational signature indistinguishable from bona fide mitochondrial mutations, and removes excess low molecule high connectedness mutations. Importantly, this option preserves the large majority of unique mutations identified by ReDeeM, maintaining the ability of ReDeeM to provide a more than 10-fold increase in variant detection compared to previous methods. Additionally, the cells remain well-connected. While there is room for further refinement in mutation calling strategies, the significant advances and biological insights provided by the ReDeeM framework are unique and remain intact. We hope that this detailed discussion and analysis enables the community to employ this approach and contribute to its further development.

Differences in nature killer cell response and interference with mitochondrial DNA induced apoptosis in moxifloxacin environment

ObjectivesAs antibiotics become more prevalent, accuracy and safety are critical. Moxifloxacin (MXF) have been reported to have immunomodulatory effects on a variety of immune cells and even anti-proliferative and pro-apoptotic effects, but the mechanism of action is not fully clear. MethodsPeripheral blood mononuclear cells (PBMC) from experimental groups of healthy adults (n = 3) were treated with MXF (10ug/ml) in vitro for 24 h. Single-cell sequencing was performed to investigate differences in the response of each immune cell to MXF. Flow cytometry determined differential gene expression in subsets of most damaged NK cells. Pseudo-time analysis identified drivers that influence MXF-stimulated cell differentiation. Detection of mitochondrial DNA and its involvement in the mitochondrial respiratory chain pathway clarifies the origin of MXF-induced stress injury. ResultsMoxifloxacin-environmental NK cells are markedly reduced: a new subset of NK cells emerges, and immediate-early-response genes in this subset indicate the presence of an early activation response. The inhibitory receptor-dominant subset shows enhanced activation, leading to increased expression of cytokines and chemokines. The near-mature subset showed greater cytotoxicity and the most pronounced cellular damage. CD56bright cells responded by antagonizing the regulation of activation and inhibitory signals, demonstrating a strong cleavage capacity. The severe depletion of mitochondrial genes was focused on apoptosis induced by the mitochondrial respiratory chain complex. ConclusionNK cells exhibit heightened sensitivity to the MXF environment. Different NK subsets upregulate the expression of cytokines and chemokines through different activation pathways. Concurrently, MXF induces impairment of the mitochondrial oxidative phosphorylation system, culminating in apoptosis.

Developing mitochondrial DNA field-compatible tests

Nucleic acid-based molecular detection of mitochondrial DNA (mtDNA) has been used for the detection of eukaryotic pathogens, genetic markers, and biomedical studies in humans and animals, forensic investigation, conservation biology, and environmental monitoring. Currently available molecular mtDNA testing is predominately dependent on relatively expensive laboratory equipment, which limits test applicability for on-site field testing. Recent technical advances in molecular testing of mtDNA using microfluidic chip technology and paper-based methods, and assays based on isothermal nucleic acid amplification show promise for field assay development. Microfluidic and paper-based components for mtDNA sample preparation and isothermal amplification establish the groundwork for targeted detection. The goal is on-site field detection mtDNA assays that combine sample processing, assay setup, and visual representation of the presence of mtDNA. This review examines advantages and disadvantages of alternative field-compatible tests for on-site detection of mtDNA. Despite challenges, developing an integrated “sample-through detection” field test platform is the future direction for mtDNA molecular detection in environmental samples.

Evaluating eDNA-based monitoring of fire salamander larvae under field conditions

Amphibians globally belong to the most threatened animal groups and monitoring their populations is of critical importance for their conservation. The fire salamander (Salamandra salamandra) is one of the European amphibian species which has been experiencing drastic regional population declines due to the spread of the invasive chytrid fungus Batrachochytrium salamandrivorans, making it a key species for monitoring efforts. Here, we evaluated how the sampling and analysis of eDNA can aid the monitoring of larval salamander populations in small streams under field conditions. Nine small streams with known adult and larval fire salamander populations were investigated in Tirol (Austria). Per steam a stretch of 30 m downstream from its source was divided into 10 m sections where salamander larvae were counted. Water samples were taken at the end of each section and filtered on site. The DNA extracted from these filters was tested by a new PCR assay developed for the detection of mitochondrial DNA of S. salamandra. This assay combines endpoint PCR with capillary electrophoresis, allowing to relatively quantify the amount of fire salamander eDNA present in the water samples. In two of the nine streams no eDNA of S. Salamandra could be detected. The outcomes of an analysis of how larval densities, discharge and volume of filtered water affected the detection of salamander eDNA will be presented. Finally, we will conclude on the practical implications of the current findings for eDNA-based monitoring of fire salamander populations.

Detection of mitochondrial DNA with 4977 bp deletion in leukocytes of patients with ischemic stroke.

BackgroundCoronary artery disease is associated with a common mitochondrial DNA alteration, a 4977 bp deletion (mtDNA4977). The role of mtDNA4977 in ischemic stroke is unknown.MethodsReal-time quantitative PCR was performed to quantify total mtDNA and mtDNA4977 in leukocytes in 283 ischemic stroke cases and 135 controls. Ratios of mtDNA4977 to total-mtDNA and total-mtDNA to nuclear-DNA were calculated. Nested PCR and Sanger sequencing were used to confirm undetectable levels of mtDNA4977.ResultsFor 191 patients and 74 control subjects in the male group and 92 patients and 61 control subjects in the female group, there were no significant between-group differences in age, cholesterol level, body mass index, stroke severity, or 4977 deletion. After adjusting for confounding factors, there was no correlation between mtDNA4977 amount and infarction risk, recurrent stroke, or stroke severity. However, mtDNA4977 was undetected in 6.94% subjects, and these individuals had a higher prevalence of stroke than those with detectable mtDNA4977 (OR: 0.181, 95% CI 0.041–0.798, p = 0.024). Additionally, mtDNA4977 status had no effect on stroke prognosis, including stroke severity and recurrent stroke.ConclusionIn conclusion, there was no apparent association between mtDNA4977 deletion and cerebral infarction. Undetectable mtDNA4977 may be a marker or risk factor for ischemic stroke.

Comprehensive Mitochondrial Genome Analysis by Massively Parallel Sequencing.

Next-generation sequencing (NGS) based on massively parallel sequencing (MPS) of the entire 16,569 bp mitochondrial genome generates thousands of reads for each nucleotide position. The high-throughput sequence data generated allow the detection of mitochondrial DNA (mtDNA) point mutations and deletions with the ability to accurately quantify the mtDNA point mutation heteroplasmy and to determine the deletion breakpoints. In addition, this method is particularly sensitive for the detection of low-level mtDNA large deletions and multiple deletions. It is by far the most powerful tool for molecular diagnosis of mtDNA disorders.

Development and Integration of Quantitative Real-Time PCR Methods for Detection of Mitochondrial DNA and Methanobrevibacter smithii nifH Gene as Novel Microbial Source Tracking Tools

A novel microbial source tracking (MST) method based on the detection of human and non-human markers was developed and applied to track the origin of fecal pollution in water systems. Mitochondrial DNA sequences were used to develop new quantitative real-time polymerase chain reaction (qPCR) assays for dog, poultry, and gull. The targets were included as part of a toolbox including human, cow, pig, and sheep assays. A primer and probe set for the detection of the human-specific nifH gene of Methanobrevibacter smithii was also designed as an indicator of human fecal contamination. The assays were tested for specificity and applied to fecal-spiked surface waters and environmental samples collected from two river catchments impacted by sources of human and non-human fecal contamination. The MST methods described were applicable to both spiked waters and environmental samples, and using the two approaches the origin of fecal pollution could be successfully determined in mixed source fecally polluted waters.

Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing

The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

Detection of mitochondrial DNA from domestic cattle in European bison (Bison bonasus) from the Altai Republic in Russia

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Detection of mitochondrial DNA from domestic cattle in bison on Santa Catalina Island

In 1924, 14 American bison (Bison bison) were introduced to Santa Catalina Island, California and sporadically supplemented thereafter with additional animals. To reduce the herd and its impact on native vegetation, over 2000 animals have been exported during the past four decades. Today, the herd is estimated to contain around 250 individuals. Genetic analysis was performed on 98 animals removed from the island in 2004. Forty-four samples (45%) had domestic cattle mitochondrial DNA (mtDNA), 12 (12%) had previously reported bison haplotypes and 42 (43%) had a new haplotype differing by one base pair from a previously reported bison haplotype. A complement of five restriction enzymes was found to be useful in identifying bison with domestic cattle mtDNA.

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection

Conclusions. In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. Objective. To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. Material and methods. This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. Results. mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.

Use of eukaryotic mitochondrial DNA to differentiate human, bovine, porcine and ovine sources in fecally contaminated surface water

A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.

A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus

In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.

Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.

The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV-1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14-bit resolution detection. Indeed, under these conditions CV-1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.

Detection and Analysis of Mitochondrial DNA Deletions by Whole Genome PCR

Southern blot analysis has been the best method available for the screening and detection of mitochondrial DNA (mtDNA) rearrangements. Recent developments in polymerase chain reaction (PCR) technology allowed the amplification of the whole mitochondrial genome (16.6 kb), making PCR a potentially useful technique for the detection of mtDNA deletions. We tested the usefulness of whole mitochondrial genome PCR by studying skeletal muscle DNA from seven patients with single and multiple deletions and controls from ages 3 to 91 years old. Specific patterns for single and multiple deletions were obtained with whole genome PCR, which were confirmed by the Southern analysis with probes hybridizing to mtDNA sequences. Amplifications from young controls (3 to 23 years old) yielded only one band (16.5 kb) while amplification from older controls revealed one or more additional smaller bands. The amplification from the 91-year-old control showed a pattern similar to amplifications from patients with multiple mtDNA deletions. Although single and multiple mtDNA deletions could be readily detected from patient samples, the high sensitivity of this method can lead to false positive results due to the presence of age-related deletions in old control samples. Despite its limitations, whole mitochondrial genome PCR can be useful for the detection of single deletions in muscle samples obtained from young individuals since the levels of age-related deletions are too low to be amplified.

Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells.

The in vivo effects of ethidium bromide on the integrity of mitochondrial DNA have been studied in a mouse L-cell system in which this DNA may be nearly exclusively radiolabelled. This allows the detection of mitochondrial DNA in the presence of contaminating nuclear DNA and eliminates the need for extensive purification of mitochondria or the use of deoxyribonuclease. The mitochondrial DNA in treated cells rapidly attains a high negative superhelix density and is not substantially nickel or degraded over the course of several days.

Demonstration of kinetoplast DNA in dyskinetoplastic strains of Trypanosoma equiperdum.

4,6 Diamidino-2-phenylindole (DAPI) forma a highly fluorescent complex with DNA which allows detection of mitochondrial DNA (K-DNA) in normal and dyskinetoplastic strains of Trypanosoma equiperdum. The K-DNA DAPI complexes in the dyskinetoplastic cells, cells lacking detactable K-DNA by other cytochemical methods, are not restricted to a single region of the organism as in the normal strain, but are seen as a row of particles. These observations support the hypothesis that the K-DNA is retained in dyskinetoplastic cells.