Name: genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing
Uploaded: 2023-02-10T13:50:00
Description: Watch this Scientific Journal Video about Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing at JoVE.com

We would like to acknowledge Silvia Marchet and Constanza Lamperti (Istituto Neurologico &#34;Carlo Besta&#34;, Fondazione IRCCS, Milan) for preparing and providing the m.3243A&#62;G cybrid cells used as illustrative examples for this protocol. We would also like to acknowledge the members of the Mitochondrial Genetics Group (MRC-MBU, University of Cambridge) for useful discussion during the course of this research. This work was supported by core funding from the Medical Research Council UK (MC_UU_00015/4 and MC_UU_00028/3). P.A.N. and P.S.-P. are additionally supported by The Lily Foundation and The Champ Foundation, respectively.

Informed consent was provided for the use of the human 3243A&#62;G cybrid cells and the immortalized m.5024C&#62;T MEFs used in this study. Ethical approval was not required in this instance as the patient cells were not collected at the University of Cambridge. The use of human fibroblasts may, however, require ethical approval. It is highly recommended to follow best practices for PCR setup when preparing the sample DNA for pyrosequencing. Frequent amplification using identical primers can lead to amplicon contaminatio...

<ol>
	<li>Taanman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome:+Structure,+transcription,+translation+and+replication.">The mitochondrial genome: Structure, transcription, translation and replication.</a> Biochimica et Biophysica Acta. 1410 (2), 103-123 (1999).</li><li>Vafai, S. B., Mootha, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+disorders+as+windows+into+an+ancient+organelle.">Mitochondrial disorders as windows into an ancient organelle.</a> Nature. 491, 374-383 (2012).</li><li>Wei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-embedded+mitochondrial+DNA+sequences+in+66,083+human+genomes.">Nuclear-embedded mitochondrial DNA sequences in 66,083 human genomes.</a> Nature. 611, 105-114 (2022).</li><li>Chinnery, P. F., Hudson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+genetics.">Mitochondrial genetics.</a> British Medical Bulletin. 106 (1), 135-159 (2013).</li><li>Ryzhkova, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+caused+by+mtDNA+mutations:+A+mini-review.">Mitochondrial diseases caused by mtDNA mutations: A mini-review.</a> Therapeutics and Clinical Risk Management. 14, 1933-1942 (2018).</li><li>Payne, B. A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+heteroplasmy+of+human+mitochondrial+DNA.">Universal heteroplasmy of human mitochondrial DNA.</a> Human Molecular Genetics. 22 (2), 384-390 (2013).</li><li>Craven, L., Alston, C. L., Taylor, R. W., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+mitochondrial+disease.">Recent advances in mitochondrial disease.</a> Annual Review of Genomics and Human Genetics. 18, 257-275 (2017).</li><li>Mok, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+cytidine+deaminase+toxin+enables+CRISPR-free+mitochondrial+base+editing.">A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.</a> Nature. 583, 631-637 (2020).</li><li>Cho, S. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+A-to-G+base+editing+in+human+mitochondrial+DNA+with+programmable+deaminases.">Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.</a> Cell. 185 (10), 1764-1776 (2022).</li><li>Gammage, P. A., Rorbach, J., Vincent, A. I., Rebar, E. J., Minczuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrially+targeted+ZFNs+for+selective+degradation+of+pathogenic+mitochondrial+genomes+bearing+large-scale+deletions+or+point+mutations.">Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.</a> EMBO Molecular Medicine. 6 (4), 458-466 (2014).</li><li>Gammage, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-complete+elimination+of+mutant+mtDNA+by+iterative+or+dynamic+dose-controlled+treatment+with+mtZFNs.">Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.</a> Nucleic Acids Research. 44 (16), 7804-7816 (2016).</li><li>Gammage, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+mitochondria+corrects+a+pathogenic+mtDNA+mutation+in+vivo.">Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.</a> Nature Medicine. 24, 1691-1695 (2018).</li><li>Bacman, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MitoTALEN+reduces+mutant+mtDNA+load+and+restores+tRNAAla+levels+in+a+mouse+model+of+heteroplasmic+mtDNA+mutation.">MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation.</a> Nature Medicine. 24 (11), 1696-1700 (2018).</li><li>Ren, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+mtDNA+SNPs+in+Chinese+Han+population+using+pyrosequencing.">Screening of mtDNA SNPs in Chinese Han population using pyrosequencing.</a> Forensic Science International: Genetics Supplement Series. 4 (1), 316-317 (2013).</li><li>Andr&#233;asson, H., Asp, A., Alderborn, A., Gyllensten, U., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sequence+analysis+for+forensic+identification+using+pyrosequencing+technology.">Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.</a> BioTechniques. 32 (1), 124-133 (2002).</li><li>Yan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+is+an+accurate+and+reliable+method+for+the+analysis+of+heteroplasmy+of+the+A3243G+mutation+in+patients+with+mitochondrial+diabetes.">Pyrosequencing is an accurate and reliable method for the analysis of heteroplasmy of the A3243G mutation in patients with mitochondrial diabetes.</a> Journal of Molecular Diagnostics. 16 (4), 431-439 (2014).</li><li>Song, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+LATE-PCR+to+allow+single-cell+analysis+by+pyrosequencing.">Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing.</a> Analyst. 138 (17), 4991-4997 (2013).</li><li>El-Hattab, A. W., Adesina, A. M., Jones, J., Scaglia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MELAS+syndrome:+Clinical+manifestations,+pathogenesis,+and+treatment+options.">MELAS syndrome: Clinical manifestations, pathogenesis, and treatment options.</a> Molecular Genetics and Metabolism. 116 (1-2), 4-12 (2015).</li><li>Minczuk, M., Papworth, M. A., Miller, J. C., Murphy, M. P., Klug, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+single-chain,+quasi-dimeric+zinc-finger+nuclease+for+the+selective+degradation+of+mutated+human+mitochondrial+DNA.">Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.</a> Nucleic Acids Research. 36 (12), 3926-3938 (2008).</li><li>Minczuk, M., Kolasinska-Zwierz, P., Murphy, M. P., Papworth, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+testing+of+engineered+zinc-finger+proteins+for+sequence-specific+modification+of+mtDNA.">Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA.</a> Nature Protocols. 5, 342-356 (2010).</li><li>Bacman, S. R., Gammage, P. A., Minczuk, M., Moraes, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+mitochondrial+genes+and+mtDNA+heteroplasmy.">Manipulation of mitochondrial genes and mtDNA heteroplasmy.</a> Methods in Cell Biology. 155, 441-487 (2020).</li><li>Gammage, P. A., Minczuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+manipulation+of+human+mitochondrial+DNA+heteroplasmy+in+vitro+using+tunable+mtZFN+technology.">Enhanced manipulation of human mitochondrial DNA heteroplasmy in vitro using tunable mtZFN technology.</a> Methods in Molecular Biology. 1867, 43-56 (2018).</li><li>Pickett, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+in+m.3243A&gt;G+mitochondrial+disease:+The+role+of+nuclear+factors.">Phenotypic heterogeneity in m.3243A&gt;G mitochondrial disease: The role of nuclear factors.</a> Annals of Clinical and Translational Neurology. 5 (3), 333-345 (2018).</li><li>Nunes, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OXPHOS+dysfunction+regulates+integrin-&#946;1+modifications+and+enhances+cell+motility+and+migration.">OXPHOS dysfunction regulates integrin-&#946;1 modifications and enhances cell motility and migration.</a> Human Molecular Genetics. 24 (7), 1977-1990 (2015).</li><li>Kauppila, J. H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phenotype-driven+approach+to+generate+mouse+models+with+pathogenic+mtDNA+mutations+causing+mitochondrial+disease.">A phenotype-driven approach to generate mouse models with pathogenic mtDNA mutations causing mitochondrial disease.</a> Cell Reports. 16 (11), 2980-2990 (2016).</li><li>Burr, S. P., Chinnery, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+single-cell+mitochondrial+DNA+copy+number+and+heteroplasmy+using+digital+droplet+polymerase+chain+reaction.">Measuring single-cell mitochondrial DNA copy number and heteroplasmy using digital droplet polymerase chain reaction.</a> Journal of Visualized Experiments. (185), e63870 (2022).</li></ol>

A critical aspect for the success of the protocol is avoiding contaminations, particularly when using low amounts of starting material. It is recommended to use a UV hood and filtered pipette tips when preparing the samples wherever possible, as well as to keep the preamplification and post-amplification areas separate. Blank measurements and samples of known heteroplasmy (such as wild-type DNA) should always be included to be used as benchmarks to check for technical or biological bias. 
A no...

The mitochondrial genome exists in the form of small (16.5 kb) circular molecules (mtDNA) present in the innermost compartment of the mitochondria named the matrix and encodes 13 subunits of the mitochondrial respiratory chain, as well as the tRNAs and rRNAs necessary for their translation in situ by the mitochondrial ribosome1. This genome represents approximately 1% of all the proteins necessary for mitochondrial function, the remainder of which are encoded by the nuclear DNA (nDNA). It is commonly assumed that mitochondria are derived from an endosymbiotic fusion event between an alpha-proteobacterial ancestor and an ancestral eukaryotic cell. Once this hypothetical symbiosis took place, the genetic information of the mitochondria was gradually transferred to the nucleus over eons, which explains the aforementioned compactness of the mtDNA when compared to the genomes of modern cyanobacteria2. Such a transfer of genes is most strongly evidenced by the existence of long stretches of nDNA that are highly homologous to the sequences found in mtDNA. These nuclear mitochondrial sequences (NUMTs) are a common source of misinterpretation during genotyping, and certain precautions must be taken to avoid nuclear biases when genotyping mtDNA3 (Figure 1A).
Another distinctive feature of mtDNA is that its copy number varies depending on the cell type, numbering anywhere from tens to thousands of copies per cell4. Owing to this multi-copy nature, mtDNA can harbor a wide range of genotypes within a single cell, which can result in a more continuous distribution of alleles in contrast to the discrete alleles associated with nuclear genes when considering the zygosity of chromosomes. This heterogeneity of mitochondrial alleles is referred to as mitochondrial heteroplasmy, which is typically expressed in the percent prevalence of a given mutation as a proportion of the total mtDNA in a given cell. Heteroplasmy can be contrasted with homoplasmy, which refers to a unique species of mtDNA being present across a cell.
Measuring mitochondrial heteroplasmy is of particular interest when quantifying the proportion of mtDNA molecules harboring pathogenic variants. Such variants come in the form of single nucleotide polymorphisms (SNPs), small indels, or large-scale deletions5. Most humans are heteroplasmic for pathogenic variants; however, they do not exhibit any clinical phenotypes, which often only manifest at higher heteroplasmy levels of pathogenic mtDNA in a phenomenon referred to as the threshold effect6. While the values associated with pathogenicity are highly dependent on the nature of the pathogenic mutation and the tissue in which it occurs, they typically lie above 60% heteroplasmy7.
There are several research areas in which mitochondrial genotyping is common. In the medical field, testing for or quantifying mtDNA mutations can serve as a diagnostic criterion for mitochondrial diseases, many of which have mtDNA aberrations as their origin5. In addition to the study of human pathogenic mutations, the prevalence of animal models harboring pathogenic SNPs in the mtDNA is likely to increase, given the recent advent of mitochondrial base editing enabled by mitochondrially targeted DddA-derived cytosine base editors (DdCBEs)8 and TALE-based deaminases (TALEDs) for adenine base editing9. This approach will be instrumental in understanding the interplay between aberrant mitochondrial genotypes and the resulting dysfunctions. There is also ongoing scientific research into remodeling the mitochondrial genome for ultimate use as a therapeutic strategy in human mitochondrial diseases via an approach known as heteroplasmy shifting. This field of research primarily involves directing mutation-specific nucleases to the mitochondrial matrix; this results in the preferential degradation of pathogenic mtDNA, leading to rescues in phenotype10,11,12,13. Any experiments involving the remodeling of the mitochondrial genotype require a robust quantitative method to assess heteroplasmy shifts.
A wide variety of methods are used to genotype mtDNA, and these vary according to the nature of the mutation. Next-generation sequencing (NGS) methods are more precise when it comes to quantifying SNPs in mtDNA; however, these methods remain prohibitively expensive for the routine quantification of mitochondrial heteroplasmy, particularly if the number of samples is small. Sanger sequencing can also allow for the detection of SNPs; however, this approach is not quantitative and often fails to detect low levels of heteroplasmy or can be inaccurate when estimating high heteroplasmies. Pyrosequencing, as an assay that involves minimal preparation and enables the rapid quantification of heteroplasmy for any mtDNA sample, is proposed as an apt compromise between these two extremes. This method has been used routinely to quantify mitochondrial SNPs by numerous researchers in various contexts, including forensic analysis14,15, clinical diagnosis16, or the genotyping of mtDNA from single cells17.
This assay involves a first PCR preamplification step of a region flanking the SNP in the mtDNA, which is followed by a sequencing-by-synthesis assay using one strand of the previously generated amplicon. One of the two primers used in the preamplification step must be biotinylated on the 5&#39; end, which will enable the pyrosequencing apparatus to isolate the single strand of DNA to be used as template for the sequencing reaction. A third sequencing primer is then annealed onto the retained biotinylated strand, which allows for nascent DNA synthesis as deoxynucleotides to be dispensed in a predefined order into the reaction chamber. The pyrosequencer records the amount of each base incorporated based on a luminescent readout, allowing the relative quantification of mutant and wild-type mitochondrial alleles upon DNA synthesis (Figure 1B). The luminescence is generated by a luciferase enzyme, which emits light in the presence of ATP that an ATP sulfurylase synthesizes de novo at each incorporation event from the pyrophosphates released by each nucleotide. These two reactions can be summarized as follows:
1. PPi (from nucleotide incorporation) + APS &#8594; ATP + sulphate (ATP sulfurylase)
2. ATP + luciferin + O2 &#8594; AMP + PPi + oxyluciferin + CO2 + light (luciferase)
Detecting adenine bases by the pyrosequencer without ATP cross-reacting with the luciferase in the second reaction is a challenge. However, this is solved by using an adenine analog for DNA synthesis, namely dATP&#945;S. Despite not being a perfect substrate for luciferase, it produces a stronger luminescence compared with the three other nucleotides, which is digitally adjusted by the pyrosequencer and set to a factor of 0.9. Due to this inherent variability, it is suggested to avoid sequencing adenine at the SNP position (see the discussion for further details).
The following protocol details the method of mtDNA heteroplasmy assessment by pyrosequencing and outlines the necessary precautions in designing the amplification primers to avoid biological or technical bias when genotyping SNPs in mtDNA. The latter involves digitally surveying and selecting the primer sets, optimizing the preamplification PCR, and finally, sequencing and refining the assay. Two applied example assays are demonstrated: first, the optimization of the most common human pathogenic variant m.3243A&#62;G18, and second, the genotyping of mouse embryonic fibroblast (MEF) cells that have undergone heteroplasmy shifting using technologies developed at the Minczuk laboratory in Cambridge10,11,12,19,20,21,22.

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			<td>KOD Hot Start DNA Polymerase</td>
			<td>Sigma-Aldrich</td>
			<td>71086</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Assay Design&#160;2.0</td>
			<td>QIAGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Absorber Strips&#160;</td>
			<td>QIAGEN</td>
			<td>974912</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Advanced CpG Reagents (4 x 48)</td>
			<td>QIAGEN</td>
			<td>974022</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Autoprep&#160;</td>
			<td>QIAGEN</td>
			<td>9002470</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Cartridge Set</td>
			<td>QIAGEN</td>
			<td>9024321</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Disks</td>
			<td>QIAGEN</td>
			<td>974901</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Magnetic beads&#160;</td>
			<td>QIAGEN</td>
			<td>974203</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Software License (1)</td>
			<td>QIAGEN</td>
			<td>9024325</td>
			<td></td>
		</tr>
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genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

This section presents an example optimization of a pyrosequencing assay for a human pathogenic mtDNA mutation, as well as sequencing data from the genotyping of heteroplasmic (m.5024C&#62;T) mouse embryonic fibroblasts (MEFs) treated with mitochondrial zinc finger nucleases (mtZFNs). Optimizing the assay for human cells and comparing two different assays demonstrates how to select the most accurate one, whereas genotyping genetically modified MEF cells in the second example serves as an applied example of detecting heter...

Watch this Scientific Journal Video about Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing at JoVE.com

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

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