Questions related to Mitochondrial DNA
I would like to determine if there is leakage of mitochondrial DNA to the cytosol in cells (after oxidative stress) by qPCR by doing nDNA/mtDNA. According to the protocol in the reference, I have extracted cytosolic mtDNA and nDNA from cells.
But, the results of my experiment were not as expected.
Do I need to ensure that the concentration of DNA in each well is consistent？ If the nDNA of each sample is diluted to the same concentration, such as 1 μg/μl, should my mtDNA change by the corresponding multiple with nDNA?
Many studies reported that the number of mitochondria differs from one cell type to another according to the energy needed. But, I can't find a clear answer about the difference in mitochondria themselves including structure, composition, mtDNA,...etc. I'm not sure if there is a more deep difference between them or not.
In other words, if mitochondria from any organ are transferred to another, can they do their work as original mitochondria regardless of the origin?
Can anyone help me with an answer?
I am attempting to isolate mtDNA from rat myocardial tissue and running into some issues. When I run what I perceive to be mtDNA on a 0.5% agarose gel with 0.5ug/mL EtBr (120V constant) I see intact bands just below the wells that don't seem to run any further. The gel is run in 1x TAE buffer. I included a picture of this. The equipment we utilize to capture images is down so I used a handheld UV back light to get it. This picture was taken about 3 hours after starting the gel run. The mtDNA I isolated has a 260/280 ratio of 1.2 and ~0.40ug/uL concentration, which I loaded 6.4ugs to see whether I could see anything at all.
I have considered three possibilities in regards to this 1) I have no mtDNA 2) the mtDNA is bound to protein thus unable to migrate further 3) the mtDNA is degraded, which may be suggested by the smearing observed.
I isolated pure mitochondria from homogenized rat myocardial tissue then proceed to use the Abcam Mitochondrial DNA Isolation kit to isolate mtDNA. Our mitochondria isolation method works as we use this method to respirate them using an oxygen probe. The steps are mainly lysing the mitochondria then precipitating out the nucleic acids. I don't think the kit does a well job of breaking down proteins as suggested by my 260/280 ratio, therefore I want to add Proteinase K into my lysing incubation. Also I avoid vortexing when isolating mtDNA to prevent any degradation.
Do you have any suggestions to determine whether I do have mtDNA? Is 1D agarose gel electrophoresis enough to run a DNA of this size? If it helps we use BioRad 5x Nucleic Acid Sample Loading Buffer and BioRad EZ Load 1kb Molecular Ruler (1-15kB).
Thank you for your time and help.
I'm looking for a method to extract DNA from desiccated crocodile scutes to be used in a genomic DNA study. Is there a best method to extract DNA from these tissues?
I am using a technique in C2C12 muscle cells called DamID to tag DNA in close proximity to the nuclear envelope in undifferentiated myoblasts and differentiated myotubes. However, following sequencing of the tag-enriched material amplified by the final PCR step, 80% of the DNA sequences identified come directly from the mitochondrial genome. So it looks like the mtDNA is being tagged and because per copy it is more numerous than any genomic sequence, gets preferentially amplified.
I have tried removing the circular and supercoiled mtDNA by;
- subtractive hybridization (purifying mtDNA, fragmenting it, biotinylating it, hybridizing it material to my sample and then pulling down the hybrid duplexes with Dynabeads)
- Nuclear isolation (hypotonic lysis, dounce homoginisation, centrifugation)
- CsCl gradients (using the density difference acquired by linear and supercoiled DNA following addition of ethidium bromide)
However, none of these have worked. Does anyone have any suggestions on easy ways to remove the mtDNA from the genomic DNA? The last thing I am thinking of trying is gel filtration?
Any help would be great!
I have a total genome of a plant sample, is it possible to isolate and sequence the chloroplast and mitochondrial genomes of the sample?
Dear all, in the lab I currently work in we intented to sequence entire mtDNA of selected mammalian species, that had not yet been annotated or deposited into GenBank. However, one stumbles upon a question, whether or not this is trully a prudent thing to do. The advent of NGS in the last decade has enabled mtDNA to be sequenced in entirety as a by-product of WGS/shotgun sequencing. Judging by complex evolutionary patterns (eg. reticulate evolution), using only mtDNA can also hardly be justified as a base for larger and well-supported phylogenetic studies, since nDNA is a must in such casses. The question therefore is, whether or not annotating single mitochondrial DNA's from a single individual of a species is still a sound final objective of a research project?
I look forward to receiving Your opinions.
I'm using the QIAGEN Multiplex PCR Kit and 10 primer pairs (product sizes from ~130 to ~900 bps) to monitor mitochondrial DNA in Candida glabrata, a close relative of Saccharomyces cerevisiae. One primer pair target the genomic DNA, and is a positive control in case the mitochondrial DNA is completely lost (this is possible in yeast). The other nine amplify nine different genes on the mitochondrial DNA. Every product has a distinct and fixed size, and they separate well in a 2% agarose gel.
Every primer pair works fine when run alone, but when run together (for 45 cycles), I only get 7 bands. The 3 missing bands are the genomic DNA product and two of the mitochondrial rRNA subunits (SSU and LSU). The 7 bands that do work are all protein-coding mitochondrial genes, which suggests a pattern, although I don't have an explanation for it.
A number of observations that might be relevant:
1) In C. glabrata, the mtDNA is present in a much larger copy-number than the gDNA - I estimate about 20-50 times more, but I could be wrong. Still, given that PCR is exponential and I'm using 45 cycles, I should still get a band from the gDNA primer pair, even if weak.
2) The LSU and SSU products are quite GC-poor (20-23%), but the other mtDNA products are not much better (~24-32%), so I don't know if that matters.
3) All the primers were designed to have a similar Tm.
4) Running the PCR in a temperature gradient didn't seem to do much.
5) In rho- petites (yeast that completely lost their DNA) I see no bands from the mtDNA primers (as expected) but the gDNA band does appear (also as expected - perhaps because the mtDNA primers don't interfere with the reaction anymore?).
I can start playing around with the primers and their mixes, but I feel there's something theoretical I'm missing. In any case, any and all advice will be appreciated.
Thank you in advance!
I want to know from the map what percentage of each maternal haplogroup each country or region has.
How can I find or create such figures?
I appreciate your help.
We want to isolate mitochondria from NIH3T3 cells. We have Sigma Protease inhibitor of calcium-activated neutral protease sourced from rabbit skeletal muscle (cat # P0787).
How much and what concentration of protease inhibitor is to be used for mitochondrial isolation?
We intend to further measure mtDNA content from isolated mitochondria.
After few years I was re-sequencing a fragment of the D-loop of dog mitochondrial DNA and I found a heteroplasmy which was not previously observed at the same position. In the attached picture there are two chromatograms of the same sequence from the same dog, from the same blood sample. The chromatogram above is from 2014 and the below one is from 2021. The isolated DNA is the same. The heteroplasmy was observed in several samples at the same position in other dogs' sequences, however in all sequences the quality is poor due to the double peaks in the baseline. I don't understand why the signal dropped only at one position, and suddenly raised in the next position.
I'm looking for protocol to extract DNA from liver tissue sample of geckos that have collected and preserved in 95% Ethanol. I also want the protocol along from DNA extract until sequencing of nuclear DNA and mitochondrial DNA. Please help
Recently, I am working on islating mitochodnrial DNA (mtDNA). I found that genomic DNA contamination is always a problem although I isolated mitochondria from cells first and digested crude mtDNA with plasmid-safe (DNase which is supposed to digest linear genomic DNA but will not touch circular mtDNA).
isolate mitochondria from cells by Magnet-anti-Tom20---> lyse mitochodnria--->digest mitochondrial lysis with plasmid-safe--->extract mtDNA
I detected mtDNA enricment through qPCR (ND1/HGB), some of the samples have 5-8 enrichemnt of mitochondria DNA after digestion (mtDNA/gDNA) , but some have no enrichment at all. I feel the mtDNA isolation method needs optimization and decide to start from digestion step.
I am looking for commonly used restriction enzymes that could digest genomic DNA but not mitochondrial genome. My purpose is to obtain pure mtDNA by removing genomic DNA as much as possible, which may be achived by using REs together with plasmid-safe.
Could you kindly suggest me in the following questions?
1. How to stably isolate pure mtDNA from human cells
2. Except qPCR, what other method I can use to confirm the enrichment of mtDNA?
2. Which REs are commonly used to fragment human genomic DNA? If I can't find the recognition sites in mitochondrial genome, does it mean that it is the RE I am looking for?
I am trying to sequence mitochondrial DNA using DNA extracted via blood DNA extraction kit. I thought of using mitochondrial specific primers to amplify the area of interest. But certain mitochondrial regions are highly homologous to chromosome 5.
The primers which i designed are specific for mitochondrial regions but they are also specific to chromosome 5 (only 2 or 3 mismatches in one of the primer ) and they give similar PCR product size too.
I am wondering if these 2 or 3 mismatches will be sufficient to amplify only the mitochondrial DNA or will it amplify both chromosome 5 and mitochondrial DNA.
I have a number of unknown samples of DNA with a list of species that may match. To go about this I want to use the c oxidase subunit 1 region of mitochondrial DNA. I need to estimate the copy number in each species, any ideas how to run a PCR for this?
I am performing sanger sequencing on human mitochondrial DNA.
When using regular sequencing method, most all the samples appear a double peak in the same position.
When using the special method designed for difficult template, almost all the double peaks disappear.
The picture shows the results of the same sample using the same primer under different methods.
All sequencing were performed by GENEWIZ
Is this a true heteroplasmy or just a method error?
I am reconstructing the demographic history of a triatomine species and would like to know what is the minimum number of mtDNA sequences that must be considering for the analysis to be efficient?
I need an appropriate protocol to extract mitochondrial DNA from any sample for forensical purposes, which would help provide an accurate analysis. If possible, kindly mention any easy way to develop a mitochondrial DNA isolation kit. Thank you.
I want to measure the copy number of mtDNA by qPCR by doing nDNA/mtDNA.
This, of course, will be after DNA extraction. The problem is that I think that I will get also the mtDNA which inside the mitochondria and I need just the mtDNA which leaks outside the mitochondria to the cytosol.
I will be happy to get some help :)
I’m studying population genetic structure of fish in Thailand. by DNA sequencing using mtDNA COI. How I can calculate the appropriate number of samples from 6 populations.
Thank you in advance.
My lab is planning to go for complete mitochondrial genome sequencing of termites. Kindly suggest which company provides the cheapest and good sequencing for insect mitochondrial genome? Kindly also share if any contact information is available with you.
I am running DIYABC for both microsatellite and mtDNA data and testing 3 scenarios. Once I start the analysis I get the following error message:
"Something happened during the reftable generation :
Program of thread 'Species_ABC reference table generation' exited (with return code -6) unsuccessfully.
Does anyone new the reason and a way to fix it?
Im looking for a human/mammalian primary cell line with a heteroplasmic mutation such as m.3243A>G. Preferably around a 60% threshold. I believe these are typically patient derived and I haven't had much luck finding a cell bank that specifies heteroplasmy.
Any advice on sourcing these or a company that stocks heteroplasmic primary cells would be greatly appreciated. Thanks for your time
Most of the literature used partial cytochrome b gene sequences for animal phylogenetic studies instead of the complete sequence. What is the reason behind this where full or complete cytochrome b gene sequence could explain more details?
Thank you very much!
What is the best technique for isolating mitochondrial DNA from the whole blood/PBMC samples?
Second, can I measure mtDNA oxidation directly (for instance by quantifying 8-oxodG) in mitochondria?
How many average genetic distance values of mtDNA control region Dloop for indicative conspecific populations or valid species in Chiroptera?. Thank you
For the past two years I have been collecting fin clippings of resident trout in the tarns and lakes of the English Lake District.
Surprisingly, despite the Freshwater Biological Association being on the shores of Windermere for decades not once was a comprehensive survey of the fish populations of the English Lake District ever undertaken.
There isn’t even a presence and absence survey let alone any attempt to see if there is any genetic divergence between isolated lakes and tarns populations and the semi-isolated drainage systems of the main lakes (interconnected by sea trout migration via the Irish Sea and Solway Firth).
At present, I am probably 2/3 in with two more years sampling the remainder of the stillwater bodies that I know, or suspect, contain trout in the Lake District catchment and I am seeking able bodied (some of the tarns are nearly 700m up) and / or currently practicing genetic analysts to share this study with.
The nature of the samples (frozen fin clippings) means that this is potentially a project that could involve researchers anywhere in the world. As for the field work component an interest in fishing is required and a healthy disregard to sudden inclement meteorological conditions is suggested.
I would like to run molecular diversity indices using Tamura & Nei as a distance method for calculating nucleotide diversity of mtDNA control region sequence.
It is difficult for me to understand directly from the Arlequin manual.
I would like to ask for some help if anyone knows how to enter my data and run using Arlequin. Thank you vm!
I would need to obtain a matrix showing the pairwise Fst values for a bunch of populations using mtDNA sequences (from NCBI). I have never done this before so I am not sure whether there is a quick/easy way to do it either using R or user friendly softwares,.
Hello everyone! I need to amplify a specific DNA fragment in order to do Sanger sequencing for the detection of a point mutation in human patients. The fragment is in an exon of my gene of interest. I designed specific primers of 20nt each, not annealing with other genes (verified by BLAST), both with the same Tm, 50% of GC and 50% of AT. I performed PCR with Phusion High Fidelity polymerase, adding firstly DMSO 5%, then Betaine 1M, and increasing Tm, but I always obtained a lighter "aspecific" band of 1500bp along with the specific PCR product of 500bp. I then decided to design new primers, but same thing happened. In electrophoresis, I obtained one band of 370bp and the same lighter band at 1500bp. Negative control is totally clean, so I don't have contaminants in my solutions. I run PCR on DNA samples from 10 different individuals and observed the same 1500bp aspecific band for all of them. I couldn't find any pseudogene or mtDNA alignement. I used RNAse when I extraxted DNA from blood. Could anyone tell me where the problem might be? I attached the picture of the electophoresis run. Thank you very much!
does anyone know whether damaged mitochondria can be repaired and recover to normal? For example, when there is mitochondria injury, like fragmentation or swelling, membrane potential decreased, ROS production, ATP production decreased, mtDNA damaged, or any other injuries happened in mitochondria. can the mitochondria be repaired again?
does anyone know any related research about this question?
thanks in advance!
The transcription of Human mitochondrial DNA ND6 to give mature mRNA for the protein NADH Dehydrogenase 6. Would any experts like to tell me what is the sequence for that mature mRNA for the protein synthesis? Many thanks.
We are looking to genotype our mice. Our mice have a mutation in their mitochondrial DNA and we want to know the level of heteroplasmy i.e. % of mutation in their mitochondrial DNA. So we cannot use standard sequencing to determine that and usually pyrosequencing is used to determine this. I cannot find a company that does this.
I am doing research where I have to quantify human mtDNA in saliva. Due to financial reasons, I do not have access to commercial mtDNA series for a standard curve. I will have to base my standard curve on total DNA. Is there an known average percentage of mtDNA when looking at the total mtDNA in human saliva?
I have mitochondrial DNA sequences to perform analysis of the genetic diversity and population structure of a fish species. What packages can I use on R? I estimate that I need to generate haplotype networks, PCA and diversity indexes.I thank you in advance for your contribution.
Hi, I'm trying to amplify bacterial DNA from plant samples for Miseq. In order to eliminate plant chloroplast I used 799f/1193r primer set, but only got mitochondrial DNA from some of my leaf samples (only one band ~800bps on the gel is visible). Just wanted to know if this happens sometimes?
I am planning to conduct an experiment to determine whether a protein of interest binds mitochondrial DNA in mouse brain tissue, but have never done chromatin immunoprecipitation so I'm hoping for some clarification. I've looked through several protocols and all are focused on nuclear DNA, but I wasn't sure how much actually needs to be changed to isolate mtDNA specifically? Will a "standard" ChIP protocol also pulldown mtDNA, and if so, would a qPCR for mtDNA-specific genes tell me if my protein is binding mtDNA?
I'm currently just planning a very rough overview of what I will need, but if anyone has a specific protocol that would be greatly appreciated too.Thank you!
I ran a three scenario analysis using DIYABC. Some output files attached. PCA looks ok and the 95% PP clearly indicates that scenario 3 is the most supported one, with no CI overlap. However, either I am not doing it well but the error is lower for scenario 2 than 3. Any idea why this happens when the remaining analyses clearly indicate that scenario 3 is the best one?
Thank you in advance
I am studying two types of Ophidiid fishes having features morphologically similar and would like to genetically classify them.
I have already investigated COI region of mtDNA, but the genetic difference is quite small (2 to 7 mutational steps between nearest and farthest haplotypes), so I am considering of conducting experiments using nuclear DNA.
However, I am not sure which genetic marker will be suitable for analysis to make a marked difference than COI.
So far, I am considering of using the RAG1 region, which has been studied in related species, or ITS, where base substitution is likely to occur because of non-coding region.
Could you tell me if there are better genetic markers that has a fast base substitution rate and is effective in clarifying interspecific or intraspecific differences?
I will be happy for your help.
I'm doing real-time PCR with two primers called MT-TL1 and 18S, for mtDNA and houskeeping gene, and I got positive control on both NCT. I tried also different primers: ND1 and B-Globoin but also in them I'm getting positive ct value on ND1 gene (mitochondrial DNA gene), the b-globolin gene was good (undetectable).
I checked my reagents and they are good, there is no contamination. On these 2 last primers shouldn't be a primer-dimer problem.
I'm getting values of: 36.95, 35.59, 35.49
I used 10uM primer conc.
I have some mitochondrial DNA data that was obtained using NGS technology. What I would like to know is which software program I can use to analyse my data.
I am currently undertaking a population genetic study on cichlids.
Looking forward to hearing from any of you.
I want to determine the phylogenetic age of the members of the Lepidoptera (Sphingidae) based on mtDNA, using BEAST software; But I do not have a good indicator. Please what is mutation rate in the mitochondrial genomes of the Lepidoptera (in million years)?
I'm going to do TEM and look at the morphology of the mitochondria, and I thought maybe I also will be able to see mtDNA which leak to the cytosol or in exosomal bubbles...Is it possible? Did it require some special staining?
Dear colleagues, in our new project, we need to isolate mitochondrial DNA from chicken blood, which have erytrocytes with mitochondria. We can also isolate the mitochondria from blood cell but we want to use a DNA isolation kit which is specific for mtDNA. I will be very happy if you can share your experiences on this issue.
Mitochondria are dynamic organelles that continously fuse and divide. This behaviour is important for content mixing and mitochondrial quality control. I am interested whether mtDNA replication and transcription rates are changed in mitochondria with altered morphology (e. g., fragmented mitochondria or hyperfused mitochondria). I didn't find much information on this issue in the literature. Maybe you know some papers that could help me with my question?
I have some question specifically about a Beast analysis I am trying to run (I am a beginner).
My dataset contains 2 mtDNA genes (COi and 16S) and 2 nuclear DNA genes (H3, 18S). I specified for - 16S the substitution model HKY+I+G and clock rate, 18S the sub. model K80+I, COi the sub. model TrNeF+I+G and clock rate, H3 the sub. model the same previous model. I have run MCMC chain length for 100 million and the ESS value for gamma.shape 3 is still below 100.
Do you have an advice how to increase this parameter?
The degradation of tissue-derived DNA without homogenization (assuming intra-cellular DNA) appeared to be faster for nuclear than mitochondrial DNA, whereas the result is reversed in homogenized tissues (assuming extra-cellular DNA), and the degradation of mitochondrial DNA appeared to be rather faster than that of nuclear DNA (Foran, 2006).
So, I want to know how physically, chemically, and biologically the cellular environment could influence the persistence of nuclear & mitochondrial DNA. Is there any other references?
Recently I've done some analysis on mitochondrial DNA and now I want to run the same analysis on only hypervariable regions of mitochondrial DNA to see whether the changes that I have observed are linked to only hypervariable regions or not. To do that, I'll need to make new fasta files from the mitochondrial DNA entries that I have and I want to know how to extract those. Any leads will be appreciated.
I have tried using the Plasmid isolation Kit as recommended by
But I could not get significant results. So, please give me some suggestions.
I need some help with this issue.
Anyone have arranged protocol?
I understand that the first step is the separation of plasma from WBC+RBC and after extraction DNA and qPCR, but it's still unclear for me how I can detect only the mt-DNA without comparing to nuclear DNA...
While performing mtDNA isolation using differential centrifugation and amplification using qPCR, I want to check if any nucDNA is also in the mix. What nucDNA specific gene/primer should I look for?
I want to use Geneland for cytochrome b sequences of the mitochondrial DNA. Could somebody share with me an example input file for DNA sequences?
Thank you very much.
We am working with mtDNA sequence analysis of skeletal remains from war. We are using Sanger sequencing and facing challenges. One is determining base call.
Using same PCR template amplified by a primer pair and performed sequencing with several primers, we observed that at some positions, the minor peaks occur in certain traces (eg C-major, T - minor) but not in others (only C) (please see figures on the attached pdf).
This also happened when we replicated PCR then sequenced the products.
Has anyone experienced this kind of situation? What are the reasons and how to solve this problem?
Thank you so much for your help.
we placed plant in a dark conditions (in growth chamber without light) before isolation of mtDNA but still we have little contamination of chloroplast DNA. Is there any other method to control it..
I want to isolate the DNA from the chloroplast of Brassica napus. In downstream, I will use that DNA to amplify my target region for cloning purposes. Presently I have isolated whole genomic DNA from it. It was thought that this DNA will contain the chloroplast and mitochondrial DNA in it as well and will be amplified by the chloroplast specific primers. But I am not getting any amplification. Now I want to isolate the chloroplast DNA and give it a try with the PCR. Please suggest for me. Thanks
I've been working with Msats in DIYABC no problem but would like to try incorporating mtDNA sequences too, however I cannot seem to get the input file right. I've been following the instructions in the manual but get an error message every time I try to upload my file. Can anyone help? Many thanks!
I have isolate the MtDNA from different Noctuidae moths. From the MtDNA i sequenced the COI gene (Cytochrome oxidase sub I). When i submit the sequence to NCBI,C they asked to submit the Coding region (CDS features). So i need to submit the CDS along with the sequence.
my median Joining network of mtDNA d-loop sequences did not share any haplotype between riverine buffalo breeds of neighboring countries, how should i explain this in my paper.
these breeds almost have same environment and ground alleviation also same.
I am comparing phylogenetic data sets in elasmobranchs from GenBank, and am under the assumption that in most species COI will be faster, or have a higher mutation rate, even though both loci are constrained by functional requirements of metabolism, since both are housekeeping genes. But certain authors have suggested that when a COI data set doesn't show differentiation, we should try adding NADH2, but I am not convinced this is a productive direction to take the project.
First off, I am not a trained evolutionary biologist but I am interested in conducting simple evolutionary analysis on a cohort of sequence data I have generated.
I have mtDNA sequences for the same drosophila strain that are separated by approximately 150 generations and I would like to determine the mutation rate. What would be the best way for mutation rate determination? Is this something that can be easily done by dividing the number of sequence changes by the length of the sequence, and then dividing that answer by the number of generation?
I am using plasmid extraction kit followed by long-range PCR but I do not observe any clear bands on the gel. The primers are designed in a way that I should get two bands of 10Kb, however, I only observe smears.