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Mitochondrial DNA - Science topic

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Hi all!
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?
Thank you.
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Katie A Burnette Hi dear, maybe you can read these articles.
Measurement of Mitochondrial DNA Release in Response to ER Stress - PubMed (nih.gov)
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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?
Many thanks.
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your question is directed more towards mitochondrial transplantation and I think you might find some answers in these articles which discuss the efficacy of such a therapeutic strategy
Best wishes
Shambhabi
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Is any isolation kit for plant mtDNA and cpDNA isolation (like genomic DNA isolation kit) available in market?
Thanks in advance for any clues......
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i recommend discontinuous percoll gradient centerfugation and use mtDNA and crDNA specific primers in your PCR amplification.
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Hello Everyone,
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.
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I tried wako and biovision kit to isolate mtDNA, but failed somehow. Only smeared band. I think maybe TBE buffer is suitable for longer running.
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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?
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This is very good and relevant question. I think same with extraction procedure tissues of animals.
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Hi,
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;
  1. subtractive hybridization (purifying mtDNA, fragmenting it, biotinylating it, hybridizing it material to my sample and then pulling down the hybrid duplexes with Dynabeads)
  2. Nuclear isolation (hypotonic lysis, dounce homoginisation, centrifugation)
  3. 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!
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I find one paper use Cas9-assisted removal of mtDNA.
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I have a total genome of a plant sample, is it possible to isolate and sequence the chloroplast and mitochondrial genomes of the sample?
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  • Do you have the whole genome sequencing data now? If you have WGS data, especially third-generation sequencing data, you can assemble the chloroplast and mitochondrial genomes by bioinformatic methods. In that case, you do not need to isolate organelle DNA by means of traditional experiments.
  • We have a lot of experience in assembling organelle genomes by bioinformatic methods, and here is our latest paper, which you can refer to.
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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.
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Mitochondrial DNA is marvelous for phylogenies. For one thing it is not subject to the confusion of recombination so what you see is similarity by descent alone.The protein coding genes are really quite tidy and phylogenetically informative for a range of ancient (COI, 1st and 2nd positions) and recent divergences (e.g. ND2, COI 3rd position) and very fast changes (control region). mtDNA alone might be somewhat biased since it is maternally inherited and the genes are all involved with respiration and metabolism. So one must also use a few messier nuclear gene to offset the bias that could result from using only mtDNA. Also the mtDNA is inherited as a single marker so represents a very nice long seuquence compared to any nuclear gene which Is subject to recombination. The best analyses will estimate a species tree from mitochondrial and well-chosen nuclear genes that will likely have slightly different gene trees from one another. Model the mitochondrial genes with a different model of sequence evolution from the the nuclear genes with using coalescent methods to coax the gene trees into a single species tree.
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Is there any paper about using Tunel staining to detect mitochondrial DNA (mtDNA) damage?
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Hello,
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!
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Hi,
I have 2 points here and i hope they help.
1. I am assuming that you are using a commercial master mix for PCR. for low GC content template, you might need to have more Mg2+ in your PCR reaction. Maybe you want to try to supplement some MgCl2 in your PCR mix. (I had the problem when I PCR low GC products from Candida albicans genome, and supplementing Mg2+ helped.)
2. Have you checked the primer dimers across the pairs of primers you have? Could it be that some of the primers form strong dimers which resulted in missing of PCR products?
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Hi everybody
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.
Best regards
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You may look at online maps by ArcGIS pro .
Or
Hope that helps!
Regards,
Satyendra
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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.
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I am using 0.02M potassium phosphate buffer to isolate azurin peptide as sec metabolite from bacteria. but couldn't get the actual amount of protease inhibitor to be used to prevent degradation of my protein.
please help me to find out the actual concentration that can be used in azurin isolation
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Hello,
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.
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I would rather go with a NUMT. By targeting mitochondrial sequences with your primers, it is likely that they also match mitochondrial fragments inserted in the nuclear genome. The fact that you find it in several dogs and that several differences seem to have already accumulated would also point in this direction.
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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
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Since DNA is insoluble in ethanol and isopropanol, the addition of alcohol, followed by centrifugation, will cause the DNA proteins to come out of the solution. When DNA concentration in the sample is heavy, the addition of ethanol will cause a white precipitate to form immediately.
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I am isolating mtDNA from human blood. Using TKM1 and TKM2 solution.
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Hi everyone,
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).
Current protocol:
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?
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Hi,
I had purified mitochondria before using iodixanol-gradient ultracentrifugation. The purified mitochondrial fraction was free from any nuclear or cytoplasmic contamination. It was further validated by PCR/qPCR using GAPDH as a genomic DNA control. I'll try that though htis is hard, but the best way to get a purified mitochondrial fraction. Alternatively I'll do ten stroke with loose followed by ten strokes with tight-fitting dounce homogenizer. After enriching mitochondria fraction, I'll do nuclear digestion. Keep in mind that if you are using cell line, it would be difficult to get nuclear DNA free mitochondria as micronuclus of the cultured cells is always a problem in such case, which you are currently experiencing. You can find the detail protocol somewhere in the internt.
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For simultaneous extraction of nuclear and mitochondrial DNA.
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Dear Jyotsna,
this is a very interesting technical question. Unfortunately I'm absolutely no expert in this field as we work in synthetic inorganic chemistry. Thus all I can do to help you at the moment is suggest to you a few potentially useful literature references which might assist you in you analysis. For example, please have a look at the following relevanr articles:
Simultaneous extraction of nuclear and mitochondrial DNA from human blood
and
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR
The second article is freely avaiable on the internet (please see attached dpdf file). The first paper has not been posted as public full ext on RG. However, three of the authors have RG profiles. Thus there is a real chance that you can request the full text directly from one of the authors via RG (although the paper is a bit older).
I hope this helps. Good luck with your research work and best wishes, Frank Edelmann
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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.
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Hi Praveenan
since mtDNA and DNA are extracted at once, I'm not sure that your primers will ensure distinct amplification, maybe you'll have a mix of both amplifications even with a smaller amount with the mismatch one.
have you the possibility to test by sequencing the results?
maybe you could increase the specificity by increasing stringency by use of DMSO at 5%, increasing Tm by 2-3°c and decreasing amount of the mismatched primer in the mix?
all the best
fred
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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?
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Hi Audrey
you need to run RTq-PCR with a housekeeping gene from which you know the copy number. analysis will follow the delta delta ct method as you can find here (https://toptipbio.com/delta-delta-ct-pcr/) and the example attached.
all the best
fred
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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?
Thanks!
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Are you sequencing both forwards and reverse and is this result seen in both directions?
You might want to consider one heteroplasmy possibility. Suppose that you have heteroplasmic mitochondrial dna with 2 significant base changes relevant to this question....one is as you show and the other is situated under the 3'end (or very close to it under the primer). Now at low annealing temperatures for both sequencing and pcr amplification the mismatched primer will produce a weaker sequence than that produced by the fully matched primer. If you now use dmso, high GC buffer or increased annealing temperature in either the pcr or the sequencing reaction then the mismatched primer will not bind and you will only get a product and a sequence from the perfectly matched primer so the second peak will vanish. I think that this is a valid result not a method error. Changing the pcr primers to a different position would clarify the situation is this instance as both low and high specificity conditions should then show both bases at your polymorphic position
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Hello!
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?
Thank you
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Hi!
Check the attached and given link.
Good Luck.
Demographic Expansion and Contraction in a Neotropical Fish during the Late Pleistocene-Holocene (https://www.scirp.org/pdf/OJS_2019081214414020.pdf)
Demographic inference through approximate-Bayesian-computation skyline plots
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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.
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If you want to amplify using mitochondrial primers for analysis you need not specifically isolate mit.DNA, even DNA isolated using normal procedure also useful for amplifying mitochndrial primers. You can check my publication availabe in RG. May I know excatly what are your plans/working!!!
Good Luck!!!
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Hi,
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 :)
Thanks
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Hi Linoy!
I would like to quantify mtDNA leakage to the cytosol of mammalian cells and I wondered if you found a good protocol..I have just tried to separate cytosolic and mitochondrial fractions using abcam kit ab65321 but I have encountered some problems..
Thank you in advance!
Ariel
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Hello,
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.
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24 individuals will be sufficient.
Kindly have a look at my publication on RG you get idea 'dentification of Local- and Habitat-Dependent Selection:Scanning Functionally Important Genes in Nine-SpinedSticklebacks (Pungitius pungitius)'
Good Luck!!
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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.
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Dear colleague,
Unfortunately I do not know.
All best wishes for your continuing successes,
Prof. Otar Shainidze
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there are many methods to mtDNA extraction from human blood , what about the positive control used as marker in gel electrophoresis
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How mtDNaA is extracted from genomic DNA for next generation mtgenomics?
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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.
not priormusmoy.fixed"
Does anyone new the reason and a way to fix it?
Thank you
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Hi Catarina,
I am experiencing the same issue with DIYABC as you described here; no matter how I adjust the parameter settings, the program run keeps stopping and giving me that error message. Do you remember more specifically what you did with the defined priors for Ne that solved this issue for you?
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Hi all,
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
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There is to my knowledge no company distributing such cell lines, but you may get lucky by contacting the different groups that have established iPSC from fibroblasts of patients with this mutations. You can find the relevant Cellosaurus entries by searching for:
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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!
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Hi!
You got the answer.
Simply to show/prove populations are phylogenetically distant from other species three regions of mtDNA viz. cytochrome b, 16S RNA and and D-loop regions will be used.
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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?
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Hi Ana,
Many thanks for your helpful response.
Best wishes,
Deniz
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How many average genetic distance values of mtDNA control region Dloop for indicative conspecific populations or valid species in Chiroptera?. Thank you
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Hi Husni,
as far I could see, there is not a "magic number" for Chiroptera. Only three species have records in genbank for the dloop region. Therefore, it's really tricky to get a safe threshold, if we can say that exist such a thing.
I'm personally not a fan of thresholds, but you could try to explore the intraspecific genetic distance of the sequences on the database. It will give you a clue about how much this sequence is instraspecifically polimorphic as well as it should overlap the between species distances.
I should also recommend you to take a look at the coalesce analysis like GMYC or the PTP (Poisson's Process Tree) and their bayesian implementation.
Good luck
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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.
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Unfortunately, due to a combination of weather and increasingly difficult waters to sample (and of course a pandemic that stopped travel at a crucial time to the Lake District) I still have some waters to sample to get a complete picture (mainly some of the remaining lakes in the valleys). As I'm no longer an active researcher (after running a music agency I'm now running a distillery) this is taking longer than originally planned. But I will get there! It occurs to me too that DNAe would work on some waters if/when enough sequences are available from the fin clippings. I envy you being able to go "fishing" in the Amazon basin. Well done!
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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!
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Are you using a Linux-based OS?
Windows OS executable version of Arlequin is very intuitive (that's why my question). If you are having trouble with your input data, try running PGDSpider to convert it from sequence data (probably FASTA?) to .arp
Feel free to write me if you have trouble getting your input file right.
Good luck!
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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,.
Thanks
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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!
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You could try increasing the annealing temperature to try to get rid of the larger band and even try a "pcr" with only one primer to see if either primer generates this band non specifically. If you really want to know what it is the purify the band and sequence it
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Is there any kit or protocol we can use to extract mtDNA from isolated mitochondria instead of from tissues or cells? Also considering extraction from inner membrane and matrix fractions.
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Hi can you share the protocol how you have isolated the mitochondria?
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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!
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mitochondria (normally) exist in form of a reticulum, not as a single organelle. when damaged, they undergo fission and one of the "daughter" mitochondria with defects undergo mitophagy, while the other "daughter" mitochondrion remain healthy and get incorporated to the reticulum. So energetically it is not worth repairing the whole organnelle.
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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.
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Yutao Liu It is great. Thank you very much.
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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.
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To detect the relative level of the mito mutation vs the reference allele, you can also use droplet digital PCR (ddPCR) with probe-targeted assays labelled with FAM/VIC to quantify the absolute copy numbers of each allele. ddPCR has been widely used to quantify the level of cell-free cancer DNA mutations in the serum/blood. If you have access to a ddPCR machine from Bio-Rad or Thermo or other providers, this could be done easily. It will cost a few hundred US $ to quantify the level of mutant mito DNA molecule. Best luck with your research.
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In order to perform mtDNA quantifification (using qPCR with a target sequence), what would be the procedural requirements? Do one needs to normalize data?
Thank you in advance!
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Degradation of RNA is not necessarily essential, because the primers stick in the target place in DNA, but it is better than the RNA be degraded.
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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?
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Hi Ilse,
I'm sure you'll get an idea when getting an eye in this paper:
fred
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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.
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Hello Sávio Guerreiro , the following article may help to address your question.
Best wishes.
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I am working with mitochondrial DNA and would like to know the amplification rate between mtDNA and nDNA.
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Generally speaking, it's easier to amplify mtDNA because each cell has only 2 copies of every nuclear gene, but will have 100s-1000s of mtDNA genomes. Plus, the small size & circular shape means that mtDNA is less likely to degrade.
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I would like to know what kits can be used to extract mitochondrial DNA from fish liver and muscle tissue?
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Great! Thank you Katie A Burnette and Svante Martinsson for your advice :)
Also, I was wondering if you have experience regarding a real-time PCR kit suitable for quantification of DNA in StepOne Plus mashine?
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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?
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I recommend the use of the 799F and 1115R to characterize the bacteria in plant leaves over other chloroplast discriminating primer sets
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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!
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Maybe PMID: 24067899 will help.
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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
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Do you mean that Type I and II errors are lower for scenario 2? Could you provide details on the prevalence of these errors for each scenario? As Jose Alberto Lopez-Aleman suggests, looking at the regression and posterior plots might give some clues.
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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?
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Hi Mita,
Given your fish species are closely related as revealed by mtDNA, it is preferable to choose hyper variable regions of nuclear genome. Markers occurring to me are microsattlites (STR) and MHC. STRs have faster mutation rate than mtDNA in animals in general. Some regions of MHC also evovled very fast due to selection. Have a look at review paper below might help.
Zhang DX, Hewitt GM (2003) Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Molecular Ecology, 12: 563-584.
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Hi
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.
Thanks.
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Signals over CT values 40 and some say 35 should be handled carefully. In my experience, some low expressed genes can be detected around 35-38 but even NTC in most cases will produce a signal above the threshold after 40. So, as suggested above the best way is to run your PCR product on a gel. You can also reduce primer concentrations to half. In some cases, it helps to reduce the background noise signals. remember primers are DNA that can attach and amplify themselves and hence produce a signal when using general binding dyes such as SYBR-green and other similar dyes.
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Hi, everyone.
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.
Regards,
Darlington
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For annotation you may use Geneious, as proposed by others. In some cases, MITOS can also do a decent job http://mitos2.bioinf.uni-leipzig.de/index.py, but in both cases you have to check the results manually.
For comparative mitogenomic and phylogenetic analyses, you may try PhyloSuite, as it was designed to handle mitogenomic data: https://dongzhang0725.github.io/
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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)?
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I would highly recommend to anyone interested in tectonic calibration to read 'Molecular Panbiogeography of the Tropics" (2012) and 'Biogeography of Australasia' (2014) as starting points. These books include hundreds of examples of tectonic correlation with extensive integration of phylogeny for an immense range of animal and plant groups, and also presents a critical account of the issues in molecular calibration. This is just the tip of the iceberg. As for molecular rates of divergence, there seem to be all sorts of models so you take your pick - assuming there is a molecular clock in any meaningful sense. Some have objected to tectonic calibration where it provides ages for a subclade that would then push the larger clade back into e.g. the pre-Cambrian. Of course they never consider that perhaps their clock model is erroneous. Primates provide an excellent example for tectonic correlation with group after group of distributions corresponding to major tectonic events of Mesozoic and Cenozoic time. Some molecular researchers fall back on the mystical 'I can't believe it' when tectonic ages considerably predate the oldest fossil, but other molecular researchers are quite happy when their fossil calibrated estimates considerably predate the oldest fossils. There is a lot of waffle thinking out there, but the fact is that the vast array of modern life shows tectonic correspondence with Mesozoic tectonics. That is the great reality. Fossil-calibrated molecular clocks were widely proclaimed to falsify that, but it was all smoke and mirrors.
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Assume that reference mitochondrial genome is available, but whole genome of organism is not.
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you can use GenomeOrganeller to filter either chloroplast or mitochondrial reads from the whole genome reads. All the best.
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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?
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Dear Linoy Israel, the only way to visualize mtDNA in the cells by EM is by DNA immunogold labeling. Good luck!
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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.
Cheers,
Demir
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Hi Demir,
You have not mentioned about what you want to do with mtDNA after isolation.
Hope this will help you.
Regards,
Jay
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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?
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Good question.
I would say YES, there is a relationship betwaeen both processes.
Have a quick look to this paper and references herein. It may shed light into your doubts.
Good luck Christian
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COI - Cytochrome C oxidase subunit I
16S rRNA- 16S ribosomal RNA
mtDNA- mitochondrial DNA
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Theoretically both markers are appropriate for species identification is most of the invertebrate groups (and both are mitochondrial!) however, as COI was selected as universal barcoding marker it has the most complete DNA library (http://v4.boldsystems.org)
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Hi all,
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?
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The first issue is why are you trying to estimate that parameter? Who cares? More constructively, I suggest you examine the trace, sometimes increasing burnin will sort things. Sometimes the chains have not converged.
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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?
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Dear Jonas,
Thank you for replying my question very much!
Your suggestion would be reasonable for me.
I'm sure to read these literatures.
Toshiaki Jo
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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.
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Akhil Kumar , hope you have already identified the hypervariable regions (coordinates). Based on the coordinates you can extract (subset) sequences from any genome using Samtools - faidx
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I have tried using the Plasmid isolation Kit as recommended by
But I could not get significant results. So, please give me some suggestions.
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Thank you William Lee for your suggestion.
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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...
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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?
Thank you.
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I would think most nuclear housekeeping gene would be good enough to be used to check for contamination.
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about cell line
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Dear Hale,
I'm the marketing manager of AcceGen. Sorry for upset you and didn't give you a satisfied experience. I've checked our database and found your inquiry record with our rep. Founded in 2016, our company and lab are still growing, and some challenging projects are beyond our ability now. However, we are always trying our best to give all the researchers the highest quality products and services. Hope we can really help you next time!
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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.
Raul
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Hola Ana.
Te pego una explicación que le hice a un colega. Fijate tambien los archivos adjuntos
You have to prepare one data set with the genetic data of each individual and then a second data set with the geographic coordinates of each individual.
For the genetic data set, you should use only the polymorphic sites and then you have to transfor, the sequences into numbers, for example: A:1, C:2, G:3 and T:4
For example:
AACCGTA would be 112234.
For the geographic data set you must put first "longitude" and then "latitude".
I am sending to you two files with information of 30 individuals.
In the GeneticData set you will see 33 variable sites of those individuals (one row per individual). It does not matter if they have the same haplotype.
In the GeographicData set you will see the geographic coordinates of the 30 individuals that belong to 5 localities. The different individuals of the same locality have the same coordinate.
I hope you understand my explanation.
Best wishes,
Raul
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Hello,
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.
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Your sequencing is probably correct. MTdna often exists in any cell as different sequences. This is known as heteroplasmy and is often associated with reduced severity of mitochondrial disorders. If some mitochondria have a different sequence at your point of interest this will lead to another peak roughly correlating with the relative number of mitochondria with the changed base
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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..
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Hello!! I'd like to ask one thing. I have isolated Chloroplast from leaves with percoll gradient. Chloroplast, observed under a microscope were very beautiful. After I extracted DNA with a DNA extraction kit and amplified with chloroplast genes. I think that in my chloroplast DNA there was nucleus contamination... how can I obtain a Ch DNA without contaminations???
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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
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Hello!! I'd like to ask one thing. I have isolated Chloroplast from leaves with percoll gradient. Chloroplast, observed under a microscope were very beautiful. After I extracted DNA with a DNA extraction kit and amplified with chloroplast genes. I think that in my chloroplast DNA there was nucleus contamination... how can I obtain a Ch DNA without contaminations???
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Hi  everyone,
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! 
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Hi Ginushika!
I have example files. If you need it, please, sent a email at carolbioms@gmail.com
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Hello
How we can do in Primer blast from NCBI that What size is made of PCR product with D-Loop Primers by using DNA samples from Cervus and Capra aegagrus?
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on NCBI primer-blast, replace the organism by "Capra aegagrus hircus (taxid:9925)" for Capra aegagrus. for the first one, you must choose among this list: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=9859
fred
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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.
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Hi
Simply go to the expasy translate tool, paste the DNA sequence and ask to translate. Select the sequence in the proper coding frame and blast it. once the selected frame gives the correct hits with complete coverage of full length gene sequence, just submit the same. if you have amplified the truncated portion of the gene, submit the truncated part of DNA sequence only that encodes the protein of interest in the proper reading frame.
Good luck
BASAVARAJ
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If I get a piece of DNA, how can I tell if the DNA is from Mitochondria of Bacteria?
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Hello
Don't worry about it. Prokaryotes don't have mitochondria at all.
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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.
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I'm assuming the lack of sharing of haplotypes isn't a deal breaker for publication. Keep in mind any given publication can't explain everything. If it were me, I'd simply tell it like it is and submit the paper.
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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.
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The genbank database has tons of elastimobranch complete mtDNA. So should be straightforward to see what the maternally inherited mtDNA has to say about the evolution of this large and important group.
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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?
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Have a look at this paper:
JW Drake, B Charlesworth, D Charlesworth, JF Crow. Rates os spontaneous mutation. GENETICS April 1, 1998 vol. 148 no. 4 1667-1686
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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.
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