Hello, has anyone experienced to extract pure mitochondrial DNA (not contaminated with genomic DNA) from filamentous fungi? There are some protocols about it, but, mostly yielding very low concentration of DNA, and sometimes contaminated with g-DNA. Recently, we obtained pure DNA expecting mt-DNA, and confirmed that g-DNA contamination is not present, however, PCR targeting mt-DNA regions did not work by using mt-DNA template, as well. I have been working and optimizing some approaches to get high quality and pure mt-DNA. I would appreciate any of your suggestions for it.
Hi Hilal,
mtDNA is always mixed with gDNA when extracting DNA. I wonder why you want to purify your targets since apparently its separation from gDNA damage it. ok there are some protocols to purify the mt DNA but even if the concentration is very low, the copy number is very high (think that it represents about 5Kb of DNA whether the genomic length is about 600.000 more). If your primers are really specific to mtDNA regions, you should simply use the mix.
you can use the UCSC (https://genome.ucsc.edu/cgi-bin/hgPcr) for higher species or NCBI primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to test in silica your PCR.
fred
Hi Frederic,
Thank you for your response. I wanna purify mt-DNA to get whole mt-genome sequence. I have some questions directing on mt-genome data. Since whole genome data is not available for the organism I study, I do not choose to go for whole genome sequence and then extract to mt-DNA. But, obtaining mt-DNA seems not very easy so far...
Hilal
Ok I see now why you want to purify it. NGS and unwon sequence reference...but take care since you'll probably have gDNA. Size selection on agarose gel?
or the Abcam society has a good kit to do it (https://www.abcam.com/mitochondrial-dna-isolation-kit-ab65321.html), just see and decide whether it could be the solution.
good luck and best year
fred
Hi Hilal
You can check this reference
http://www.fgsc.net/fgn44/baidyaroy.html
Ref.2
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=2ahUKEwi50YCSptnfAhWPNOwKHRWuCNsQFjACegQIBxAB&url=http%3A%2F%2Fmic.microbiologyresearch.org%2Fcontent%2Fjournal%2Fmicro%2F10.1099%2F00221287-94-1-204%3Fcrawler%3Dtrue%26mimetype%3Dapplication%2Fpdf&usg=AOvVaw0GHTx21-4LMN4eAarVReR4
Ref.3
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=2ahUKEwi50YCSptnfAhWPNOwKHRWuCNsQFjAEegQIAhAB&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F8087879&usg=AOvVaw1ypu2boNCJue4gP0Vv9zKp
Thank you Laith for your suggestions..I had tried isolating mitochondria by using a commercial kit then went for DNA extraction following regular dna extraction protocol as well as plasmid isolation protocol. But, it yields very low concentration of DNA and include some g-DNA contamination, still. Unfortunately, methods I tried so far, can not provide high amount of mt-DNA which even can not be detectable on agarose gel. I surprise on gel images on references, maybe I should try some old manuals. I have some pure mt-DNA samples with low concentration but I suspect if it will be good enough for NGS reads.
Hi Hilal,
in your case I would imagine a"blind" protocol: size selection with a reference marker and after WGA (Whole Genome Amplification). you therefore should have enough mtDNA to see it on gel (it is considered to see a DNA band on gel at 50ng). WGA kit can be obtained by Sigma (https://www.sigmaaldrich.com/technical-documents/articles/biology/complete-wga-kit.html).
fred
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