mtDNA - included in isolated gDNA? - (Dec/07/2009 )
i try to get some cells without mtDNA. So now i have to proof that all of the mtDNA is gone. I thought, i could do this by extracting whole DNA of a cell and then control the presence or absence by PCR with specific Primers on mitochondrial Genes. What i don't know: is mtDNA isolated by normal DNA extraction protocols (in detectable amounts if present)?
Or do u have any suggestions for another control?
Thx & cheers
Wow this is no easy task. I'd use sperm cells, but I guess you can't
I work with mtDNA and we simply extract all the DNA. Then we use primers mito specific.
If you want to use PCR as your control, make sure you don't target a pseudogene.
I remember a colleague, a looong time, ago who was using a protocol that separates mt from nuclear cells. If nobody responds, I'll ask.
He he , yes a kind of complicated topic. As earlier i was working with bacteria, there it was possible to do colony-pcr. Is this possible with eukaryotic cells too, i.e. crush an aliquot of cell culture and use it directly as a template for pcr?
Up to now i saw only some suggestions for isolating first the mitochondria and after this isolate the DNA from there. But in my opinion i think there must be an easier way!?
By looking at PubMed, I found this:
Biochem Biophys Res Commun. 1997 Oct 9;239(1):257-60.
Isolation and characterization of mitochondrial DNA-less lines from various mammalian cell lines by application of an anticancer drug, ditercalinium.
Inoue K, Takai D, Hosaka H, Ito S, Shitara H, Isobe K, LePecq JB, Segal-Bendirdjian E, Hayashi J.
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Since ethidium bromide was not effective in mouse cell lines for isolating mitochondrial DNA (mtDNA)-less cells (rho zero cells), we examined whether an anticancer drug, ditercalinium (DC), which has been shown to exclude mtDNA from mouse cell lines, could be effective in various mouse and human cell lines. We found that after DC treatment rho zero cells could be isolated from all cell lines of mouse or human origin tested. Moreover, these rho zero cells maintained ability to receive exogenously imported mtDNA and allow its replication and gene expression. These observations suggest that DC eliminates mtDNA from mouse and human cells without affecting the property to receive exogenous mtDNA. Therefore, DC could be applicable to cell lines expressing various differentiated phenotypes for studying whether mtDNA plays a significant role in expression of phenotypes by manipulating mtDNA elimination and reintroduction.
I guess you could use PCR and use a rho zero cell as a control?