I have extracted gDNA from an infected tissue. I need to sequence the gDNA from the pathogen (not the tissue), but I cannot culture the pathogen in any media (its a rickettsiae bacterium). I have rickettsiae and tissue gDNAs in my sample.
Hi
I am also doing some work on rickettsia but mainly on its proteome. I would imagine the bacterial genome would be smaller in size than that of the host. Assuming the DNA has not been fragmented and the there is no RNA contamination, how about you try running the extract on agarose 0.7-0.8% or higher for greater separation. if you see two bands, you could excise these and do a gel extraction and clean up. After this you could use gene specific primers to validate whether it is that of the parasite or host.
Best way would be if you could fractionate the parasite from the host cell.
Dear Marcel,
I think you should amplify the genomic DNA of rickettsiae with specific PCR primers then you can sequence the PCR product which specifically belongs to the pathogen.
Yours,
Heidar
Hi!
There are some things I can suggest:
Just sequence at high enough coverage and you will get both host and pathogen. Usually when assembling you will get a very good bacterial genome, because it is smaller and will have more coverage. Alternatively you can map your reads to the genome of related bacteria and the host and then assemble the bacterial genome from the reads that only mapped to the rickettsiae reference.
Youd could to rolling circle WGA after sucrose floating the (smaller) rickettsiae genome from your total gDNA.
You can try digestions with DpnI and DpnII, which either cut or don't cut bacterial DNA. But this depends on the methylation status of your DNA.
Cheers
Philipp
Thank you Heidar
I want to sequence the entire genome of the bacterium. But if I have gDNA from another organsm, I will be having erroneous sequences, probably not belonging to the bacterium that I want to study.
Regards
It is really not my topic but it this is very interesting work. Is it posible do a cell fractionation to separe both organisms, similarly if you want to get mitochondria?
The best! =)
You can get some ideas for purification of genomic DNA from those works on genome sequencing. I think it would be quite difficult to separate DNA from different organisms once you have performed the extraction, it is always easier to obtain your bacterial cells and then extract DNA. Here two different approaches:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516817/
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008361
Dear Marcel, good day
Firstly you must amplify the pathogen DNA using specific primer
the second step you must clean up the products
finally the sequencing can performed
Hi
I am also doing some work on rickettsia but mainly on its proteome. I would imagine the bacterial genome would be smaller in size than that of the host. Assuming the DNA has not been fragmented and the there is no RNA contamination, how about you try running the extract on agarose 0.7-0.8% or higher for greater separation. if you see two bands, you could excise these and do a gel extraction and clean up. After this you could use gene specific primers to validate whether it is that of the parasite or host.
Best way would be if you could fractionate the parasite from the host cell.
Hi!
Give a bit clear information about question so that we can advise better.
Vast literature is available. No idea exactly what species you are working with. Seems variety of species are reported. I suggest clone your PCR product, may be a bit tedious then go for sequencing you can find out whether any differences in the seq.s, all are same or any variation in the clones that you have selected.
Good Luck!!
I agree with Heider Sharafi. Designed primers specific to rickettsiae sequence and amplify genomic DNA.
Hi,
If you're doing next-gen sequencing, you don't need to separate the host and pathogen genomes, as Phillip Shiffer suggested, because the assembly of the two genomes will be separate.
If you still want to separate the pathogen genome from the host, you could try doing what Lusisizwe Kwezi suggested and purify the genome from agarose gels. However, this will only work if the ricketsia genome is small enough to run into a regular agarose gel, which I think is unlikely. You might have to do pulsed-field gel electrophoresis (PFGE) to get the big DNA pieces to even enter the agarose gel, which will require a fair bit of optimisation. Unless you are familiar with PFGE, I would stay away from it.
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