I want to get pure genomic fraction and pure episomal fraction without cross-contamination. I want to use them in PCR. I am working with herpesvirus which has quite a large genome. Any suggestions?
I think that the possibility of getting a completely virus-free (i.e. not single virus molecule left in the genomic fraction) genomic fraction from Herpes-containing cells is very low if not impossible. Shearing during preparation will always leave some linear viral DNA in the prep which you won't be able to remove completely. If anyone has a better answer I also would be interested to hear it.
Pulse-field agarose gel electrophoresis would be perhaps the best bet to try a physical separation using very thin gels and lysing the cells in the gel.
What experiment are you planing with the DNAs? Do you need to recover viral DNA from cells?Could you use instead pure virus and non infected cells as your source of DNA?
Thank you for your answers.
The problem is that I am working with latently infected cell lines. Therefore there should not be whole virions only latent viral genome. My plan is to investigate whether the viral genome can be integrated in the cell genome or it is only in form of episome. This is the reason why I want to get completely separated fractions.
If you have access to whole-genome or -transcriptome sequencing, such as DNA or RNA-Seq, there are bioinformatics methods to determine the specific integration sites of viruses: http://www.ncbi.nlm.nih.gov/pubmed/23162058
If you do a Southern blot labelling with virion DNA you should be able to distinguish viral integration from episomal form at low cost and effort. Estimating copy number would also help. Concatenates might also be integrated which might be taken into consideration as a complication in interpreting the Southern blot.
I used FIGE many years ago to separate and map out episomal HHV-6 DNA, it works.
Can you not try Alu-PCR as used for detecting integrated HIV?
Eons ago in a netherworld, I used to treat an "episomal" prep of HIV (9kbp) with Plasmid-Safe DNase to get rid of genomic DNA. Not sure if it will work with herpesviruses >150 kbp).
other refs
http://www.pnas.org/content/107/12/5563.full
http://www.bushmanlab.org/assets/pdf/publications/Butler,etal.NatMed7.pdf
Ian
Ian, you gave useful references. Is there also an original scientific publication (rather than the 'sales promotion' advert protocols of Companies) on the development and testing of the properties of the Plasmid-safe DNAse?
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