Dear Researchers,
Can you please clarify my doubt on the possibility for viral DNA extraction from blood/serum will be of only pure viral DNA or it would carry both viral and human DNA.
Is it good to go for viral dna extraction from viral cultures like lymphocyte cultures, if so the possibility of lyphocyte DNA as same above?
I need a Viral DNA for my NGS work?
Can you suggest me a best way?
Dear Nandagopal,
For the first question about human DNA traces in patients plasma my answer is absolutely: YES. I had many years of experience with plasma/serum extractions for viral diagnostics in hepatitis patients. I extracted DNA (for HBV analysis) and RNA (for HCV), both of them for quantitative and qualitative analysis.
I used several methods, for DNA I used column extraction methods, from different manufacturers, and finally I used the Boom method, because of its time effectiveness. I can say that all automated methods are more reproducible, than manual ones. For my purpose I preferred PLASMA. Most of viral kit manufactures recommend serum, probably because that from the same sample you can do antibody titers. But for my purpose I used plasma, because of MICROPARTICLES (microvessicles) released from activated (inflammatory) cells or destroyed (infected) cells. In serum I did not expected as much microparticles as in serum (because of coagulation). But it is completely different topic.
I always bio banked eluted DNA , in case, because sometimes in the same patients I had to do some human gene polymorphisms.
It is my experimental prove that you can see human DNA traces in plasma samples.
Probably you can avoid more DNA if you will use SERUM, and you will utltacentrifuge them before extraction.
I hope that I helped you.
Ewa
Dear Dr. Ewa,
Thank you very much for sharing your experience, Since am working in NGS, i want to do sequencing of a viral genome directly from serum/plasma,
But NGS will generate both viral as well as human genome, that could be discriminate by Bioinformatic tools as suggested by Dr. Artur. I too knew this.
I just want to make sure, is there any possible way to avoid or reduce the amount of Human DNA from viral DNA.
I thought to do a gel electrophoresis after the extraction of DNA from serum/plasma
and can see two bands, one in above which is Human genome and the below one Viral genome.
So that i could elute the lower band and go for purification and do NGS.
Is it right way or can miss some genome while purification or elution, is my worry.
Thank you very much for your kind answers doctors.
Dear Chia julius, sir,
Am not bothering when its PCR, where we target our gene by Primer specific.
I was bothered about NGS where Human DNA will overlook the viral genome with higher output of Human contamination and lesser coverage on viral genome.
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