The aim of our researches is to do DAF (RAPD with very very short primers 5-mers and 6-mers) on noroviruses, rotaviruses and adenoviruses. We have serious problems with noro and rota to do PCR with specific primers according to mutations in their small genoms in our geographic region in comparision to another reserches described in articles. Specific primers from articles doesn’t work. OK, so we decided to do DAF. We choose this very short primers because these viruses have very small genoms. We want to have as good polymorphism as it is possible. We wrote a program in PERL which search in whole genome of virus places which fit to sequence of primer so in this way we found five pentamers and four hexamers. This primers theoretically do very good polymorphism in different genomes of this viruses found in genbank (NCBI). Simple but we have now very serious problems..and we are looking for miracle. Maybe someone on this forum can help us. Please if you know somethink which can help us - write sth.
OK, how we do it (noroviruses and rotaviruses, because adenoviruses are DNA viruses and this procedure not contain RT step and contains DNA isolation step, other steps look similar):
Samples of stool from patients with viruses – RNA isolation using Qiagen kit (we choose good samples, we know that viruses are there, we choose another 2 methods to be sure) -> checkpoint: it works, we have RNA on agarose gel -> Reverse Transcription (Fermentas RevertAid), one cycle with random hexamers -> checkpoint: how? We don’t know if cDNA is there, it should be..-> PCR (finally, we think in this step of our researches there must be somethink wrong.. When we do PCR with specific primers we have fragments of DNA in different positions in different samples – sometimes we have more then one, we think it means that it works, we have cDNA but we don’t have sequences exacly fit to our specific primers. But when we do PCR with one primer, pentamer or hexamer, we have nothing on a agarose gel - gel is empty).
Pentamers: AATGC, CCTGA, GGTCA, AAGCC, TGACT
Hexamers: AATGCA, TGCAAT, ATATGC, ATCATG
We checked them using spectrophotometer – overything is OK.
Finally, we use 2 ul of primer (2 x 100 pmol) according to article we found about doing PCR with hexamers (they also use this method to do PCR with viruses but it was plant RNA virus, not human).
2xPCR MasterMix from Fermentas (we checked volumes – 25ul and 50 ul) – in article they use mix done by themselves, we also checked it..
We checked many combinations of volumes of DNA template. We also checked many templates (RNA viruses – norovirus, rotavirus, DNA viruses - adenovirus, moulds – Fusarium poae, Fusarium culmorum) but we have nothing. Moulds template (from another reserches) have very good quality and there is a big concentration of DNA.
95oC – 3 min.
94oC – 60 sec
30oC – 60 sec
72oC – 90 sec
72oC – 5 min
We checked many combinations of parameters and volumes of reagents, template, primers but it doesn’t work, even with good DNA template from moulds. Please help us to find the reason why it doesn’t work. It is very very very important to us.
If it will be necessary, our e-mail: siemensc75 (@@@@) gmail.com