RAPD and viruses - RAPD using very very short primers (Aug/16/2008 )

Hi,

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 phorum 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).
Our primers:
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).

PCR mixture:
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.

PCR parameters:
95oC – 3 min.

35 cycles:
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

umm.... i don't think you can use primer (5-6bp) this short for PCR. Taq need at least 16 bp for normal extension. Anything below 14bp and i don't think you can even extend the primer.

I've heard that S-Tbr polymerase can extend short primers 9 to 10 bp.

But 6bp.... could you show us the paper that reported extension with 6bp? I am curious. Could they be using Klenow? I know you can extend hexemer primers with klenow... but I always though that didn't extend very far.

Thanks!!!

Paper that reported extension with 6bp:

Restriction primers as short as 6-mers for PCR amplification of bacterial and plant genomic DNA and plant viral RNA.
Ryu KH, Choi SH, Lee JS.

Department of Horticultural Science, College of Natural Science, Seoul Women's University, Korea. ryu@swu.ac.kr

Amplification of DNA or RNA sequences using the polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) requires primers of an appropriate length to be designed. Two hexamer restriction primers, denoted as E101 and H301, which correspond to sequences of EcoRI and HindIII recognition sites, respectively, were selected and used as primers in PCR and RT-PCR. We first applied the restriction primers to the plasmid DNA and bacterial (Pseudomonas) and plant (Cymbidium) genomic DNAs. We observed positive DNA amplifications with the recombinant plasmid DNA and bacterial and plant genomic DNAs. Purified viral RNA was used for template in the RT-PCR with the primers and successful DNA amplification was obtained. These results suggest that the 6-mer restriction primers can be useful for new applications in PCR.


link to the first site of article:
http://www.springerlink.com/content/51361t...text.pdf?page=1

perneseblue I never heard that primers lenght influence to the kind of polymerase we are using. It is interesting. Probably i will try it.
But authors of the article said:
"and 2,5U Taq DNA Polymerase (Perkin Elmer)". I am afraid that they used ordinary polymerase.

bob1 but I have genomes ~7500bp or only a little bit more. It is impossible to find sequences fit to 7bp primer, I think using 10-mer it will be a lottery. I found that 6-mers primers are quite good (a few places where they fits) and 5-mers are really good. You suggest that with 10-mers (even my program don't find that it will work) it will be working? In which way?

Thanks for the paper, sadly I don't have excess to more than the preview.

RAPD... PCR with 10bp primers using Taq . Usable sub-optimum amplification. Looks like you learn something new everyday .

I think you can drop the melting temperature time for 94C - 60sec, down to 94C-30sec, maybe even 20sec.

And here is a link to the S-Tbr polymerase.
http://www.pubmedcentral.nih.gov/articlere...i?artid=2227730