Problem amplifying viral gene - (Mar/06/2013 )
Hello friends,
I want to amplify a gene from PPV virus. The gene size is 1350 bp but after pcr i see very faint band of this size and intense band 1/2 size of this band
I tried altering pcr conditions but not success.
I gel extracted the 1350 bp band and did pcr again and got same result.
then I saw the purified pcr still has about 30-40% intense 700bp band.
I sliced only the 1350 bp band but i dont know why the 700bp still persists in the purified sample.
Because of many problems I changed the primers and order new primers that are expected to amplify 1530bp band
but here again the pcr failed i got pcr amplification of 420 and 380 bp.
I used Viralgene spin ViralDNA/RNA isolation kit. I havent treated with RNase - could this be the reason ???
could the 1/2 size Dna be single strand of 1350 bp product ??
Could my sample have other contaminants ??
Thank you for your time
PPV is RNA virus. You need to synthesize cDNA. Do you? you didn't mention in your post.
First of all, PPV can stand for Plum pox or Porcine parvovirus, I suspect you are talking about the later.
Not familiar with viral expression, but RNA of this gene should be the same as the single stranded DNA, but since you didn't do any RT, it wouldn't amplify. Only thing it can do is inhibit the reaction in the excess.
Since as I said I have no idea about this virus life cycle, it's maybe possible that the DNA undergoes some editing. Or what you see is unspecific product. If you can't get rid of it, you can try to sequence it to see what it really is.
Also Pfu is proof-reading but less eficient polymerase, you may try to amplify it with some other to see if your product is more distinct. For cloning/sequencing you can use a mix of proofredaing and normal polymerase.
And no, ssDNA can't have a product of half a length. In PCR all products become double-stranded anyway.
I am talking about porcine parvovirus. I am going to use normal taq as suggested above
Also i read that the rna formed from this virus undergoes alternate splicing could it affect my pcr in anyway ??
No, RNA splicing should not affect PCR, since RNA is not amplified with DNA polymerase. Unless you do reverse transcription of RNA in some step, RNA will not amplify.
But question is since viruses are kind of quirky, and the way they replicate their DNA, maybe there could be some portions of (+) strands or fragments, I have no idea. If it's specific (contains the exact sequence of your primers) then it's probably more possible in viruses than other organisms, if it's unspecific, you should be able to get rid of it, by changing conditions of primer sequences.
Another thing to check is, if you know the sequence of the bit you are trying to amplify, is there an alternate binding site for either of the primers?