Hello Mahboodeh,

Several reasons may explain why you get no amplified DNA, unfortunately!

Pfu pol or Taq pol are not 'that' different. So the "no-amplification result" you get should be explained in another way.

The most ferquent cause of "no amplification" in my lab is a bad (or degraded) template DNA. I would thus recommend that you check the quality of your template DNA.

The second most frequent reason is a not very good accuracy in the two Tm of the pair of primers you use for PCR amplification. It is an uneasy task to correct this, because there will be as much "recipees" as many people answering you! However, I do recommend you to try this "rceipee": make a dual amplification in which the two first steps use an annealing temperature of 42°C (and not 50-65°C). Then the annealing step of all remaining cycles are at the normal temperature. We frequently get an amplification by doing this in cases there were no amplifcation systematically.

Another try is to add an "helper" to the PCR mix, usually we had PerfectMatch from Stratagene. It yields astonishing results most of the time but not always.

Best wishes and good luck for your experiments,

Prof. P. Urban

I think you may need to redesign the primers from the consensus sequences. May be you have different copy from the gene.  

Could you please contact to Dr Zohreh Sharifi at Iranian Blood Transfusion Organisation.

I hope, you get the  good answer.

First, you should have a fair idea about the HBV genotype prevalent in your region. Second, redesign your PCR primers. Third, use a mix of Taq and Pfu polymerases.