Hepatitis B genotyping - (Aug/26/2006 )

Hi,

We are doing hepatitis B genotyping in our lab. using nested PCR as methodology (1st and 2nd PCR reaction).
After we proceed the extraction procedure and the PCR amplification steps in the 1st round PCR reaction, Products on the gel were confusiing, results from many samples showed no bands, and that samples with known high titer viral load (done by previous quantitative PCR procedure) showed no bands while some of those with low titer showed faint band, providing that these samples are from chronic hepatitis patients and all of these samples have previous high viral load done by Cobas Amplicor method.
- we use different extraction method (Gentra, High pure and phenol-chloroform extraction method).
- Optimization for the PCR steps was put in consideration, we play with the annealing temp., the conc. of primer, the conc. of MgCl2, unfortunately no products .


Hint: the sequences of PCR primers used in this study for the 1st round PCR were in the pre-S1 through S genes:
5'-TCA CCA TAT TCT TGG GAA CAA GA-3' (nt 2823-2845, universal, sense)
5'-CGA ACC ACT GAA CAA ATG GC-3' ( nt 685-704, universal, antisense)
==============> (1063 bp)
Where could be the problems .

Help will be highly appreciated

Did you run a positive control? it is necessary for situations like these as some researcher don't run them at all, if you did run a positive control, does it show the band of interest? if it does you have problem with your sampling.... If not your problem might be with the primer you are using....

friley