positive DNA probes

From:Patty Kubier <pkubier@hotmail.com>

We perform HPV ISH(screening and subtypes)in our laboratory using 
biotinylated probes.  We have often found that even if we get a strong 
signal with the Pan HPV, the signal for the pos. DNA probe on the same 
tissue is often very weak.  I understand that the purpose of the positive 
DNA probe is to demonstrate the accessibility of the DNA content in the 
tissue and that if weak staining is observed (possibly due to over fixation) 
the test can not be considered valid.  However, I would think that if the 
Pan HPV gives a strong nuclear signal then the Pos. DNA should also give a 
strong nuclear signal. The pos. DNA probe is supplied in a ready to use 
form.  The pos. DNA, neg. probe and HPV's are all handled in the same manner 
for the entire procedure.  The procedure includes a 1 hour hybridization 
step in which the Pan HPV and the subtypes work very well but the pos. DNA 
signal continues to be extremely weak.  I have increased the hybridization 
time to 2 hours in order to get a stronger pos. DNA signal which has helped 
a little; but, it seems to me that the problem may be with the concentration 
of the pos. DNA probe since the other probes work very well at a 1 hour 
hybridization step (the manufacturer's recommendation). I would appreciate 
any feedback from other laboratories that could share their experience 
regarding the use of biotinylated positive DNA probes. Thanks for your help.

    Patty Kubier

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