Genechip p53 sequencing

[Chandi Griffin]

Hello Emanuela,

I have performed GeneChip analysis using the p53 arrays and fixed DNA 
with success in the few analysis I've had occasion to perform, however I 
found that in some cases the initial multiplex PCR rxn was insufficient, 
and a second amplification was needed. I would imagine the extent of 
fragmentation suffered by the final sample may be the determining factor 
with respect to the need for re-amplification. Affymetrix in fact does 
point out that DNA isolated from fixed tissue may in fact require a 
second round of amplification, so even the company is aware of the issue 
and has presumably performed some level of in-house R&D to address the 
matter. Overall it is my experience that for all the p53 multiplex rxns. 
it is very useful to run a sample of a known quality multiplex rxn as a 
standard in electrophoresis to which one can compare the fixed samples. 
With the reference intensity provided one can gain some sense of the 
extent to which the PCR was a success. 

I hope this helps, and good luck. 

P.S. Do you per chance know a dear friend of mine -Dr. Alberto Sechi, he 
is based in Udine?

Chandi Griffin
Mol. Biol. GCRC Core
SFGH -U.C.S.F.
email: aniya at itsa.ucsf.edu




In article <3A9D1656.B07A4E0E at cmns.mnegri.it>,
 Emanuela Guerra <guerra at cmns.mnegri.it> wrote:

> I would like to know if p53 sequencing using DNA chips works with
> formalin-fixed, paraffin embedded tumor samples - the DNA I get from
> these is very heavily fragmented, and I wonder if it amplifies well
> enough in the initial multiplex PCR...Anyone can help?
> Emanuela
> 
> Emanuela Guerra
> DCBO
> Consorzio Mario Negri Sud
> S. Maria Imbaro (Ch)
> Italy
>