RT-PCR

Holger Bohnemeier Bohnemeier at ukbf.fu-berlin.de
Fri Nov 3 11:43:24 EST 1995


Hi you all!

I've certain problems with my RT-PCR. I want to amplify a part 
of the GAPDH-cDNA sequence performing first a RT with total mRNA
and later continuing with a PCR and specific GAPDH primers.
The primers are designed to overspann several introns even 
overlapping an intron (i.e. the 3' part begins in one exon whereas
the 5' part ends in the following one), so I can differenciate 
genomic DNA and cDNA. RT-PCR works fine. I get the amplicon that
I expect for cDNA.
The problem: even if I don't use reverse transcriptase (RT) in my
assay, I get the same PCR-product as done with RT, expecting not 
to have any PCR product. Faults like contamination or changing samples
are excluded.
So I supose a) the sequences published for GAPDH mRNA (cDNA) and
               the corresponding genomic DNA are not correct
               (there might be polymorphisms laking 4 introns?),
            b) my Taq-Polymerase (GIBCO) has a slight RT-activity
               producing few cDNA molecules during the first steps of
               PCR.
My question:
   Has anyone encountered with similar problems? Are there any ideas
   about this phenomenon (for me)?

Thanks in advance for your answers!



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