RT activity in Taq pol?

Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Sep 2 11:13:41 EST 1994


Does anyone know if Taq has ever been observed to have any reverse
transcriptase activity? I have been using a one-step RT-PCR amplification,
and have noticed something a little strange. I do not know the intron/exon
splice boundaries of the gene I am amplifying, so I am not sure if my
primers fall outside an intron-containing DNA sequence. The problem is, I
need cDNA fragments which do NOT contain introns. Therefore, each time I do
an amplification of total RNA with Taq polymerase, I set up a negative
control reaction the same as the amplification reaction, with Taq, primers,
etc., but with no reverse transcriptase. This is to make sure the products
obtained by PCR have not been amplified from contaminating genomic DNA in
the RNA prep. Furthermore, I have digested the RNA with RNAse-free DNAse I
to eliminate any genomic DNA, and have run the RNA on a gel to make sure I
see no extra bands.
    Most of the time (but not always), after doing the RT-PCR I get a band
in the RT- negative control, which is the same size as the bands in the
Taq/RT positives. The PCR product I get is the size I expect with no
introns, but I am guessing that the products I am observing are amplified
from contaminating genomic DNA in the RNA prep, even though I digested the
RNA with DNAse I, since I get the same size product in the RT- negative
control. An alternative explanation is that the Taq polymerase is making the
first strand DNA copy of the mRNA in question. Is this possible (I don't
think so)? So, does anyone know if Taq has ever been observed to have any
reverse transcriptase activity?

Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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