DNA-dependent activity of RT?
(by aawara from FEMA-trailer.org)
Tue Mar 20 07:22:20 EST 2007
In <etobva$as0$1 from news.uni-kl.de>,
Simone Marker <marker from rhrk.uni-kl.de> wrote:
> does anyone know if reverse transcriptases normally used for RT-PCR have a
> DNA-dependent DNA-Polymerase activity beside the RNA-dependent
> DNA-synthesis? I use MMulV H- from finnzymes or cMaster from Eppendorf.
They do. There is no retroviral RT that does not have DDDP activity.
The enzyme most commonly used in RT-PCR kits is an RNaseH(-) derivative
of Moloney murine leukemia virus RT.
> RNA samples are acceptable free of genomic DNA (according to the noRT
> control template) but nevertheless I wondered if my RT-PCR products really
> originate from only reverse transcribed RNA and not from residual small
> amounts of "RT-amplified" genomic DNA...
Viral RTs are thermolabile. In a RT-PCR, at best RT will double the
amount of genomic DNA if there was any contaminating your sample. If
you're concerned about contaminating genomic DNA, just leave out the RT;
Taq will effectively amplify it. Another control that you could use is
to treat your sample with RNaseA.
> I know that there is a DNA-dependent RT activity in the reverse
> transcriptases of some viruses, but I don't know if I have to take that into
> consideration with commercial RT.
DDDP activity is in the commercial enzymes, but is unlikely to lead
to spurious signals from genomic DNA.
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