DNA-dependent activity of RT?

Aawara Chowdhury via methods%40net.bio.net (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:

> Hi,
>
> 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.

> My 
> 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.

AC
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