Summary for "Non-specificity for Primer Extension?"

Steve Rodems smrodems at students.wisc.edu
Mon Apr 4 22:50:09 EST 1994


In article <94093.215839LIU at QUCDN.QueensU.CA>, <LIU at QUCDN.QueensU.CA>
wrote:

>     I am trying to locate the 5' ends of viral DNA polymerase transcripts on
> a virus genome by primer extension (PE) assay, but suffering from the
> non-specificity of the assay system. I observed 2 PE products in both mock
> and virus infected samples.
> 

Maybe I'm missing something but these are virus infected *cells* so host
derived RNA will also be present in infected cells.  Thus, a transcript can
be extended from both mock and infected cells if the primer happens to
hybridize to it.

> Various solutions to this.
> Try adding actinomycin D, i believe this prevents extension products acting
> as primers and causing non-specific extension.
> Also verify the pe products using ribonuclease protection, s1
> hybridization, or RT-pcr analyses.

I believe actinomycin D is an inhibitor of RNA polymerase II.  This is
included so that any RNA products synthesized in the tube are derived from
the RT and not from RNA polymerase.  I don't know where the RNAP II would
be coming from since most protocols call for guanadinium treatment to
release RNA which I believe has its way with most proteins.

-- 
Steve "Some day I will get the hell out of Wisconsin" Rodems

"Then I am here for the Lee family renioun ... shur-wajo-shur"



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