Help: SP6/T7 specificity

[Dr. Peter Gegenheimer]

On Fri, 15 Aug 1997 22:45:15, Peter Schuchert 
<schuchert at ubaclu.unibas.ch> wrote:

> How specific are SP6 and T7 RNA polymerase concering the other
> promoter sequences?
> 
> I use RNA trancripts as in situ probes, generated from an cDNA
> with flanking T7 and SP6 promoters. The antisense probe gives
> me a very strong signal in the expected cell population. However,
> also the sense probe gives a signal in the same cells, although 
> much much weaker.
> I inerpreted this that by generating my sense probe with SP6,
> also some transcripts are formed from the T7 site and producing.
> 
> Has anyone made similar observations? 
> 
>  
> -------------------------------------------------------
> Dr. P. Schuchert
> University Hospital INSEL
> Dept. Gastroenterology,  MEM-B814
> CH-3010 Bern Switzerland
> Tel 0041 31 632 84 92    Fax 0041 31 632 96 65
> http://www.unibas.ch/dib/zoologie/research/schuch.html
> -------------------------------------------------------
According to published reports, there is no detectable 
"cross-transcription" among T3, T7, and SP6 RNA polymerases. It is 
certainly conceivable, though, that certain specific clones might 
show some cross-txpn (maybe < 1%).  

_IF_ your plasmid has been linearized, it's much more likely that 
the SP6 polymerase is transcribing the plasmid's opposite strand 
non-specifically, by initiation at the end of the DNA. This is a 
characterized phenomenon for templates containing 3'-protuding 
sticky ends, as the polymerase will "sit down" on the protruding 3' 
end and start copying it (moving from 3' to 5' end of the strand 
being copied, i.e. 5' to 3' with respect to the strand being 
synthesized). I suspect the reference for this is in one of the 
original papers from Krieg & Melton on SP6 RNAP (Nucleic Acids 
Research, late 80's??).

Hope this helps.

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