T7 transcribed RNA probes

Duke Groebe drg at prophet.pharm.pitt.edu
Fri Jan 19 09:42:53 EST 1996


> : >From: Torsten Boerchers <borcher at uni-muenster.de>
> : >Subject:T7 transcribed RNA probes
> : >Date: 14 Jan 1996 13:45:46 GMT
> : >Message-ID:<4db1ea$1cpi at majestix.uni-muenster.de>
> 
> : >Hello Northern or in situ hybridization specialists
> : >
> : >Anybody else using T7 RNA polymerase to transcribe
> : >RNA from a DNA template (e.g. linearized plasmid with T7-
> : >promoter) who also observes multiple bands in a gel or blot?
> : >
> : >We use this technique for generation of DIG labelled 
> : >RNA probes and would like to know whether some 
> : >people have explanations for this behaviour or 
> : >are aware of published evidence.
> : >
> : >In some manuals it is emphasized not to allow 34overhangs
> : >in the restriction made for linearization. The overhang 
> : >could be an initiation point for the T7 RNA polymerase. 
> : >Any mechanism for this known?
> : >
> : >Thank you for any help
> : >
> : >Torsten


It had been my experience (awhile ago) that T7 RNA polymerase will add a
random nucleotide or two (preferably A, I think) to RNA transcripts from
either cut plasmid or from synthetic DNA templates.  There is no way around
this particular problem.  In addition, the polymerase "stutters" at tracts
of polyA and poyU (again, if memory serves), resulting in transcripts of
different lengths due to the different length of the polyN region.  Check
out early T7 polymerase stuff -

Milligan, J.F., Groebe, D.R., Witherell, G.W., & Uhlenbeck, O.C.
"Oligoribonucleotide Synthesis using T7 RNA Polymerase and Synthetic DNA
Templates" Nucleic Acids Res. (1987) 25, 8783-8798

Groebe, D.R. & Uhlenbeck, O.C. "Characterization of RNA Hairpin Loop
Stability" Nucleic Acids Res. (1988) 16, 11725-11735

Milligan JF.  Uhlenbeck OC "Synthesis of small RNAs using T7 RNA
polymerase" Methods in Enzymology (1989) 180:51-62. 



Duke

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