how to determine protected species size exactly in RPA ?

[Dr. Peter Gegenheimer]

On Tue, 7 Oct 1997 15:35:04, Colin Dolphin <c.t.dolphin at qmw.ac.uk> 
wrote:

> In the past, I have used RPA to determine the transcriptional start
> point(s) (TSP) of a gene by running the resulting protected species on a
> sequencing gel alongside a DNA ladder. However, as the ssDNA fragments do
> not migrate equally with their ssRNA countertparts this makes accurate
> sizing of the protcted species difficult. Having perused some papers on
  
  ** RNA molecules normally run about 5% SLOWER in 7-8M urea gel 
than do ss-DNA fragments of the same chain length. In other words, 
an RNA species which co-electrophoreses with a 200-nt DNA marker 
will actually be about 190 nt (0.95 * 200)long. This isn't perfect, 
however. 

> So, how can I determine exactly the size of the protected species in RPA
> ? Is there a way of generating an RNA sequence ladder - say from
> pBluscript using T7 pol and deoxyNTPs ? (I think there are a few old refs
> to such a technique and I believe Pharmacia may once have provided some
> sort of kit).

 ** This is reasonable if T7 RNAP will incorporate the dNTPs (it 
will not extend a dNTP under normal conditions). Guido Krupp has 
published several papers on incorporation of modified NTPs by T7 
RNAP.  However, if it worked, but I doubt whether you'd get a 
uniform ladder extending over 100's of bases. Furthermore, the RNA 
fragments (terminated with 3-OH) will not co-migrate with the 
products of RNase treatment (terminated with 3'-OPO3 or 2',3' cyclic
PO4).

The simplest approaches, and ones that I know work, are:
  For relatively small protected fragments (say <100 nt), heat your 
labelled probe for varying times (say 1, 2, 5, 10 min) at 90 deg. C 
in 50 mM bicarbonate buffer at pH 10. This generates a ladder of 
3'-P-terminated fragments.

  For longer markers, digest the plasmid encoding your probe RNA 
with various restriction enzymes which linearize it at different 
distances downstream of the T7 promoter. After deproteinization, 
these templates can be mixed together and transcribed to give a 
mixture of RNA markers. They'll be 3'-OH terminated, and won't give 
single-base resolution. 

If you really want to experiment, you can try 1) making cold marker 
RNAs from a transcription reaction primed with free guanosine or ApG
(5-8X excess over [GTP]). The RNAs will start with a 5'-OH which can
be kinased whenever you like. 2) Make cold marker RNAs by 
transcription, then 3'-end-label with RNA ligase and [32P]pCp. Might
be less efficient than kinasing, but will give 3'-P-terminated 
markers. 3) For the courageous: use nuclease P1 instead of RNase to 
digest the hybrids. P1 leaves RNAs with 3'-OH (and 5'-P). It's a 
member of the S1 nuclease-like enzymes, but is much cleaner and 
functions well at neutral pH (50 mM NH4OAC, pH not adjusted), up to 
50 deg.C, and without any added metal (the catalytic Zn ion is 
tightly bound). 

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| Dr. Peter Gegenheimer       |  Vox: 785-864-3939  FAX: 
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