David F. Spencer
dspencer at is.dal.ca
Thu Jul 17 09:56:11 EST 1997
In article <33CCE30A.518 at molbio.uoregon.edu>, Matt Thomas
<Thomas at molbio.uoregon.edu> wrote:
> Does anyone know of conditions where RNase T1 will cut other then after
> a G? IT is advertised that it cuts only after G's but I have some
> strange digestion patternes which suggest sometimes it cuts weird. The
> DNA sequence that I make the RNA is fine, and I'm checking the RNA
> directly with primer extension, but I want to rule out that it is the T1
> that is doing strange things.
In the right (or wrong, depending on your perspective) conditions T1 will
do what is called overcutting. This feature was actually exploited by the
original RNA "sequencing" techniques, T1 oligonucleotide cataloguing and
fingerprinting. I did a little of this in late '78 - early '79 while
getting set up for "modern"
RNA sequencing using enzymatic and/or chemical degradation of end-labelled RNA.
Normally T1 works best at about pH 5.0 and with lower quantities of enzyme.
If you use excess amounts of T1 and neutral or above pH (we did the
reaction in water I believe) then T1 will cut (still as an endonuclease)
after anything but 'C's'. Therefore you will get oligos beginning with 'C'
(or 'C's') and ending in any of the other 3 nucleotides. The only exception
will be the original 5' end of the target RNA (or RNA's) which won't change
You may be trying to force complete digestion of your RNA by using too much
enzyme and have inadvertently produced overcutting. You should also know
that if your goal is to digest RNA to completion (i.e. after every 'G')
using T1 you will quickly discover that this never works; you will always
end up with some partials. The best way to get maximal T1 digestion is to
use lower amounts of enzyme for long periods of time. T1 is extremely
tough and will survive many hours (maybe days) at 65 C in 8M urea; the
normal buffer is 20 mM citrate-Na, pH 5.0. I assume you are aware that T1
leaves 3' (occasionally 2'-'3 cyclic) phosphates and 5' hydroxyls so if the
digested RNA is run on a gel its mobility may be slightly different from
what you expect.
David F. Spencer, PhD
Dept. Of Biochemistry
Halifax, Nova Scotia
dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca
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