Dr. Peter Gegenheimer
PGegen at kuhub.cc.ukans.edu
Sun Mar 30 02:59:45 EST 1997
In <5hcqh8$gcl$1 at d2.tufts.edu>, dsarraci at emerald.tufts.edu (David Sarracino) writes:
> I was trying to sequence a RNA strand and was curious if any RNA
>single strand specific, non sequence specific, exonucleases existed,
>either 3'-5' cutting of 5'-3' cutting.
Both Ribonuclease II (RNase II) of E. coli, and polynucleotide phosphorylase (PNPase), from many bacteria, are _relatively_ non-sequence-specific 3'-->5' exonucleases. PNPase is commercially available, I believe. (Note: the reaction is RNA(n)+Pi-->RNA(n-1) + ppN.)
I see no a priori reason, however, why you need an RNase, unless you have DNA present which must be protected. Snake venom exonuclease (3'-->5') and spleen exonuclease (5'-->3') are commercially available, extremely well-studied, and have been extensively used for direct RNA and DNA sequencing in the "classical" period (B.G.S., before Gilbert & Sanger--although it was Sanger who used them for RNA sequencing). Both exonucleases require a free terminal hydroxyl (at the end from which they start). The only drawbacks are that commercial prep'n's have not been squeaky clean (phosphatase contamination) and they don't work well for long stretches of RNA. Indeed, I once planned a method of RNA sequence determination based on spleen and venom exo degradation, but gave it up after learning of the enzymatic problems.
(Another technique, if your RNA is 5' end-labelled, is wandering spot sequencing with P1 nuclease (a single-strand specific, non-base-selective, RNA/DNA endonuclease). A very limited digestion with P1 will give you a ladder of cleavage products, which can be separated by 2-D electrophoresis/chromatography. Of course, this is much more work than simple base-specific gel labber sequencing.)
Hope this helps...
| Dr. Peter Gegenheimer | Vox: 913-864-3939 FAX: 913-864-5321 |
| Departments of Biochemistry | PGegen at UKans.edu |
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