Use of vaccinia virus poly(A) polymerase for RNA 3'-end labeling with a chain-terminating nucleotide or a short 3' homopolymer tract
Conditions are described for the 3'-end labeling of RNA with 32P 3'-dATP (3'-deoxyadenosine-5'-triphosphate), a chain-terminating nucleotide, using the poly(A) polymerase (PAP) encoded by vaccinia virus. Reaction time, divalent cation species and concentration, and the requiremen...
| Veröffentlicht in: | BioTechniques. - 1991. - 19(1995), 3 vom: 02. Sept., Seite 416-20, 422-5 |
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| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1995
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Research Support, Non-U.S. Gov't Technical Report Journal Article Cations, Divalent Deoxyadenine Nucleotides Deoxyadenosines Phosphorus Radioisotopes Manganese 42Z2K6ZL8P RNA mehr... |
| Zusammenfassung: | Conditions are described for the 3'-end labeling of RNA with 32P 3'-dATP (3'-deoxyadenosine-5'-triphosphate), a chain-terminating nucleotide, using the poly(A) polymerase (PAP) encoded by vaccinia virus. Reaction time, divalent cation species and concentration, and the requirement for both subunits of the PAP were investigated. In the presence of Mn2+, vaccinia PAP is able to tail RNA primers with tracts of 3'-oligo(U), oligo(C) and oligo(G). Conditions for the addition of labeled 3'-homopolymer tracts were characterized. The use of low nucleotide concentrations in this study revealed an apparently fixed divalent cation concentration optimum of 0.1 mM, distinct from the previously noted requirement for a 1:1 divalent cation:NTP complex. This indicates a possible requirement for multiple divalent cations in nucleotidyl transfer by vaccinia PAP |
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| Beschreibung: | Date Completed 18.01.1996 Date Revised 23.11.2016 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |