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...

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Veröffentlicht in:BioTechniques. - 1988. - 19(1995), 3 vom: 02. Sept., Seite 416-20, 422-5
1. Verfasser: Thomson, J G (VerfasserIn)
Weitere Verfasser: Gershon, P D
Format: Aufsatz
Sprache:English
Veröffentlicht: 1995
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Research Support, Non-U.S. Gov't Technical Report Cations, Divalent Deoxyadenine Nucleotides Deoxyadenosines Phosphorus Radioisotopes Manganese 42Z2K6ZL8P RNA 63231-63-0 mehr... Polynucleotide Adenylyltransferase EC 2.7.7.19 cordycepin GZ8VF4M2J8 Magnesium I38ZP9992A 2'-deoxyadenosine triphosphate K8KCC8SH6N
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245 1 0 |a Use of vaccinia virus poly(A) polymerase for RNA 3'-end labeling with a chain-terminating nucleotide or a short 3' homopolymer tract 
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520 |a 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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