Thomson J G, Gershon P D
Institute of Biosciences and Technology, Texas A&M University, Houston, USA.
Biotechniques. 1995 Sep;19(3):416-20, 422-5.
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.
本文描述了使用痘苗病毒编码的聚（A）聚合酶（PAP），用链终止核苷酸32P 3'-dATP（3'-脱氧腺苷-5'-三磷酸）对RNA进行3'末端标记的条件。研究了反应时间、二价阳离子种类和浓度以及PAP两个亚基的需求。在Mn2+存在下，痘苗PAP能够用3'-寡聚（U）、寡聚（C）和寡聚（G）片段对RNA引物进行加尾。对添加标记的3'-同聚物片段的条件进行了表征。本研究中使用低核苷酸浓度揭示了一个明显固定的二价阳离子最佳浓度为0.1 mM，这与之前提到的对1:1二价阳离子：NTP复合物的需求不同。这表明痘苗PAP在核苷酸转移中可能需要多种二价阳离子。
