Theus S A, Liarakos C D
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock 72205.
Biotechniques. 1990 Nov;9(5):610-2, 614-5.
We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.
我们开发了一种检测方法，该方法使用苯基硼酸琼脂糖柱色谱法来测量用于克隆cDNA体外转录的RNA聚合酶的加帽效率。在帽类似物存在的情况下，通过用SP6或T7 RNA聚合酶进行体外径流转录合成带帽的32P标记的卵清蛋白mRNA，并用T1和T2核糖核酸酶完全消化。通过在苯基硼酸琼脂糖上进行色谱分离得到的3'-核苷单磷酸（NMP）和帽结构，两者之间的放射性比率用于估计转录本加帽的程度。
