3'-END LABELING OF RNA WITH RECOMBINANT YEAST POLY(A) POLYMERASE
3'-END LABELING OF RNA WITH RECOMBINANT YEAST POLY(A) POLYMERASE
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DOI：
10.1093/nar/21.12.2917
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发表时间：
1993-06-25
影响因子：
14.9
通讯作者：
KELLER, W
中科院分区：
文献类型：
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作者：
LINGNER, J;KELLER, W
两种常用的末端标记RNA分子的方法是用多核苷酸激酶标记5 '末端和用pCp和T4 RNA连接酶标记3'末端。我们在这里表明，RNA的3 '-末端也可以用链终止类似物虫草素5'-三磷酸（3 '-脱氧-ATP）标记，它是由聚（A）聚合酶添加的。对于合成的RNA，当核苷酸以限制浓度使用时，40%的虫草素被掺入，并且当虫草素5 '-三磷酸过量时，基本上所有的RNA在其3'-末端被修饰。反应在几分钟内完成，RNA产物是均匀的，适合于序列分析。标记效率因不同的RNA分子而异，与RNA连接酶不同。Poly（A）聚合酶优先标记较长的RNA分子，而T4 RNA连接酶更有效地标记较短的RNA分子。
Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.