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Author:
Date:
2020-10-20
[Abstract] Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications ...
[摘要] [摘要] 对DNA结合蛋白和DNA切割蛋白的生化研究通常得益于放射性标记与两个DNA末端之一的特异性连接。在许多情况下，有必要进行两种版本的同一实验：一种是5′DNA末端标记，另一种是3′DNA末端标记。虽然均匀的5′-放射性标记可以通过单个激酶催化磷酸化步骤完成，但现有的3′-放射性标记程序通常会导致探针的异质性，从而妨碍了下游实验中精确的DNA片段鉴定。我们提出了一种新的方案，利用廉价的DNA寡核苷酸修饰（2′-O-甲基核糖核酸和核糖核酸糖取代）、两种酶（T4多核苷酸激酶和T4 RNA连接酶2），将32P磷酸有效地连接到任意序列的DNA寡核苷酸的3′端，以及DNA和RNA对氢氧化物处理的差异敏感性。该方法制备的放射性探针分子具有均一性和氧化剂相容性，可用于DNA酶特性和DNA足迹分析中的精确切割位点定位。
[背景] ...
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