End-labeling oligonucleotides with chemical tags after synthesis.
End-labeling oligonucleotides with chemical tags after synthesis.
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DOI：
10.1007/978-1-62703-113-4_14
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Ryder, Sean P
中科院分区：
文献类型：
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作者：
Zearfoss, N Ruth;Ryder, Sean P
许多确定核酸功能的实验策略都需要用放射性同位素或化学标签对核酸进行标记。标记能够实现核酸检测，提供有关其状态的信息，并且可以作为一种手段，借此从混合物中纯化核酸及相关因子。放射性磷酸盐通常在合成后利用酶催化反应添加到寡核苷酸的5′或3′末端。相比之下，化学标签通常在合成过程中添加，或者利用在合成过程中引入的反应性基团添加。在此，我们介绍了将化学标签在合成后与未修饰的RNA或DNA寡核苷酸偶联的实验方案。该方法可用于将荧光染料和生物素基团连接到寡核苷酸上，并将寡核苷酸固定在固体支持物上。用荧光染料标记的寡核苷酸在基于凝胶和酶标仪的检测中都很容易被检测到，而生物素或树脂偶联的寡核苷酸是亲和纯化的有用工具。荧光末端标记相较于放射性标记具有若干优势，它减少了与放射性相关的危害，产生一种不会衰变的标记分子，同时提供了许多操作所需的灵敏度。
Many experimental strategies for determining nucleic acid function require labeling the nucleic acid with radioisotopes or a chemical tag. Labels enable nucleic acid detection, yield information about its state, and can serve as a handle by which the nucleic acid and associated factors can be purified from a mixture. Radioactive phosphate is commonly added to the 5′ or 3′ end of an oligonucleotide post-synthesis using enzyme-catalyzed reactions. In contrast, chemical tags are usually added during synthesis or using reactive groups that are incorporated during synthesis. Here, we present protocols for post-synthetic conjugation of chemical tags to unmodified RNA or DNA oligonucleotides. The approach can be used to attach fluorescent dyes and biotin groups to oligonucleotides, and to immobilize oligonucleotides to a solid support. Oligonucleotides tagged with fluorescent dyes are readily detected in both gel- and plate reader-based assays, while biotin or resin conjugated oligonucleotides are useful tools for affinity purification. Fluorescent end-labeling provides several advantages over radioactive labeling, reducing radioactivity-associated hazards and yielding a labeled molecule that does not decay, while providing the sensitivity required for many procedures.