Nichols Nicole M, Tabor Stanley, McReynolds Larry A
Curr Protoc Mol Biol. 2008 Oct;Chapter 3:Unit3.15. doi: 10.1002/0471142727.mb0315s84.
T4 RNA ligase 1 catalyzes the ATP-dependent covalent joining of single-stranded 5'-phosphoryl termini of DNA or RNA to single-stranded 3'-hydroxyl termini of DNA or RNA. T4 RNA ligase 2 also catalyzes the joining of a 3'-hydroxyl terminus of RNA to a 5'-phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double-stranded substrates. A truncated form of T4 RNA ligase 2 requires a pre-adenylated substrate for ligation. This unit describes specific reaction conditions, as well as applications such as radioactive labeling of the 3' termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning.
T4 RNA连接酶1催化DNA或RNA的单链5'-磷酸末端与DNA或RNA的单链3'-羟基末端的ATP依赖性共价连接。T4 RNA连接酶2也催化RNA的3'-羟基末端与5'-磷酸化的RNA或DNA的连接；与T4 RNA连接酶1不同，该酶更喜欢双链底物。T4 RNA连接酶2的截短形式需要预腺苷酸化的底物进行连接。本单元描述了具体的反应条件，以及诸如RNA 3'末端的放射性标记、寡脱氧核糖核苷酸和寡核糖核苷酸的环化、寡聚物连接和切口修复、创建杂交和嵌合DNA/RNA分子以及miRNA克隆等应用。
