Cannistraro V J, Wice B M, Kennell D E
J Biochem Biophys Methods. 1985 Aug;11(2-3):163-75. doi: 10.1016/0165-022x(85)90052-1.
T4 polynucleotide kinase has been used to end-label specific RNA purified by multiple hybridizations to nitrocellulose-bound DNA. The pico moles of ends of a specific mRNA transcribed from the chromosome, even from several liters of Escherichia coli, give concentrations perhaps 2000-fold below the Km value of the kinase-RNA substrate. In such a reaction, optimal incorporation was observed with increasing ATP concentration to greater than or equal to 7 microM (greater than or equal to 15 mCi of carrier-free [32P]ATP in a 300-500 microliter reaction). The unreacted ATP (greater than 150-fold excess) could best be eliminated by multiple gel filtrations rather than by precipitation, ion exchange chromatography or dialysis. The [5'-32P]RNA was digested with T1 or pancreatic RNase and the [5'-32P]oligonucleotides separated by size in a 20% polyacrylamide gel. Oligonucleotides of a specific size were separated sufficiently by a second dimension electrophoresis on cellulose acetate. We have used partial alkali digestion in sequencing the purified oligonucleotides. As opposed to other digestions, alkali produces 5',3'-diphospho-oligonucleotides whose mobilities can differ from those of the monophosphates, e.g., much longer running times in conventional homochromatography.
T4多核苷酸激酶已被用于对通过多次与硝酸纤维素结合的DNA杂交纯化的特定RNA进行末端标记。从染色体转录的特定mRNA的末端皮摩尔数，即使是来自几升大肠杆菌的，其浓度可能比激酶-RNA底物的Km值低2000倍左右。在这样的反应中，随着ATP浓度增加到大于或等于7微摩尔（在300-500微升反应中大于或等于15毫居里的无载体[32P]ATP），观察到最佳掺入。未反应的ATP（过量超过150倍）最好通过多次凝胶过滤而不是沉淀、离子交换色谱或透析来去除。[5'-32P]RNA用T1或胰核糖核酸酶消化，[5'-32P]寡核苷酸在20%聚丙烯酰胺凝胶中按大小分离。特定大小的寡核苷酸通过在醋酸纤维素上的二维电泳充分分离。我们在对纯化的寡核苷酸进行测序时使用了部分碱消化。与其他消化方法不同，碱产生5',3'-二磷酸寡核苷酸，其迁移率可能与单磷酸寡核苷酸不同，例如，在传统的同系层析中运行时间长得多。
