Cannistraro V J, Strominger M B, Wice B M, Kennell D E
J Biochem Biophys Methods. 1985 Aug;11(2-3):153-61. doi: 10.1016/0165-022x(85)90051-x.
Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.
考虑了通过与固定在硝酸纤维素上的DNA连续杂交来纯化特定原核RNA的方法，并特别考虑了与T4多核苷酸激酶反应中后续末端标记相关的问题。(1)激酶抑制剂可能与质粒相关，但通过聚丙烯酰胺凝胶电泳分离DNA片段可将其去除。(2)残留的可溶性丙烯酰胺会污染DNA并阻止其有效保留在硝酸纤维素上，可通过DE52柱层析将其去除。(3)短的变性DNA需要高盐(0.9 M)才能与硝酸纤维素结合，但在这些盐浓度下会迅速复性，除非在4℃以1 ml/min的流速以小于或等于0.3微克/毫升的浓度加入。(4)杂交反应的动力学是DNA长度、浓度和温度的函数。(5)甲酰胺是比12 M尿素或8 M盐酸胍更有效的变性剂，可从滤膜上去除杂交RNA，但会导致DNA从硝酸纤维素上大量释放，同时也是激酶反应的另一种强效抑制剂。通过在沸水中洗脱，可大大减少DNA和其他激酶抑制剂的释放。
