Hamelin R
INSERM U 248, Faculté de Médecine Lariboisière-Saint-Louis, Paris, France.
Anal Biochem. 1988 Dec;175(2):500-6. doi: 10.1016/0003-2697(88)90574-x.
A simple method for mapping RNA on a Northern blot with a mixture of end-labeled DNA fragments is described. The DNA fragments are labeled either in 5' or in 3' directly after digestion by restriction enzyme(s) and used without any further purification step as probe to hybridize a Northern blot. After autoradiography, the DNA fragments hybridized to each mRNA species are recovered by heating the nitrocellulose and analyzed on denaturing polyacrylamide or agarose gels. This method indicates which DNA fragment hybridizes with which mRNA species and requires far fewer different manipulations than successive hybridization of a Northern blot with several nick-translated purified DNA fragments.
本文描述了一种使用末端标记的DNA片段混合物在Northern印迹上对RNA进行定位的简单方法。DNA片段在经限制酶消化后直接在5'或3'端进行标记,无需任何进一步的纯化步骤即可用作探针与Northern印迹杂交。放射自显影后,通过加热硝酸纤维素膜回收与每种mRNA杂交的DNA片段,并在变性聚丙烯酰胺或琼脂糖凝胶上进行分析。该方法可表明哪个DNA片段与哪种mRNA杂交,并且与使用几个缺口平移纯化的DNA片段对Northern印迹进行连续杂交相比,所需的不同操作要少得多。