Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-chloroform extraction step during 5′-end labeling with polynucleotide kinase and γ-³²P]ATP; (b) ZnSO₄ inactivation of RNase T₁ results in a highly efficient procedure for 3′-end labeling with T4 ligase and 5′-³²P]pCp; and (c) a rapid 4-min procedure for variable quantity range of ¹²⁵I and RNA results in a qualitative and quantitative sample for high-molecular weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.