The use of nuclease P- | in sequence analysis of end group labeled RNA

A method is described for the direct sequence analysis^of 20-25 nucleotides from the termini of 5'- or 3'-end-group [ P] labeled RNA. The method involves partial endonucleolytic di-1 gestion of the labeled RNA with nuclease P ^ (from Penicillium citrinum) followed by separation of the partial digestion pro-ducts by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the ter-minal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequences of 5'-end group labeled oligonucleo-tides, containing modified bases, derived from T ^ or pancreatic RNase digestion of tRNA