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Nucleic Acids Research, 1977, Vol. 4, No. 12 4091-4108
© 1977

Articles

The use of nuclease P1 in sequence analysis of end group labeled RNA

M. Silberklang*, A.M. Gillum+ and U.L. Rajbhandary

Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, USA

Received September 2, 1977.

A method is described for the direct sequence analysis 20–25 nucleotides from the terinini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinuim) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal 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 tbe sequences of 5' group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.

*Present address: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, Calif. 94143.

+Present address: Department of Pathology, Stanford University School of Medicine, Palo Alto, Calif. 94305.


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