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Nucleic Acids Research, 1977, Vol. 4, No. 6 1957-1978
© 1977

Articles

Use of specific endonuclease cleavage in RNA sequencing

Ramesh C. Gupta and Kurt Randerath

Department of Pharmacology, Baylor College of Medicine, Texas Medical Center Houston, TX 77030, USA

Received March 18, 1977. Nonradioactive RNA fragments may be sequenced by incorporation of (3H)-label into 3'-terminal positions, controlled digestion with specific ribonucleases, and separation according to size of the digestion products on polyethyleneimine- (PEI-) cellulose thin layers. This combination of techniques allows one to measure accurately distances of specific cleavage sites from the labeled terminal positions. The cleavage specificities of RNases T1, U2, and A are utilized to identify the positions of G, A, and pyrimidine residues respectively. C and U may be distinguished by mobility differences on PEI-cellulose thin layers at pH 2.6. The procedure is simple, rapid, and highly sensitive; as little as 0.5 – 1 µg of a RNA of the size of tRNA will be needed to sequence all fragments in a complete RNase digest.


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