Use of specific endonuclease cleavage in RNA sequencing.
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 microgram of a RNA of the size of tRNA will be needed to sequence all fragments in a complete RNase digest.
D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli; D008722:Methods; D009843:Oligoribonucleotides; D012343:RNA, Transfer; D012260:Ribonucleases; D015003:Yeasts
D009843:Oligoribonucleotides; D012343:RNA, Transfer; D004720:Endonucleases; D012260:Ribonucleases