Nucleic Acids Research, 1977, Vol. 4, No. 8 2527-2538
© 1977
Articles |
Mapping adenines, guanines, and pyrimidines in RNA
Department of Biochemistry and Molecular Biology, Harvard University Cambridge, MA 02138, USA
Received June 3, 1977. The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeled RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.
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