Nucleic Acids Research, 1978, Vol. 5, No. 1 37-56
© 1978
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Sequence analysis of 5'[32P labeled mRNA and tRNA using polyacrylamide gel electrophoresis
*Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139 +;V. A. Hospital, and Departments of Biological Chemistry and Medicine, College of Medicine, University of Cincinnati Cincinnati, OH 45267, USA
Received November 2, 1977. Sequence analysis of 5'-[32P] tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described.
5'-[32P] Labeled rabbit ß-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1c-RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2 RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60–80 nucleotides from the 5' -end of the RNA determined.
Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.
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