RNA sequence determination in the nanogram range by a combination of in vitro labeling procedures. |
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Authors: | H Domdey H J Gross |
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Institution: | Max-Planck-Institut für Biochemie, D-8033 Martinsried bei München, West Germany |
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Abstract: | Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′-32P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T1 and RNase A, respectively. These digests were again 32P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments. |
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Keywords: | BD cellulose benzoylatedDEAE cellulose |
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