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Nucleic Acids Research, 1990, Vol. 18, No. 4 879-884
© 1990

MOLECULAR BIOLOGY

RNA sequencing using fluorescent-labeled dideoxynucleotides and automated fluorescence detection

Günter J. Bauer

Max-Planck Institut für Biophysikalische Chemie, Abteilung Biochemische Kinetik Postfach 968, D-3400 GOttingen, FAG

Received November 9, 1989. Revised January 3, 1990. Accepted January 3, 1990.

Although dideoxy terminated sequencing of RNA, using reverse transcriptase and oligodeoxynucleotide primers, is now a well established method, the accuracy is limited by sequence ambiguities due to unspecific chain termination events. A protocol is described which circumvents these ambiguities by using fluorescence labels tagged to dideoxy nucleotides. Only chain terminations caused by dideoxynucleotides were detected while premature terminated cDNA's remain undetectable. In addition, the remaining multiple signals at nucleotide positions can be assigned to sequence heterogeneities within the RNA sequence to be determined.


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