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Is it possible to sequence rRNA directly, that is, using the ribosome rather than the DNA from the nucleus? For example, this paper, Complete nucleotide sequence of a 16s rRNA gene from E. coli, suggests that it uses DNA to find the sequence. Would it be possible to isolate the rRNA and derive the sequence from that?

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As far as I know it'spossible to reverse transcribe a rRNA gene, you may not get the best yields though. The secondary / tertiary structures will be a problem, however, there are reverse transcriptase's commercially available that can handle secondary structure: Thermo-X™ Reverse Transcriptase from Invitrogen or Sensiscript™ from Qiagen.

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  • $\begingroup$ Weirdly enough, Thermo-X is discontinued according to the website … $\endgroup$ – Konrad Rudolph Jul 20 '12 at 12:55
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    $\begingroup$ @KonradRudolph: Oh perhaps these can be used as alternatives then Thermoscript or Affinity Script $\endgroup$ – GWW Jul 21 '12 at 20:27
  • $\begingroup$ This answer does NOT answer the question, which is whether it is possible to sequence rRNA directly, rather than as DNA. $\endgroup$ – David Jan 20 at 22:12
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Unequivocally, YES.

Before DNA sequencing was used to deduce the sequence of cloned or reverse-transcribed RNA molecules, slower and more cumbersome direct sequencing of RNA was performed using the method of Sanger et al. (Sanger, F., Brownlee, G. G. and Barrel B. G. (1965) J. Mol. Biol. 13, pp. 373–398). This used ribonucleases in a manner somewhat similar to pre-Edmann protein sequencing.

Because of its abundance and ease of purification ribosomal RNA was a suitable species for this approach, and Carbon et al reported the complete sequence of E. coli 16S rRNA in 1978 (Carbon P., Ehresmann C., Ehresmann, B. and Ebel J.P. (1978) FEBS Letters 94, pp. 152-156).

Sigh! How little do modern molecular biologists know about the history of their subject.

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  • $\begingroup$ In addition to that most people doing (m)RNA/transcriptome sequencing nowadays actually worry about sequencing rRNA - there is so much more rRNA in cells that it needs to be thoroughly removed before you can even sequence any other RNA species. $\endgroup$ – Nicolai Jan 21 at 9:05

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