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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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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, 2019 at 9:05

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