Reverse Transcriptase

Michael van Waes bi__mvw at SELWAY.UMT.EDU
Thu Apr 7 18:41:52 EST 1994

>Can somebody help me? I have started sequencing ribosomal RNA from yeast. I
>have used several methods but now, I use the "Promega" protocol published
>in "Protocols and applications guide 1991". I have made lot of sequence
>gels but in all cases I see only just 120 nucleotides. I changed ddNTP/dNTP
>ratio, quantity of AMV RT (2 times more) but it didn't improve situation.
>In all my sequence gels I see on the top 4 lines in the same positions
>indicating that my reaction always stops. From the literature, I know that
>it is perhaps something with my RT. What can I do?
Well, welcome to the rRNA sequencing world!!! I know lots of people do it
without blinking, but it takes a lot of time.It is no doubt muuch more of
an art than sequencing other RNAs. You have two problems: The secondary
structure, and the modified bases. RT has a terrible time getting past two
methylated As in tandem at the 3' end, and all the other methylated bases
on the SSUrRNA. There's a BioTechniques article 13(5), Nov, 1992 describing
a protocol to sequence T7-transcribed 16S rRNA. This works very well for
unmodified RNAs, and it works also with methylated 16S, as long as your primer
is 5' of the two hellacious As (I am not sure where they fall in yeast), but
apparently it is about 120, according to your results :-). Another protocol
can be found in just about any of Noller's papers; and they use mostly native
RNAs. Regardless, you'll always get full stops throughout your gel, and there
is no way around it that I know of.

Cheers and Good Luck!!

		Michael vW.

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