RNA sequencing with dye-deoxy-NTPs

colossus... s535290 at aix1.uottawa.ca
Sat Nov 15 15:55:15 EST 1997


> >Don't bother doing that. Just use do your first step of cDNA synthesis 
> >using RT, then sequence using the standard Taq-based kit. Just make sure
> >that the sequencing primers are complementary to the cDNA, and not to the
> >RNA. This is sort of like RT-PCR. I've never done it myself, but I'm
> >assuming that it should work.
> 
> have a look for early papers on sequencing 16s rRNAs. Alwys used to be
> done with RT. The RT will incorporate the dye terminators but I doubt
> the ratios supplied in cycle sequencing kits will work properly. RT is
> more error prone than Taq.
> 
> Duncan 

I think that is precisely the problem. ddNTPs are incorporated by RT,
but I don't think the dye terminators would, at least not with the
kinetics required. This is why the old kits required tons of dye
terminators. Taq affinity for the dye terminators was really low.
The engineered Taq has higher affinity for the dye terminators and can
thus handle not having a vast excess of dye terminators in the mix. 
Who knows how RT will perform on any of the commercially available cycle
sequencing kits. 

I think for automated sequencing of RNAs, you can do a cDNA
synthesis step first, and then follow that up with straight sequencing
of the cDNA with any standard kit.

Ed




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