Assume we are given an RNA sequence of, say, 300 bps.
AACAGCUAGCUGAUCGAUGCUAGCUG....ACACUGUACG
and now we want to reverse transcribe either the 5' or 3' end in vivo, but not the entire transcript, but rather just a part up to a certain point.
This has to be sequence specific, i.e., no or just few other RNAs in the cells should be affected. Also, we would be allowed to add "start" and "termination" sequences, i.e., assume we would like to amplify the 5' end of the aforementioned sequence and the start and termination sequence would look like "agucc" resp. "ugccaa", then it would be possible for us to extend the above sequence like
aguccAACAGCUAGCUGAUCGAUGCUugccaaAGCUG....ACACUGUACG
or so to ensure that just the right part gets reverse transcribed (either with or without the start or termination sequences, that would not be so important).
Do you know a way to achieve that?
Many thanks in advance!
Some kind of context would help! If you could clearly describe your end goals, helping you out would be a little easier.
To perform RT on only a specific sequence you need to have a sequence specific primer. You can't just 'insert' sequences into RNA from which you can then prime the RT reaction. Also, If you could let me know what a termination sequence is I would be grateful?
Some clarity please.
Yeah...context would be great.
Not sure why you don't just do general RT, and then PCR up your fragment with primers that add the 5' and 3' sequences you want. Unless you're trying to control copy number or something else.
But... Sequence specific RT primer (these sometimes suck) with a second primer/probe for where you want the RT to end. The second primer will have a 3' block on it (missing OH) so that it's unable to be used by RT as a primer, but will instead act as a road block (maybe). Would need to use Tth polymerase as your RT enzyme here, as it has no strand displacement. Other RT enzymes are strong strand displacers.
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