cloning/amplifying attenuate RNA transcripts - (Feb/09/2006 )
I want to visualise the lengths of attenuated mRNA transcripts that have been produced in cells following a drug treatment. I have an idea involving first-strand cDNA synthesis and then Southern blotting.
However, what would be better would be if it was possible to clone that sequence into a plasmid and then simply get it sequenced professionally. The problem is that whilst I know the exact sequence at the beginning of my cDNA molecule of interest I have no idea what the 3' sequence is (this is what I am trying to find out).
Anyone got any idea how I could clone this if I only know the sequence at one end? I'm not sure it's possible.
there's probably an easier way, but here's a thought (is this what you were already talking about?)
turn your RNA into cDNA
do a Southern
screen a library for overlapping clones
I am assuming your organism's genome is not yet sequenced?
That's pretty much what I had in mind though I don't really need to screen against a library. I'm doing this in human and I know the gene of interest already. I simply need to get as much detailed information on the exact base pair sequence at the attenuation site.
The idea is to determine consensus binding sites for novel DNA-binding compounds in intact cells.
Hi, I am not sure how much this may help but...
What about using a gene-specific primer (from your known sequence) to prime cDNA synthesis for RACE then do TA cloning and screen for different sized inserts and sequence? The only problem is that there are multiple reasons (other than reaching the end of an intact mRNA) that could lead to different sized inserts..
Here is where my knowledge fails me.. does attenuation produce capped mRNAs?? then you could combine cDNA synthesis with a gene specfiic primer and adapter primer addtion requiring only capped mRNAs to be amplified then TA clone... If there is not capping then you can use the additon of adapter primers not requiring capping, but in this case things like degraded mRNAs or early termination of the RT would not be easily distinguishable from the attenuation??
Does that make sense to you?
Hope that helps you, and good luck....
Sorry, I just delete my former post which state using 5’ and 3’RACE, since I feel misunderstand the concept of attenuation site.
While I still think you might perform 3’ RACE, instead of normal RNA, consider of using PAP treated RNA which might make attenuated mRNA transcripts can be reverse transcripted using polyT anchor primer. Then run gel check the size or clone into plasmid for sequencing.
I not sure my answer is make sense to your case...
Thanks for all the advice. I'll look the techniques with which I am unfamiliar.
Just to explain. The RNA transcripts are not capped in any way. What is happening is that transcription occurs as usual with the RNA polymerase migrating along the DNA strand. However, it will eventually meet a covalently-attached small molecule which causes the polymerase to dissociate and hence an attenuated RNA transcript is produced with absolutely no additional modifications.
I a trying to determine where (at what exact sequence) that small covalently-bound molecule has attached itself to the DNA.
Apart from PolyA tail technique, ligating an adaptor to your RNAs’ 3’end by RNA ligase might be another choice. Using primer based on adaptor sequence for reverse primer in 3’RACE, you will find exact ending of you target RNA after sequencing.