For cloning purpose when we design primers, we need to deal with the upstream and downstream sequence of the cDNA. could you please suggest a software which would be appropriate in doing that?
Hi Saugata,
I presume this is in an organism with a sequenced genome? If so, you can usually retrieve the upstream/downstream sequences (in different size windows such as 500bp, 1000bp etc.) from either an organism-specific datase (e.g. PomBase, FlyBase) or from a genome repository, e.g. UCSC genome browser, NCBI or similar.
If you're looking at an organism without a sequenced genome, things are obviously a little more complicated. If you're interested in 5'/3'UTRs, you could do a RACE (rapid amplification of cDNA ends) approach on the transcribed RNA. If you're interested in regions beyond the transcribed region, you've probably got to create a library of genome fragments and use colony PCR or something to select and sequence the constructs containing your gene of interest (there may be better ways to do this - it's not particularly my area).
Hope that helps!
Dan
Thank you very much for your answer Dan.
I'm not looking for retrieving information from a non sequenced genome and your answer is very well satisfy my requirement. I'm also aware of those processes.
Here my query is for satisfying different purpose. Actually, when new students start their journey in our group, they are not ready to handle those situation wise individualistic treatments. I was thinking about a software which could retrieve nucleotide sequence upstream or downstream, so that the concerned student wont have to go through the complex process.
Please see the attachment for example, in case of m.tuberculosis genes. I want to have a software like this which would work for any gene.
I only work in multicellular organisms but have found both EST datasets and predicted UTRs to be particularly unreliable and if it's necessary to have the correct UTR, I simply clone it with RACE. UCSC annotated genes usually take into account all available data and therefore can give a broad idea of the possibilities except many of its transcripts I have never managed to detect and remain curious about its false positive rate. If your species has a Refseq genome, this is usually the most trustable, but if your species isn't frequently checked and relies too much on EST datasets, again the UTRs are very often misannotated, particularly for long 3' UTRs. RNAseq data is very helpful. An older dataset called DBTSS did a pretty good job of getting some UTRs but struggled with longer transcripts. If your species has been subjected to CAGE analysis such as by the RIKEN group, those datasets are quite reliable. But a real problem is that both alternate TSSs and 3' UTRs are a frequent method of tissue specific regulation so there's no guarantee the UTR is the one used in your tissue if you're in a multicellular organism, and for many of my previous research projects, those differences have profound implications on experimental designs.
Alternatively, just to get a ball park of where a UTR might be, I've marched realtime PCR primers along where I think it might be to see where the signal drops off. However, this method requires considerable optimization (i.e. good quality standard curves usually with gDNA) to make sure the relative amplifications are correctly quantified. However, for genes that were hard to clone or I wanted to be assured that there were not some lesser abundant UTR expressed at an appreciable level, this can be helpful. Marching your reverse primer along the UTR in RACE can also give some reassurance to see the product size get smaller and disappear for primers beyond the UTR, which this method doesn't require actual sequencing of the clone.
Hi Saugata,
Jacob's answer is very thorough and covers a lot of good ideas. The only minor addendum I can make is to mention the cRACE (circularised RACE) protocol, which allows you to describe the 5' and 3' UTRs simultaneously; it also lets you do this in a paired-end fashion, so you can correlate alternative 5' ends with corresponding 3' ends etc.. I used this technique a lot during my Ph.D. and, while the circularisation step can be a little finicky, but it's powerful and useful. Let me know if you'd like the protocol.
cheers,
Dan
You may use Artemis genome browser:
http://www.sanger.ac.uk/science/tools/artemis
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