It is best to have the full length UTR for reasons that you have already mentioned, but having smaller fragments does not hurt, it depends on what you are trying to extract out of these experiments.

We initially had the same problem as you did, cloning large DNA fragments especially 3`UTR's was challenging. However, we significantly modified our cloning protocol and now we can clone large fragments (upto 10kb) pretty seamlessly. The following are two important modifications that we made,

a. If you gel extract your DNA fragment before ligating it into your vector, crystal violet staining and visualizing under ambient light is preferable to EtBr staining and visualizing under UV.

b. Using high transformation efficiency competent cells that can accommodate large plasmids, like XL10 gold or sometimes even SURE2 (from stratagene / agilant), helps tremendously.

Best of luck!!

ok eleonora thanks for the heads up!

M

Hi Marilena,

I also tried to clone several Kb long 3' UTRs in the past , but never succeeded...

I got the feeling that 3' UTRs can be more challenging to PCR up and clone due to the AT rich environement that often does not allow for good primer design, especially at their 3' end. I used different high fidelity polymerases, but no luck....

I was trying to clone rat sequences, which are much less annotated than other organisms, especially outside of ORFs.

Your concerns re: 2D or 3D structures that may regulate the miRNA function cannot be completely ruled out, but you can just do the best that you can :-) Hypothetically, there may be miRNA function-regulatory sites all around the place, including ORF and 5' UTRs....So keep that in mind, but do what is the most pragmatic possibility and eventually discuss this in your publication, if appropriate.

Cut and paste jobs as suggested by Rafal could be a possibility, although I also finally opted for the limited cloning of some hundred bp for my reporter assays.

Good luck!

We usually clone the full 3'UTR, however when this is too big (7/8Kb) we split it in two parts. People publish the cloning (and the response to miRNAs) of fragments as small as 200 nt!!!

If you consider to study the overall regulation by multiple miRs you need to clone the full length. Otherwise if you want to show the response for a particular miR then I will suggest to clone smaller fragment. For functional regulation you need to study the target mRNA or protein upon over expression of your miR of interest. UTR regulation by RNA binding proteins, silencing complex and poly A binding protein are of great interest now a days may be secondary structure is playing a major role. For that you need to do RNA IP and reporter assays (according to the function of the target) to support your hypo. Please let me know you like it or not.

It is best to have the full length UTR for reasons that you have already mentioned, but having smaller fragments does not hurt, it depends on what you are trying to extract out of these experiments.

We initially had the same problem as you did, cloning large DNA fragments especially 3`UTR's was challenging. However, we significantly modified our cloning protocol and now we can clone large fragments (upto 10kb) pretty seamlessly. The following are two important modifications that we made,

a. If you gel extract your DNA fragment before ligating it into your vector, crystal violet staining and visualizing under ambient light is preferable to EtBr staining and visualizing under UV.

b. Using high transformation efficiency competent cells that can accommodate large plasmids, like XL10 gold or sometimes even SURE2 (from stratagene / agilant), helps tremendously.

Best of luck!!

Thanks all for your comments!

Hi, I just wanted to add one more comment. The luc assays are really to confirm that given miRNA does bind tSpecifically to it's respective target sequence, nothing more. You're right that the presence of the entire 3'UTR might affect the binding, but unless you have prior evidence (e.g. RNA folding, however inaccurate they might be) that access to the target sequence is hampered by secondary structures, if the miRNA binds to the target you should see some effect. It might be that the effect in the whole 3'UTR is different (either stronger or weaker) compared to the short sequence only.

Generally the 3'UTR is in one exon (maybe last exon), if so, you can PCR amplify the 3'UTR from genomic DNA and it should be easier. I will suggest using Herculase for the PCR amplification especially for the size of 4.6 kb. If there are other splicing sites in the 3'UTR, then 2 step PCR can be applied or cloning the smaller piece of 3'UTR containg the miRNA binding site(s) may be considered.

I agree with others that using the natural (i.e. full length) 3'UTR is preferable. Otherwise, how will you know if your observations are valid? One question though, if these very long 3' UTRs are so difficult to amplify, how have they been mapped in the first place? Is PCR amplification rather than RT extension and subcloning the problem? I'd be careful of very long region URTs identified by computational approaches only.

I´m trying also to clone several 3´-UTR, my problem is not the amplification, I succeed to do it by PCR. My real problem (is making me crazy) is to transform with the plasmid with 3-UTR. I can not get colonies. Any ideas what is happening?

One shot clonning we do in our lab by clontech/takara hd infusion kit. You will have clones in one week