Uridines in linker design for miRNA cloning. - Why uridine is often used in the 3´-Linkers. (May/10/2007 )

Hi everyone!

By reading different papers about novel methods for cloning of miRNA I have observed that 3´-Linkers are designed with three uridine bases at the 5´-arm. The first uridine is phosphorylated.
I cant find any explanation for why the linker is a RNA/DNA hybrid.

Mostly these linkers are used in direct Reverse Transcription without any 5´-Linker ligated to the RNA of interest.
The 5´-Linker is aligned to the ccc- overhang produced by the RT enzyme and the transcription is continued there after.
This is used in Smart and Super Smart PCR cDNA synthesis kit.

Will this cloning approach work with a 3´-Linker designed without these three uridines?



Example of adoptors used:
5´-pUUUaaccgcatccttctcx-3´ x-inverted deoxythymidine
5´-(Pu)uuAACCGCGAATTCCAG(idT)-3´ idT-inverted deoxythymidine

Related Papers:
http://nar.oxfordjournals.org/cgi/content/full/gkl653v1
http://www.pubmedcentral.nih.gov/articlere...gi?artid=519194

Thanks for any help.

Hi.

I got some response about my qestions, and want to share it with you.

The uridine bases at the 5´ end of the linker are there in order to make it easier for the RNA-ligase to preform the ligation.
Using a 3´-Linker without uridines can result in lower yield of ligated product.

Thats all information that I got about it.