cDNA or genomic DNA for over expression in Arabidopsis - (Feb/07/2012 )
I have to over express quite a few genes in Arabidopsis. I am planning to go ahead with cDNA of gene. But I am little worried if I will be in trouble because some genes has some regulatory sequences in introns as well. So is it safe to use genomic DNA? Anybody faced any problem by using cDNA ? Please let me know if you can help with some suggestions. Thanks in advance.....
I our lab we’ve used both cDNA and CDS for overexpression in Arabidopsis, and both seem to work fine.
The cDNA shouldn’t contain much in the way of regulatory elements, as all of the introns will have been spliced out of the mature mRNA that is used as the template (there may be a small percentage of cDNA sequences that were synthesised from immature RNA and thus contain introns, but this should be a very small minority).
You will have the 5’ and 3’ untranslated regions that may contain some regulatory sequences, but I doubt that they would have much effect on overexpression.
My personal preference is to use the CDS, because then you know you’re working with the protein coding sequence of the gene and nothing else.
I also like using the CDS because then you can sub-clone into vectors with C- and N-terminal fusions without having the UTR sequence in-between.
Hope this helps!