Are the UTR (5' and 3') regions of cDNA of an eukaryotic (Human) protein necessary to express them in prokaryotes (e.coli)? Am planning to do RNA isolation and obtain cDNA from them. To amplify cDNA using RT-PCR, should I design the primers including the 5' and 3' UTR regions along with coding region? Please help.
(this cDNA will be then transformed and expressed in E.coli using expression vectors)
Necessary? No.
Helpful? Possibly (but unlikely).
Actively detrimental? Also possibly (and more likely).
Most UTRs influence targeting, stability and regulation of a given transcript, and eukaryotic systems are vastly more sophisticated than prokaryotic systems.
Also, eukaryotic systems are monocistronic, so the UTR influences a single transcript. Prokaryotic polycistronic RNAs are necessarily not as nuanced.
It is far more likely that the UTRs will be detrimental (or just pointless) in a prokaryotic system, so as your first approach I would tend to omit them. Besides, if you're hoping to tag your protein (his or flag or HA or whatever), then you'll have to omit at least one anyway.
Hi Brindha,
the design of primers to do qRT-PCR assays will be only on exons. one of the primers need to be across a splice junction, while the amplicon needs to be the most 3' of the gene you can (RNA degradation starts at 5'end) and its length must be around 100-150bp to respect the delta delta Ct rules.
fred
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