[Automated-sequencing] Re: T3 and T7 primers
Phillip San Miguel
(by pmiguel from purdue.edu)
Thu Oct 16 09:16:15 EST 2008
Buffet, Eleonore wrote:
> Hi I'm a student having a problem with some
> extra research I am conducting. I have come across numerous definitions
> as to the function(s) of T7 and T3. Could you please provide your expert
> opinion as to which are false?
> To transfer a plasmid to other bacteria
> Used as a priming site (my personal preference)
> Can be used to make ssRNA copies of DNA
> A promoter for cloned DNA expression
> Viral RNApol promoter
Your post is (at least partly) on topic for bionet.genome.autosequencing
so I'll answer.
As a sequencing core facility we are frequently bedeviled by these
promoters being used as sequence priming sites. I don't find T3 to be
much of a problem. But there are many variants of T7. Because some
vectors/constructs use one sequence and other use others, we always ask
what vector is being used before picking a T7 primer.
The problem arises because T7, T3 and SP6 were not originally designed
to be sequence priming sites. Rather they are sites where the RNA
polymerases of these respective phages will bind prior to initiation of
transcription. So, if you look at the T7 genome, you will find that the
T7 site is not perfectly conserved. Apparently T7 RNA polymerase is
forgiving of these minor difference.
However, DNA polymerases nearly always must be primed to function. If
the last base of your primer does not match the last base of the priming
site, the reaction will generally fail. (Here having a "dirty" DNA prep
could actually help. Contaminating nucleases could chew back the priming
oligo so it could be extended. Of course the sequence will likely be of
poor quality in such a situation. But arguably better than nothing.
Anyway, to answer your question... 4 of the 5 possibilities you present
are true. "Viral RNApol promoter" is the best, in my opinion, because it
describes their primary (evolved) function--the other 3 correct answers
are derived characteristics. The answer I can't make fit is "Transfer a
plasmid to another bacterium". Though it wouldn't surprise me if,
somehow, this one could be true also.
Good luck with your "research",
PS Yeah, I know it is bad form to answer what is obviously a student
homework assignment. But the traffic in this group is so low I couldn't
resist the temptation.
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