themoon at mars.com
Thu Jul 27 23:52:58 EST 2000
i dont know much im new to all this too. but are you talking about when u have
some cDNA and want to amplify it? so you know the sequence of the coding
strand, its given. the other strand is not given. so you want to design primers
to amplify part of the sequence? you can look in the coding strand and since
the sequence is right in front of you you just find the part you want to prime
and that gives you the sequence of the first primer. the other primer is going
to go on the other strand, and you dont have the sequence for that, but it can
be figured out from the fact that its complementary to the one you have. so you
write that out and find the part you want to prime (the other side of the
sequence you want to amplify). and you have to invert it because the other
strand runs 5'-3' in the other direction. is that what youre talking about? the
only reason you have to translate to complementary sequence and invert for the
second primer is that you were only given one strand's sequence. if you were
given both, the situation would be the same for both primers....
is this what youre talking about? if not, ignore all this crap....
> Hi everybody!
> First excuse me for this very basic question, but I'm a beginner with the
> PCR technique.
> It is possible to find the location of upper and lower primers in the gene
> sequence in order to know if or not they will generate a PCR product
> included in one exon or covering two exons.
> For the upper primer, we have just to find it directly in the gene sequence,
> but for the lower primer it is necessary to "translate" the primer sequence
> in complementary bases and after to invert it before searching it in the
> gene sequence. Why the upper ca be find directly and not the lower ?
> I can't stay more time without undertanding this basic point of PCR
> technique .
> Thanks in advance !
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