Technically you're right, but..
When the primers bind, polymerases tend to bind pretty quickly to the
loose 3' end of the primer, and immediately start extending. Sure,
they're not very fast at that temperature, but they have long enough to
make that short primer long enough that the melting temperature is much
higher by the time the next step rolls around.
Secondly, the melting temperature isn't the absolute temperature beyond
which no DNA is bound; it's calculated as something of a midway point
between "No dissociation" and "All primers dissociated", so there's a
lot of wiggle-room. I also imagine that once a primer has bound, it has
a certain intrinsic stability that helps keep it there a while longer.
Of course, longer primers usually still work fine and have a higher Tm.
On 27/01/12 16:43, Jonathan Nesser wrote:
> Sorry to start yet another new thread for such a small question, but I
> don't want to pollute other threads with off topic posts... I've been
> trying to understand why primer melting temperatures have to be in the
> 55-65C range when PCR cycles call for 94C for DNA melting... I've tried to
> find an explanation for this but haven't been able to... Wouldn't that
> result in the primers splitting off from the template DNA before you reach
> the extension cycle of 75C (or around there)? I'm sure I'm wrong because
> obviously these PCR programs work, but I don't know WHY I'm wrong, and
> would like to understand. Thanks again for explaining something that is
> probably so common knowledge that nobody feels the need to address it in
> writing :)
>
> Jonathan Nesser
> jonathan.nesser@gmail.com
> diybioandneurosci.blogspot.com
>
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