Introduction of restriction sites via extended primers?

Peter Frank peter_frankde at
Fri Aug 13 13:07:19 EST 2004

"salli" <nospam at> wrote:

>I don't understand the last bit, where you say that AATT contributes less to
>annealing that G and C? This is of cause correct, however this overhang is
>NOT going to anneal to template - so do you include it when calculating
>annealing temp?

I am not the one who gave the answer but the one who started the
thread but I think what Kyle meant was that AATT contributes less to
the melting temperature of the original primers annealing to each
other. For the primer-template hybrids the overhang will be irrelevant
and not affect the annealing temperature. However, pimer dimers may be
more likely with GC-rich overhangs than with AT-rich overhangs because
as you know GC base pairs cause stronger binding than AT base pairs.


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