primer annealing (View forum version)


Posted 07 January 2010 - 10:23 AM

Dear all,

I was wondering why higher annealing temperatures give less aspecific binding of primers?

I would rather think that the higher the temperature, the more energy there is, the easier a primer would bind wrong bases.


Posted 07 January 2010 - 11:18 AM

the easier a bad match can release.


Posted 07 January 2010 - 03:29 PM

Look at it like this...

if you are climbing a tree - is it easier to hold on when it is still (low energy) or when it is shaking in the wind (high energy)?

In this metaphor you holding on is equivalent to the chemical bonds between molecules and still and shaking are cold and hot respectively.


Posted 08 January 2010 - 11:28 AM

I see thanks.

However one more question: the annealing of the primer against the template doesnt require any extra energy then (you do not need to input heat to make them bound?)
This seems logical, knowing that the more energy the less mismatch you would have.
(the worser the binding is, the more mismatch, the easier it is to remove the bindings)

Or ?


Posted 10 January 2010 - 04:22 PM

Its not quite that simple I'm afraid. The reactions are largely not controlled by exo- or endo-thermic properties. The pairing is by hydrogen bonding - purine binding to pyrimidine - and stacking interactions stabilize the DNA. It is more that the mis-matches are energetically unfavourable because of inter-molecular distances.

At higher energy, only the ideal interactions will hold together, as the molecules move around more, whereas at lower temperatures the DNA can mis-pair through any interaction doing the job.