PCR Design

Jose de las Heras josenet at tiscali.co.uk
Mon Oct 9 05:35:43 EST 2006

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Two questions:

Is the Tm (melting temperature) of a primer calculated on the subsequence of 
the primer that binds to the template DNA? Why? because I have added 
restriction sites at the 5' end of the primer, which does not bind to the 
template DNA.
How is the Ta (annealing temperature) calcuated? from what I read, it should 
be 5 deg C less than the lowest Tm of the primer pair... but why?



regarding the melting temperature when you add restriction sites... sure, 
the first cycle you don't get annealing over that region... but on 
subsequent cycles you will.
I normally take into account the whole oligo (typically 14-16 or so bp plus 
the recognition site I want to add)

as for the annealing temperature... I'm not sure anybody does any "real" 
calculations. The Tm should be calculated considering things like salt 
concentration etc, and it would represent the temperature at which 50% of 
the sequences are annealed. With that in mind, if you use a slightly lower 
annealing temperature on the PCR, you're favouring annealing... the cost is 
increasing possibility of mispriming... but if the primers are well designed 
it's not normallya  problem. Conversely, you can go above the calculated Tm 
and still get a product. Sometimes a particular sequence is hard to amplify 
cleanly, and doing  a few cycles at higher Ta helps improve things a lot...

I odn't think it's an exact science, although you can calculate away your 
Tms, Tas and % of annealing with your particular buffer, in practice it's 
often easier use just the Tm as a guide... and optimise around it by trial 
and error. Most PCRs will work fine without much fiddling, and it's 
reccomeded to start witha  Ta a few degrees below Tm... and see what 
happens. Then change if necessary depending on the result.


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