Designing primers

Dave Knorr dak at apldbio.com
Wed Feb 21 15:55:08 EST 1996


In article <hasoar-1802961550460001 at lucky118.nuts.nwu.edu> Haunani A.
Soares, hasoar at nwu.edu writes:

>You may have heard that you should avoid AT pairing at the end of the
>primer. A GC pair would be a better anchor because it forms 3 hydrogen
>bonds whereas the AT pair forms 2 bonds.

On the other hand, "false priming" can occur if the 3' terminus of the
primer can anchor it to the template.  I have been designing primers by
computer (a largely theoretical exercise) that are more stable at the 5'
terminus and will therefore only prime synthesis if the correct 3' match
is made.  The idea is that you may take a hit in yield, but the
specificity of the priming should increase.  Using the correct
amplification conditions results in a robust amplification.
>
>Finally, if you want BOTH your primers to perform optimally under one
>reaction condition, it makes sense that both primers have similar Tm's. 
>I'm sorry to say that I've not tried PCR with primers that have very
>different Tm's.  I'll bet it would be very inefficient program.  

Although I didn't design it that way, I have performed PCR with two
primers calculated to have a 25 degree (C) difference in Tm.  Amazingly
it worked, although it looked to me to be similar to an asymetric
amplification.

Dave Knorr



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