Who is wrong with primer design? [long]
dknorr at lynxgen.com
Tue Aug 1 19:25:12 EST 2000
Thanks for the little summary. I shifted away from designing 3' GC clamps a
while back. I have designed dozens of PCR and TaqMan systems using a
combination of software packages. I first let the software choose a set of
primers and then I fine tune them. With Oligo(tm), it's fairly easy to
observe the predicted stability of the selected oligo. I try to set
stability from 5' high to 3' low, and therefore disregard the specific
nucleotides at either end. This (I suppose) leads to increased specificity,
but usually at some loss of yield. Finding these regions is not always
easy. I have also used another fine product called Amplify (thanks Bill
Engels, U of Wisc.) to help predict spurious products and to keep track of
my primer designs.
Another idea that I have adopted in designing PCRs comes from the folks at
PE. I hold the buffer conditions and the thermalcycling conditions
constant, and design the primers into that system. The criteria I set for
primers then is a certain predicted Tm, and a stability profile. it's
fairly straightforward, if a little tedious. I have a friend who has also
designed many PCR systems. His trick? He just looks at the sequence and
scribbles out a set of primers. Works pretty well most of the time.
As they say, "YMMV"
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