Gregory P. Riddick
gr3k at Virginia.EDU
Sat Mar 12 19:31:49 EST 1994
Our lab has been working on ampliflying the APOE gene.
Although we are getting the correct product of 220 bp,
anomalous products are showing up in the 75-130 bp range. We
have systematically varied [Mg], taq conc., primer conc.,
Dntp's, cycling time, and annealing temperature. A new set of
reagents has also been tried. We are using a P-E 9600 machine.
The primers are GC rich with 73% and 61%, so the annealing
temperature is set at 65 deg. Secondary structure must be a
problem because the wanted product only shows up when 10% DMSO is
The unwanted products show up without DNA template added, so I assumed that
contamination must be a problem. But new reagents didn't solve
the difficulty and a PCR of B-globin gene with the same
reagents was very clean.
Is it possible that the primers for APOE are self-priming?
They have 2 homologous regions of 5 bp (all GC), so I guess
it's a possibility. If so, how could I correct for this (aside
from finding new primers)?
If anyone has had experience amplifying APOE or has insight
into this problem, your help would be greatly appreciated.
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