APOE Pcr

Timo Hiltunen hiltunen at convex.csc.FI
Mon Mar 21 02:23:59 EST 1994


Hi!

I have had similar kind of false bands with GC-rich primers, and
I think they might arise from DNA polymerase bound DNA.
I have overcome the problem with a high annealing tempeerature and
a low number of cycles.

Timo Hiltunen
Univ. of Tampere
Finland


In <1994Mar13.003149.3674 at Virginia.EDU> gr3k at Virginia.EDU 
(Gregory P. Riddick) writes:

>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
>added.
>  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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