Plant DNA PCR problems

Volker Knoop knoop_v at mpimg-berlin-dahlem.mpg.de
Mon Jun 19 14:51:40 EST 1995


Hi netters and colleagues,

   I've followed the discussion about PCR errors/mistakes/mutations in
this group lately and I wonder whether the inherent Taq pol (or whatever pol)
error rate is really the problem. Having prepared DNAs and RNAs of quite a lot
of plant species and amplifying part of a mitochondrial gene, we generally
do not
find any discrepancies between DNA and cDNA sequence (except for the C to U
type of plant mitochondrial RNA Editing in certain position and that is exactly
what we are looking for).
   Recently however, the nucleic acid preparation of a hornwort really gave us
hard time. The DNA looked perfectly nice on a gel but even after purifying it
in a CsCl gradient it was not amplifiable. The stuff even inhibited other
amplifications. The trick was finally to dilute the preparation (and thus
maybe dilute an apparent inhibitor) to finally obtain a product.
   Now that we've sequenced this, however, we find lots of discrepancies
in the sequences of different clones. My feeling is that something
copurifying with DNA
(or even a covalent modification?) inhibits the PCR reaction and/or makes
Taq pol more error prone. This is definitely not a problem of our reaction
setup since the same works perfectly with about two dozen other species.
   Ideas, comments, similar observations?

Volker.

-- 
Volker Knoop
IGF Berlin Dahlem
14195 Berlin
phone +49-30-830007-48 Fax -36
e-mail: KNOOP_V at MPIMG-BERLIN-DAHLEM.MPG.DE



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