TAQ contaminated with DNA? (fwd)
rapr at MED.PITT.EDU
Wed Aug 24 14:11:13 EST 1994
> We are not talking a lot of DNA with the enzyme ie 10-20 genome
> equivalents. ie roughly I think 0.001pg. How can you measure DNA at
> those levels?
The answer IS (not obviously? :-)) via PCR!!!! But "10-20 genome equivs"
is not a meaningful number without "per something" e.g. "per unit of Taq".
And here's an interesting botch I recently did: I used PCR primers obtained
from a commercial source of human YAC strains to be used as positive controls
to show that the strains did in fact have YAC vector in them. The primers
gave real nice bands on the "no-DNA" controls. Turns out that the primers
were from a region of the YAC vector that was originally pBR322 vector,
and various additional control expts showed that the source of the contam.
pBR322 sequences was almost certainly the Taq itself, which I presume was
grown as a recombinant using a vector with pBR322 fragments in it. Dumb,
Along similar lines, while trying to use ribosomal RNA near-universal
primers for diagnostics development, I had to jump through every hoop on
the planet to get clean "no DNA" controls. Ultimately the solution to
that problem, for me, was to give the project to someone else. But in
the meantime I had found that Promega RNase-free DNase I could be used
to cleann up the PCR master-mix (excluding only primers and target DNA)
using a complicated Calcium-addition (activates and stabilizes DNase I)
then equimolar EDTA-addition (chelates calcium and magnesium to heat-
destabilize the DNase) prior to 65 degree 10 minute inactivation of
DNase. By a plasmid-supercoil nicking assay, DNase I turned out to be
remarkably heat-resistant unless there was no free Ca or Mg present.
I didn't publish that since there seem to be so many ways (see Biotech-
niques) to deal with endogenous DNA contamination already, but anyone
who feels like torturing himself with universal primers in PCR should
be aware that DNase I can be part of the apparatus.
rapr at med.pitt.edu
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