Question re DNA stability
RAYMOND CHAWLA (BIOCHEMISTRY) PG
RCHAWLA at ollamh.ucd.ie
Tue Sep 15 06:54:21 EST 1998
Hello fellow scientists,
I have a very perplexing problem and would be grateful for any
assistance/advice you can offer me.
I generate a 265 bp DNA fragment from the lac Z gene by PCR using a
plasmid as my source of template DNA. However when I perform
chemiluminescent detection of this fragment using a probe that is
exactly the same fragment but labelled with DIG I observe 2
fragments. The second fragment is exactly half the Mw. I proved
that this second band is not a PCR artifact by gel-excision, etc. I
have ascertained that it is in fact ssDNA as I performed a Southern
blot where I did NOT denature the DNA. In this case only ssDNA could
be detected. Why is the DNA denaturing?
There is an even more interesting twist to this story!! When I
perform 2-fold dilutions, with autoclaved HPLC grade water, of the PCR
mix and load the same onto an agarose gel I observe an increase in
the amount of ssDNA as the dilution factor increases!! When I
perform the dilutions in either TE buffer, pH 8.0 or Tris-HCl buffer,
pH 8.0 (i.e. TE buffer without the E) I no longer observe ssDNA. In
other words the buffer is able to cause the DNA to re-anneal.
I have seen images from other workers who also used this fragment.
They have observed the fragment but never bothered to try and explain
what it was. I can only think of two possible avenues to proceed
with this matter. The first is to use a different source of water
for both PCRing and diluting. However as this effect has been seen in
other labs I don't think this will help. The second idea is to
determine if the DNA is bending, etc. Is there some physical
sequence which if present in my particular sequence can cause it to
be prone to spontaneous denaturing?
Any references that might put me in the picture will be greatly
appreciated! I really don't want to be re-inventing the wheel and I
presume this effect has been seen before. (I can make copies of my
images available should this be necessary.)
Many thanks in anticipation of any help you may be able to afford me!
University College Dublin
rchawla at ollamh.ucd.ie
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