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!

Kind regards,

Raymond Chawla
Post-Graduate Student
Biochemistry Department
University College Dublin
Ireland

rchawla at ollamh.ucd.ie



More information about the Methods mailing list