Dyes and supercoiled DNA

Andy Bates bates at liv.ac.le
Mon Mar 6 04:35:25 EST 1995


In article <3j5v0m$7g3 at moe.cc.emory.edu>
Brad Thomas <bthomas at gmm.gen.emory.edu> writes:

> Are there dyes that can distinguish between supercolied DNA and relaxed
> circles and linear DNA in gels?

Brad,

The answer to your question is no, as far as I know, although
intercalaters like ethidium bromide bind with different affinity and
stoichiometry to supercoiled relaxed and linear DNA.  The best way to
distinguish between them is to do it by relative mobility, either with
EtBr in the gel, or without.  In the absence of EtBr (be careful to
wash your tanks out), supercoiled plasmid DNA will run ahead of relaxed
DNA since it is more compact.  Relaxed should run at approx. the same
position as nicked (or open circular) DNA (the normal contaminant of a
supercoiled plasmid prep) and will normally consist of a set of
topoisomer bands.  Linear normally runs between the two, usually close
to the nicked position. In the presence of saturating ethidium bromide,
it is difficult to distinguish between supercoiled and relaxed DNA, as
they both have high mobility.

If you need to carefully distinguish between different levels of
supercoiling, or positive and negative, etc, then the situation gets
more complex and you will need to start running gels with defined
levels of intercalater, usually chloroquine rather than ethidium
bromide, as it binds rather less tightly.

Get back to me privately if you need more advice, but at the risk of an
outrageous plug, this is discussed in more detail in 'DNA Topology' by 
Bates and Maxwell, IRL Press, 1993.  It may be in the library, so you
won't have to buy it :-).  Some discussion is in other lab manuals as
well.

Good luck, 

Andy Bates.
____________________________________________________________
Dr. Andrew D. Bates             Tel:   (+44) (0)151 794 4322
Department of Biochemistry      Fax:   (+44) (0)151 794 4349
University of Liverpool         Email: bates at liv.ac.uk
PO Box 147
Liverpool
L69 3BX, UK



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