Circular DNA & EthBr in electrophoresis
fujimoto at u.washington.edu
Wed Dec 13 16:28:49 EST 1995
zjons at vetbio.unizh.ch (Zophonias O. Jonsson) writes:
>In article <Pine.SOL.3.91.951212122819.20606A-100000 at tin>, "Mr. I.S.
>Viney" <iviney at hgmp.mrc.ac.uk> wrote:
>> I think this is an expected anomaly, the forms which migrate slightly
>> differently are topoisomers. Pretreatment with EtBr destroys the
>> differences in mobility, maybe someone else (better versed in DNa
>> topology!) can explain this clearly.
>EtdBr is an intercalating dye and in the process of intercalating it leads
>to "positive" supercoiling in contrast to the normal negative
>supercoiling. The bulk and positive charge of EtdBr slows down the DNA on
>gel and drowns out the difference between different topoisomers at high
>concentrations. At very low concentrations the differe may actually be
>increased. Then of course linear and nicked DNA can bind more EtdBr than
>covalently closed ds DNA circles, that's why nicked and linearized
>plasmids run slower than intact ones on gel. That about summarizes it.
A closed circular DNA at native superhelical density is more "compact"
than a linear DNA of the same length. The superhelical stress causes
the DNA to twist up into something resembling a rod. (To see this take
a piece of flexible tubing, and while holding the ends together slowly
twist one end. The tube will go from a circle to a figure 8 and then
to an interwound configuration.) As a result the supercoiled DNAs will
migrate more rapidly than the linear form in the _absence_ of an
intercalating dye. A nicked circle DNA cannot navigate the pores of
a gel as easily as a linear DNA and so migrates slower.
As intercalating dyes are added to the gel, the superhelical stress
in the supercoiled DNAs are relaxed. This starts to convert the
topology of the supercoiled DNAs into that of a nicked circle.
Ordinarily, although the different topoisomers of a circular DNA
have different topologies, they are not different enough be resolved
on a gel. However, as the concentration of the intercalating dye is
increased, and the superhelical stress in the DNA becomes small, the
differences in topology become large enough to be observable in a gel.
If you start with a sample with near zero superhelical density (as
I believe the original poster did) the differences in topology can
be resolved in a gel in the absence of an intercalator. As the gel
is saturated with an intercalator, the DNA will become wound up and
it may be that it is wound up enough (into an interwound configuration)
that the gel can no longer resolve the differences in their toplogies.
One final note, at low concentrations, the binding constant of an
intercalating dye should be larger for binding to a supercoiled DNA
than to the same DNA in its linear or nicked circular form. The
relaxation of the superhelical stress makes a favorable contribution
to the free energy of binding.
fujimoto at denali.chem.washington.edu
>For more take a look at:
>Keller, W. (1975) PNAS 72:1787-1791 or
>Foglesong, P.D. and C. Reckord (1992) BioTechniques 13:402-404
>or if you are really into it
>Bauer, W and J. Vinograd (1968) J.Mol.Biol. 33:141-171
>Zophonias O. Jonsson
>Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75
>Universitat Zurich-Irchel Fax: (41-1)-362-05-01
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