Biology Reference
In-Depth Information
Programmed Electrophoresis
Even though the two complementary strands of most plasmids have very
similar molecular weights, they may have different folding configurations
when separated from each other. Therefore, after running agarose gel
electrophoresis in one direction for a certain period of time, we can reverse
the electrodes and run it in the other direction. The duration of each run can
vary by changing the current or voltage. In addition, the condition of the
buffer can also be adjusted. The configuration of one of the two
complementary circles may then move faster. Thus, current, tune, pH,
direction, and other factors of the run can be altered in order to get the best
separation of the two bands. Depending on the physical length of the
agarose gel, we can have a programmed electrophoresis for plasmid
molecules of different molecular weights. If this idea works, the separated
bands can again be concentrated and analyzed, or be hybridized with various
probes.
Restriction Enzyme Activities
It has been noted that most restriction enzymes can digest linearized DNA
molecules faster than DNA supercoils. A careful quantitative analysis of
these enzymatic activity may support the idea that linearized DNA
molecules are in the conventional double helical configuration and that
restriction enzymes usually bind to double helices. On the other hand, DNA
supercoils can exist in the four-stranded structure as discussed before, and
may not bind these enzymes effectively. However, local conversion from
one structure to the other in equilibrium can give rise to reduced rates of
digestion.
Binding of Topo-isomerase I
During recent years, topo-isomerase I is commercially available.
Presumably, in the near future, other topo-isomerases, as well as gyrases,
helicases, etc. will also be available to the molecular biology community.
Their interactions with DNA supercoils and linearized DNA molecules can
then be investigated carefully. For example, in the presence of mRNA,
intact pUC19 molecules give two bands on agarose gel electrophoresis.
