An alternative explanation assumes reptation, which is an end-to-end movement of the DNA through the
pores of the gel. This theory explains the observed movement of DNA through gels more accurately, espe-
cially at high voltages where mobility becomes independent of molecular mass. In practice, the analysis of
observed mobility in gels is made by comparison with and extrapolation from known standard samples.
The topological conformation of a DNA sample can have a marked effect on the rate of migration and the
response of the sample to gel concentration and applied voltage. Relaxed circular DNA and supercoiled
DNA migrate anomalously in agarose gel electrophoresis in comparison to linear DNA of similar molecular
weight. The mobility of supercoiled DNA (Section 2.3.5) is also dependent on the linking number; as the
linking number increases, so does the mobility of the sample. In addition, its mobility changes with the
concentration of added ethidium bromide because increased intercalation of ethidium changes the number
of superhelical turns.
The shape of linear DNA also has a strong effect on electrophoretic mobility. Bends or curvature in the
DNA helix have the effect of slowing the migration relative to non-bent DNA. The extent of retardation
depends on the end-to-end distance of the DNA and so is dependent on the location of the bend within the
DNA helix (Figure 11.13). A qualitative theory, derived by Lumpkin and Zim, supports the general rule
that mobility is related to the mean square end-to-end distance. In addition to the above examples, many
elegant experiments in electrophoresis have been performed to examine the properties of poly(A) tracts,
extra-base bulges, protein binding (gel-shift) and Holliday junctions.^27
The fact that DNA of very high molecular mass does not separate well under normal electrophoretic
conditions has led to the development of pulsed field electrophoresis. There are several variations of the
technique, but in general the use of a voltage pulse with a resting period in between pulses changes
the mobility of large DNA fragments and this can be used to separate DNA species of 1000 kbp or larger. The
use of an alternating polarity field, with the positive pulse being longer than the negative pulse, enables
the efficient resolution of very large DNA fragments. The application of inhomogeneous, perpendicular
electric fields has been used to separate whole yeast chromosomes. In all of these applications, the size and
duration of pulses can be tuned to obtain the specific resolution required.
11.4.4 Microcalorimetr y
Calorimetry is a technique in which the heat of a reaction is measured. There are several types of calorimetry
available that utilise a variety of different physical principles. Two direct methods are available for measuring
Physical and Structural Techniques Applied to Nucleic Acids 443
Figure 11.13 The electrophoretic mobility of curved DNA is dependent on the exact location of the bend in the DNA
helix