Instruction manual

presence of two or more melting domains. This is caused by the melting of the first domain
which leads to an almost complete stop in the gel migration. This can be resolved by increasing
the run time so that higher melting domains can be seen.
Fig. 3.2. Perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular
to the electrophoresis direction. Mutant and wild-type alleles of exon 6 from the TP53 gene amplified
from primary breast carcinomas and separated by perpendicular DGGE (0-70% denaturant) run at
80 V for 2 hours at 56°C. The first two bands on the left are heteroduplexes and the other two bands are
the homoduplexes.
3.3 Parallel DGGE
For parallel DGGE, the boundary of denaturant concentrations are determined to be above
and below the melting of a given domain as seen in the perpendicular denaturing gel. Examples
for determining the denaturing concentrations are shown in Figure 3.3. Typically, a
difference of 25–30% stock denaturant from top to bottom, which is centered at the melting
point, is used.
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If the melting of two or more domains is seen in the perpendicular gel, then
two different gels with different gradients can be used to maximize separation.
Fig. 3.3. Example of a perpendicular denaturing gradient gels used for determining the denaturant
concentration range in a parallel DGGE gel. The DNA fragment melts at a denaturing concentration of
50% and a range of 35–65% denaturants can be used.
In parallel DGGE, the denaturant concentration increases from the top of the gel to the
bottom of the gel. With the parallel gel, it is possible to run more samples under the optimal
conditions. Gel combs are used to form wells in the gel and depending on the number of samples
needed to run, different combs with different number of wells can be used. Parallel gradient
0% 100%
Denaturant
Electrophoresis
35% 65%
50%
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