Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 15)

Section 4
Denaturing Gel Electrophoresis (DGGE, CDGE, TTGE)
4.1 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing Gradient Gel Electrophoresis (DGGE) is an electrophoretic method to identify
single base changes in a segment of DNA. Separation techniques on which DGGE is based were
first described by Fischer and Lerman.
2 In a denaturing gradient acrylamide gel,
double-stranded DNA is subjected to an increasing denaturant environment and will melt in
discrete segments called "melting domains". The melting temperature (T
m
) of these domains is
sequence-specific. When the T
m
of the lowest melting domain is reached, the DNA will become
partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in
a polyacrylamide gel. Since the T
m
of a particular melting domain is sequence-specific, the
presence of a mutation will alter the melting profile of that DNA when compared to wild-type. DNA
containing mutations will encounter mobility shifts at different positions in the gel than the
wild-type. If the fragment completely denatures, then migration again becomes a function of
size (Figure 4.1).
Fig. 4.1. An example of DNA melting properties in a perpendicular denaturing gradient gel. At a low concen-
tration of denaturant, the DNA fragment remains double-stranded, but as the concentration of denaturant increases,
the DNA fragment begins to melt. Then, at very high concentrations of denaturant, the DNA fragment can completely
melt, creating two single strands.
In DGGE, the denaturing environment is created by a combination of uniform temperature,
typically between 50 and 65 °C and a linear denaturant gradient formed with urea and
formamide. A solution of 100% chemical denaturant consists of 7 M urea and 40% formamide.
The denaturing gradient may be formed perpendicular or parallel to the direction of
electrophoresis. A perpendicular gradient gel, in which the gradient is perpendicular to the
electric field, typically uses a broad denaturing gradient range, such as 0–100% or 20–70%.
2
In parallel DGGE, the denaturing gradient is parallel to the electric field, and the range of
denaturant is narrowed to allow better separation of fragments.
9 Examples of perpendicular
and parallel denaturing gradient gels with homoduplex and heteroduplex fragments are shown
in Figure 4.2.
11
Single strands
Double strand
Partially melted
“wild type”
Partially melted
“mutant”
* Wild Type
Mutant
*
0% 100%
Denaturant
Electrophoresis
Double strand
Single strands
Denaturant
Wild Type
Mutant
100% 0%
Partially melted
"wild type"
Partially melted
"mutant"
Electrophoresis