Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 12)

Section 4
Using WinMelt/MacMelt for Denaturing Gradient Gel
Electrophoresis
Calculating a melt profile for a wild-type or known sequence using the WinMelt/MacMelt
software is a good initial step in mutation detection using denaturing gel electrophoresis. The
term melt refers to the change in the structure of DNA from an orderly helix to a disordered
structure without base-pairs. The program calculates the midpoint temperature (T
m
) at which
each base-pair is at 50/50 equilibrium between the helical and melted states. This data is then
plotted, T
m
versus the sequence base, to create a melting profile. The goal of mutation
detection by denaturing gel electrophoresis is to manipulate the sequence so that the region of
interest is in the lowest melting domain. This requires the proper placement of GC clamps to
attain this. The GC clamp is typically 40 base-pairs in length and creates a region of high
melting temperature.
2, 3
After analyzing a sequence with MacMelt or WinMelt, the DNA samples are run on a
denaturing gradient gel for screening mutations within the sequence. Denaturing Gradient
Gel Electrophoresis (DGGE) is an electrophoretic method to identify single base changes in
a segment of DNA. The separation techniques on which DGGE is based were first described
by Fischer and Lerman.
4 DGGE samples can be run on the Bio-Rad DCODE denaturing gel
electrophoresis system for mutation detection.
When running a denaturing gradient gel, both the mutant and wild-type DNA fragments
are run on the same gel. This way one can detect a mutation by seeing a band shift on the gel.
The mutant and wild-type fragments are typically amplified by the polymerase chain reaction
(PCR) to make enough DNA to load on the gel. Optimal resolution is attained when the
molecules do not completely denature. The addition of a 30–40 base-pair GC clamp to one of
the PCR primers ensures that the region screened is in the lower melting domain and the
DNA will remain partially double-stranded.
Other methods that use the melt profile information from MacMelt or WinMelt include
Constant Denaturing Gel Electrophoresis (CDGE) and Temporal Temperature Gradient Gel
Electrophoresis (TTGE). CDGE is a modification of DGGE where the denaturant
concentration found to give optimal resolution from a parallel or perpendicular DGGE gel is
held constant.
5 A CDGE gel can be used to rapidly screen samples for the presence of a
mutation. TTGE exploits the principle upon which DGGE is based, without the requirement
for a chemical denaturing gradient. A gel containing a constant concentration of denaturants
is used and during electrophoresis, the temperature is increased gradually and uniformly. The
result is a linear temperature gradient over the time course of the electrophoresis run. Thus,
a denaturing environment is formed by the constant concentration of denaturants in the gel in
combination with the temporal temperature gradient.
A sequence from the betaglobin gene (exon 1) will be used to illustrate how the software
works in proper placement of GC clamps and visualizing difference in the melt profile of
wild-type and mutant sequences. The 299 base-pair betaglobin sequence is first imported into
the project. Figure 4.1 shows the Sequence Display window for the imported sequence.
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