Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 16)

Fig. 4.2. A. 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.
B. Parallel denaturing gradient
gel in which the denaturing gradient is parallel to the electrophoresis direction. Mutant and wild-type alleles of
exon 8 from the p53 gene separated by an 8% acrylamide:bis (37.5:1) gel with a parallel gradient of 40–65% denaturant.
The gel was run at 150 V for 2.5 hours at 60 °C in 1x TAE buffer. Lane 1 contains the mutant fragment, lane 2 contains the
wild-type fragment, lane 3 contains both the mutant and wild-type fragments.
When running a denaturing gradient gel, both the mutant and wild-type DNA fragments are
run on the same gel. This way, mutations are detected by differential migration of mutant and
wild-type DNA. 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 and the region screened is in the lowest
melting domain. The addition of a 30–40 base pair GC clamp to one of the PCR primers insures
that the region screened is in the lower melting domain and that the DNA will remain partially
double-stranded.
34 An alternative to GC clamps is using psoralen derivative PCR primers called
ChemiClamp primers. 10 Because ChemiClamps covalently link the two DNA strands at one
end, they should not be used when isolating a DNA fragment which is going to be sequenced
from a gel. The size of the DNA fragments run on a denaturing gel can be as large as 1 kb in
length, but only the lower melting domains will be available for mutation analysis. For complete
analysis of fragments over 1 kb in length, more than one PCR reaction should be performed.
11
The thermodynamics of the transition of double-stranded to single-stranded DNA have been
described by a computer program developed by Lerman. 12 Bio-Rad offers a Macintosh ®
computer program, MacMelt ™ software, which calculates and graphs theoretical DNA melting
profiles. Melting profile programs can show regions of theoretical high and low melting domains
of a known sequence. Placement of primers and GC clamps can be optimized by analysis of
placement effect on the DNA melting profile.
The method of creating heteroduplex molecules helps in detecting homoduplex mutations.
This process is typically done when it is not originally possible to resolve a homoduplex
mutation. Analysis of heteroduplex molecules can, therefore, increase the sensitivity of DGGE.
Heteroduplexes can be formed by adding the wild-type and mutant template DNAs in the same
PCR reaction or by adding separate PCR products together, then denaturing and
allowing them to re-anneal. A heteroduplex has a mismatch in the double-strand causing a
distortion in its usual conformation; this has a destabilizing effect and causes the DNA strands
to denature at a lower concentration of denaturant (Figure 4.3). The heteroduplex bands always
migrate more slowly than the corresponding homoduplex bands under specific conditions.
12
A B
0%
40%
70%
65%