6 Denaturing Gradient Gel Electrophoresis
87
dient gels, as shown for parallel and perpendicular DGGE in Figure 1 and
Figure 2. To improve electrophoretic detectability of mutations, heteroduplex molecules are generated by mixing normal and mutated DNA fragments prior to electrophoretic analysis. Subsequently, these mixtures are
denatured and reannealed. The resulting heteroduplex molecules contain
base mismatches at the site of the mutation. The reduced binding energy of
mismatches leads to a reduced melting temperature and to greater migration differences upon electrophoresis.
The gradient needed for melting analysis can be generated by casting a
P AG with a gradient for denaturing substances such as urea and formamide
(DGGE), or by generating a physical temperature gradient along the gel
using a heat exchanger attached to the gel (temperature gradient gel electrophoresis, TGGE).
Suitable DNA fragments for analysis can be generated by restriction enzyme digestion or PCR, the latter being used most commonly. The fragments should be in the size range of 100 to 400 bp. If PCR is used to generate
fragments, the melting proflle can be altered by
(i) varying the location of the primers, or
(ii) adding a GC clamp, i. e. a sequence of 40 nt of G and C to the 5' end of one
primer. The GC clamp has a very high melting temperature ( 1 00°C} and acts
as a clamp to hold tagether the double strand at one end, since it does not
melt under normal gel conditions. This modifies the melting proflle such
that long domains (more than 100 bp) of uniform melting temperature are
created. Theoretically, these domains are langer and easier to analyze than
the naturally occurring domains in a given DNA fragment. However, in our
experience, a drawback is that PCR with GC-clamped primers sometimes
proves difficult with low yields of product.
V ery recently, psoralen phosphoramidite has become commercially
available. Psoralen phosphoramidite can be added to AA dinucleotide tails
at the 5' ends of primers during synthesis in an oligonucleotide synthesizer.
When used in PCR, the photo-reactive moieties are introduced into the PCR
product. The two strands can then be covalently linked with 10 to 20 minutes ofUV irradiation (360 nm). The covalent band seems to replace the GCclamp and exerts the same impact on the melting profile (Costes et al. 1993}.
Moreover, PCR with psoralen-labeled primers results in higher yields of
product, and synthesis of primers is less costly.
The following protocol provides a general outline of the use of DGGE for
analyses of PCR products. DGGE may be set up in two different ways: In
perpendicular DGGE where the temperature gradient is perpendicular to
the direction of electrophoresis whereas in parallel DGGE the gradient is
parallel to the direction of electrophoresis. Perpendicular DGGE is useful
87
dient gels, as shown for parallel and perpendicular DGGE in Figure 1 and
Figure 2. To improve electrophoretic detectability of mutations, heteroduplex molecules are generated by mixing normal and mutated DNA fragments prior to electrophoretic analysis. Subsequently, these mixtures are
denatured and reannealed. The resulting heteroduplex molecules contain
base mismatches at the site of the mutation. The reduced binding energy of
mismatches leads to a reduced melting temperature and to greater migration differences upon electrophoresis.
The gradient needed for melting analysis can be generated by casting a
P AG with a gradient for denaturing substances such as urea and formamide
(DGGE), or by generating a physical temperature gradient along the gel
using a heat exchanger attached to the gel (temperature gradient gel electrophoresis, TGGE).
Suitable DNA fragments for analysis can be generated by restriction enzyme digestion or PCR, the latter being used most commonly. The fragments should be in the size range of 100 to 400 bp. If PCR is used to generate
fragments, the melting proflle can be altered by
(i) varying the location of the primers, or
(ii) adding a GC clamp, i. e. a sequence of 40 nt of G and C to the 5' end of one
primer. The GC clamp has a very high melting temperature ( 1 00°C} and acts
as a clamp to hold tagether the double strand at one end, since it does not
melt under normal gel conditions. This modifies the melting proflle such
that long domains (more than 100 bp) of uniform melting temperature are
created. Theoretically, these domains are langer and easier to analyze than
the naturally occurring domains in a given DNA fragment. However, in our
experience, a drawback is that PCR with GC-clamped primers sometimes
proves difficult with low yields of product.
V ery recently, psoralen phosphoramidite has become commercially
available. Psoralen phosphoramidite can be added to AA dinucleotide tails
at the 5' ends of primers during synthesis in an oligonucleotide synthesizer.
When used in PCR, the photo-reactive moieties are introduced into the PCR
product. The two strands can then be covalently linked with 10 to 20 minutes ofUV irradiation (360 nm). The covalent band seems to replace the GCclamp and exerts the same impact on the melting profile (Costes et al. 1993}.
Moreover, PCR with psoralen-labeled primers results in higher yields of
product, and synthesis of primers is less costly.
The following protocol provides a general outline of the use of DGGE for
analyses of PCR products. DGGE may be set up in two different ways: In
perpendicular DGGE where the temperature gradient is perpendicular to
the direction of electrophoresis whereas in parallel DGGE the gradient is
parallel to the direction of electrophoresis. Perpendicular DGGE is useful