6 Denaturing Gradient Gel Electrophoresis
95
ing temperature" of this domain can be calculated from the concentration
of denaturants at the point of inflection of the curve. As a rule of thumb, in a
gel heated to 60°C, 3o/o denaturants equal an increase in temperature of 1 oc.
Therefore, a gradient gel with 0-80% denaturants will cover a temperature
range from 60°C (i. e. buffer temperature) to 87°C. Once the melting temperature has been determined, parallel DGGE may be used to screen large
numbers of samples. In our experience it is best to have a range of denaturants of 30% from top to bottom, i. e. ±15% flanking the melting temperature of the domain to be analyzed.
It is essential that the DNA fragment reaches the region ofthe gelthat con- Running time
tains enough denaturant to melt the domain of interest. If the gradient is
narrow and the domain of interest is the first to melt, this may be achieved
with 500 Vh. Gels can be run at low voltage overnight, e.g. at 25 V for 16
hours. Short runs during the day time, e.g. 200 V for 2.5 hours, may be preferable since everything is under immediate control. In addition, bandswill
be sharper. It may be necessary to performtest runs in which one fragment
is applied to the gel at 0, 1, 2, 3, etc. hours after initiation ofthe run. The ideal
running time is achieved when the fragment reaches its melting temperature and gives a well-focused band in the gel.
Wehave used two different methods for staining of DGG. A very simple Staining
method is staining with ethidium bromide. The gel is immersed in 1 J..Lg/
ml ethidium bromidein water for 15 to 20 min, rinsed with water, then
photographed using a UV transilluminator. Since ethidium bromid is carcinogenic, it must be discarded properly (follow the rules of your laboratory's safety guidelines). Silver staining is moretime consuming but also
more sensitive. There are good kits available commercially ( e. g. BioRad).
Follow the manufacturer's instructions.
References
Abrams ES, Stanton VP (1992). Use of denaturing gardient gel electrophoresis to study
conformational transitions in nucleic acids. Meth Enzymol 212: 71-104.
Myers RM, Maniatis T, Lerman LS {1987). Detection and localization of single base
changes by denaturing gradient gel electrophoresis. Methods Enzymol155: 501-527.
Lerman LS, Silverstein K {1987). Computational simulation ofDNA melting and its application to denaturing gradient gel electrophoresis. Meth Enzymol155: 482-501.
Costes B, Girodon E, Ghanem N, Chassignol M, Thuong NT, Dupret D, Goossens M
(1993). Psoralen-modified oligonucleotide primers improve detection of mutations
by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping. Hum Mol Genet 2: 393-397.
95
ing temperature" of this domain can be calculated from the concentration
of denaturants at the point of inflection of the curve. As a rule of thumb, in a
gel heated to 60°C, 3o/o denaturants equal an increase in temperature of 1 oc.
Therefore, a gradient gel with 0-80% denaturants will cover a temperature
range from 60°C (i. e. buffer temperature) to 87°C. Once the melting temperature has been determined, parallel DGGE may be used to screen large
numbers of samples. In our experience it is best to have a range of denaturants of 30% from top to bottom, i. e. ±15% flanking the melting temperature of the domain to be analyzed.
It is essential that the DNA fragment reaches the region ofthe gelthat con- Running time
tains enough denaturant to melt the domain of interest. If the gradient is
narrow and the domain of interest is the first to melt, this may be achieved
with 500 Vh. Gels can be run at low voltage overnight, e.g. at 25 V for 16
hours. Short runs during the day time, e.g. 200 V for 2.5 hours, may be preferable since everything is under immediate control. In addition, bandswill
be sharper. It may be necessary to performtest runs in which one fragment
is applied to the gel at 0, 1, 2, 3, etc. hours after initiation ofthe run. The ideal
running time is achieved when the fragment reaches its melting temperature and gives a well-focused band in the gel.
Wehave used two different methods for staining of DGG. A very simple Staining
method is staining with ethidium bromide. The gel is immersed in 1 J..Lg/
ml ethidium bromidein water for 15 to 20 min, rinsed with water, then
photographed using a UV transilluminator. Since ethidium bromid is carcinogenic, it must be discarded properly (follow the rules of your laboratory's safety guidelines). Silver staining is moretime consuming but also
more sensitive. There are good kits available commercially ( e. g. BioRad).
Follow the manufacturer's instructions.
References
Abrams ES, Stanton VP (1992). Use of denaturing gardient gel electrophoresis to study
conformational transitions in nucleic acids. Meth Enzymol 212: 71-104.
Myers RM, Maniatis T, Lerman LS {1987). Detection and localization of single base
changes by denaturing gradient gel electrophoresis. Methods Enzymol155: 501-527.
Lerman LS, Silverstein K {1987). Computational simulation ofDNA melting and its application to denaturing gradient gel electrophoresis. Meth Enzymol155: 482-501.
Costes B, Girodon E, Ghanem N, Chassignol M, Thuong NT, Dupret D, Goossens M
(1993). Psoralen-modified oligonucleotide primers improve detection of mutations
by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping. Hum Mol Genet 2: 393-397.