Chapter 6
Denaturing Gradient Gel Electrophoresis
KAI-OLAF NETZER
lntroduction
Denaturing gradient gel electrophoresis (DGGE) (Abrams and Stanton
1992; Myers et al. 1987) is a technique suitable for detecting point mutations
as well as small deletions and insertions of several base pairs in double
stranded DNA. The method is based on the analysis of strand separation
(denaturation) ofDNA molecules. The process of strand separation by disruption ofhydrogen bonds is referred to as "melting" ofthe DNA, while the
"melting point" is defined as the temperature or the denaturant concentration at which half of all possible hydrogen bonds are disrupted. It appears
that melting occurs in domains, i. e. stretches of several tens of base pairs
melt at the same temperature. Several factors influence the melting of a given DNA sequence: temperature, length, and base composition. GC-rich
sequences have higher melting temperatures than AT-rich sequences.
Moreover, since hybridization depends on hydrogen-band formation,
the presence ofhydrogen-bond disrupting agents (denaturants) in the medium will also affect the melting point. Computer algorithms are available
that can predict melting properties ofDNA sequences (Lerman and Silverstein 1987). The resulting melt maps can be used todetermineoptimal experimental conditions.
Melting of a DNA fragment within a gel matrix greatly alters electrophoretic mobility. In polyacrylamide gels (PAG), mobility decreases exponentiallywith the length of the melted sequence. Introduction of a sequence
change in a melting domain usually alters the melting temperature by several tenths of a degree Celsius. Therefore, sequence changes within a melting domain can be visualized by differential migration in denaturing graKai-OlafNetzer, Krankenhaus Köln-Merheim, Medizinische Klinik I, Ostmerheimer Str.
200, Köln, 51109, Germany (phone +49-221-8907-2200;/ax +49-221-8907-2335; e-mail
kai.netzer@uni -koeln.de)
Denaturing Gradient Gel Electrophoresis
KAI-OLAF NETZER
lntroduction
Denaturing gradient gel electrophoresis (DGGE) (Abrams and Stanton
1992; Myers et al. 1987) is a technique suitable for detecting point mutations
as well as small deletions and insertions of several base pairs in double
stranded DNA. The method is based on the analysis of strand separation
(denaturation) ofDNA molecules. The process of strand separation by disruption ofhydrogen bonds is referred to as "melting" ofthe DNA, while the
"melting point" is defined as the temperature or the denaturant concentration at which half of all possible hydrogen bonds are disrupted. It appears
that melting occurs in domains, i. e. stretches of several tens of base pairs
melt at the same temperature. Several factors influence the melting of a given DNA sequence: temperature, length, and base composition. GC-rich
sequences have higher melting temperatures than AT-rich sequences.
Moreover, since hybridization depends on hydrogen-band formation,
the presence ofhydrogen-bond disrupting agents (denaturants) in the medium will also affect the melting point. Computer algorithms are available
that can predict melting properties ofDNA sequences (Lerman and Silverstein 1987). The resulting melt maps can be used todetermineoptimal experimental conditions.
Melting of a DNA fragment within a gel matrix greatly alters electrophoretic mobility. In polyacrylamide gels (PAG), mobility decreases exponentiallywith the length of the melted sequence. Introduction of a sequence
change in a melting domain usually alters the melting temperature by several tenths of a degree Celsius. Therefore, sequence changes within a melting domain can be visualized by differential migration in denaturing graKai-OlafNetzer, Krankenhaus Köln-Merheim, Medizinische Klinik I, Ostmerheimer Str.
200, Köln, 51109, Germany (phone +49-221-8907-2200;/ax +49-221-8907-2335; e-mail
kai.netzer@uni -koeln.de)