Temperature gradient gel electrophoresisTemperature Gradient Gel Electrophoresis
Denaturing Gradient GelElectrophoresis (DGGE)
are forms of electrophoresis where there is a temperature or
chemical gradient across the gel. TGGE and DGGE are useful for analyzing nucleic acidssuch as DNA and RNA, and sometimes for proteins.
TGGE is one of a family of electrophoretic methods for separation of nucleic acids like
DNAor RNAthat rely on temperature dependent changes in structure; the original
method was DGGE, which is almost identical. DGGE was invented byLeonard Lerman,
while he was a professor at SUNY Albany
.While the same equipment can be used for analysis of proteins, to generate similar looking patterns, the fundamental principles are quite different for proteins and nucleicacids (Thomas E. Creighton of the MRC Laboratory of Molecular Biology, Cambridge,England was the first person to do this
).Since a gradient of denaturant and a gradient of temperature are linearly related, the twotechniques are, from a theoretical standpoint, almost identical. Thus, it stands to reasonthat understanding TGGE would best be accomplished by first considering the principlesunderlying DGGE. TGGE was first developed by Lerman and Andersen (unpublished,communication to the author), using a beryllium Oxide plate as a thermal diffuser (BeOhas a very high thermal conductivity) and by Roger Wartell of Georgia Tech. Extensivework was done by the group of Riesner in Germany. Commercial equipment for DGGE isavailable from Bio-Rad, INGENY and CBS Scientific; a system for TGGE is availablefrom Biometra.
Temperature gradient gel electrophoresis
To understand T/DGGE, there are two fundamental points. The first is how the structureof DNA changes with temperature; the second is how these changes in structure affect themovement of DNA through a gel. We start with a double stranded DNA molecule of afew hundred basepairs in length. At room temperature, in the presence of at least a mM of salt, the double stranded form is quite stable, and we can consider the molecule to be twostrings tightly wrapped about each other so that there are effectively two ends. DNA is anegatively charged molecule (anion) and in the presence of an electric field, will move tothe positive electrode. A gel is a molecular mesh, with holes roughly the same size as thediameter of the DNA string. In the presence of the electric field, the DNA will attempt tomove through the mesh, and for a given set of conditions, the speed of movement isroughly proportional to the length of the DNA molecule — this is the basis for sizedependent separation in standard electrophoresis. As one raises the temperature, the twostrands of the DNA start to come apart; this is melting. At some high temperature, the twostrands will completely separate. However, at some intermediate temperature, the twostrands will be partly separated,with part of the molecule still double stranded and partsingle stranded, just as if one took a piece of string and partially unravelled some of thestrands; one could do this from one end, to make a y shaped structure with 3 ends, from