Temperature gradient gel electrophoresis

Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA, and (less commonly) proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pare sequence. DGGE was the original technique, and TGGE a refinement of it.

DGGE was invented by Leonard Lerman, while he was a professor at SUNY Albany.

The same equipment can be used for analysis of protein, which was first done by Thomas E. Creighton of the MRC Laboratory of Molecular Biology, Cambridge, England. Similar looking patterns are produced by proteins and nucleic acids, but the fundamental principles are quite different.

TGGE was first described by Thatcher and Hodson and by Roger Wartell of Georgia Tech. Extensive work was done by the group of Riesner in Germany. Commercial equipment for DGGE is available from Bio-Rad, INGENY and CBS Scientific; a system for TGGE is available from Biometra.

DNA has a negative charge and so will move to the positive electrode in an electric field. A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string. When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly inversely proportional to the length of the DNA molecule (shorter lengths of DNA travel faster) — this is the basis for size dependent separation in standard electrophoresis.

However, in TGGE, there is also a temperature gradient across the gel. At room temperature, the DNA will exist stably in a double-stranded form. As the temperature is increased, the strands begin to separate (melting), and the speed at which they move through the gel decreases drastically. Critically, the temperature at which melting occurs depends on the sequence (GC basepairs are more stable than AT due to stacking interactions, not, as commonly thought, due to the difference in hydrogen bonds (there are three hydrogen bonds between a cytosine and guanine base pair, but only two between adenine and thymine)), so TGGE provides a "sequence dependent, size independent method" for separating DNA molecules. TGGE not only separates molecules, but gives additional information about melting behavior and stability (Biometra, 2000).

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