Temperature gradient gel electrophoresis
The Temperaturgradientengelelektrophorese ( TGGE, english temperature gradient gel electrophoresis ) and the Denaturierungsgradientengelelektrophorese ( DGGE, Eng. Denaturing gradient gel electrophoresis ) are gel electrophoretic method for the separation of charged biomolecules. Using a temperature gradient or a chemical gradient across the length of the polyacrylamide gel. DGGE and TGGE which are for the separation of nucleic acids such as DNA or RNA and less frequently used for proteins. Alternative methods include, for example, DNA sequencing, SSCP, heteroduplex EMSA and denaturing HPLC.
Temperaturgradientengelelektrophorese
TGGE which is a gel having a temperature difference over the length of the gel. From a certain temperature double-stranded DNA melts into two single strands. The melting point is dependent on the DNA sequence and its base-pairing and can be calculated approximately. From the melting temperature, the rate of migration of the DNA in the gel drops significantly. The gradient can be used for electrophoretic migration direction (English parallel TGGE ) or orthogonal (English Perpendicular TGGE ) are applied in parallel. Orthogonally oriented gradients allow the identification of the melting point and the optimum temperature range for a sharp separation as possible. In parallel to the temperature gradient electrophoresis, separation is performed by means of different melting points, and only after the initial molecular weight. This allows both mutations and secondary structures are detected. A variant of the previously used TGGE hybridization to heteroduplexes to amplify the differences in electrophoretic mobility.
Denaturierungsgradientengelelektrophorese
In the DGGE gel containing a chemical gradient of a chaotropic agent. The abolition of secondary structures is carried out by the increasingly denaturing concentration of chaotropic agent. The denaturation is carried out usually not continuously across the gradient, but in discrete steps. Characterized mutations such as SNPs can be compared in the two DNA sequences. The disadvantage of the chemical gradient is its poor reproducibility and heteroduplexes an occasional blurred separation.
History
The DGGE was developed from 1979 by Leonard Lerman and Stuart Fischer at SUNY Albany. The separation of proteins by DGGE was first described by Thomas E. Creighton at the MRC in Cambridge. The TGGE was developed in 1981 by Thatcher and Hodson and by Roger Wartell.