Temperature Gradient Gel Electrophoresis

Brief Description:  

TGGE is a method of electrophoresis used to separate proteins or nucleic acids (DNA and RNA) across a temperature gradient. TGGE is based on the principles that the structure of proteins and DNA changes with temperature and that these changes can be characterized by their movement trough a gel. Nucleic acids melt when heated, forming a gradient of entire strands to partially separated strands to completely separated strands. The speed across a temperature gradient is inversely proportional to the degree of separation. And the exact temperature of the denaturation of nucleic acid depends on its specific sequence.

TGGE is a type of Denaturing Gradient Gel Electrophoresis (DGGE) and they are at times used interchangeable in the literature, although the techniques to differ somewhat. TGGE is often associated with PCR analysis. Specific regions of DNA or RNA can be isolated by PCR techniques the products are then analysis be TGGE. One of the most common regions in the 16S rRNA gene isolated by PCR and then analyzed by gradient gel electrophoresis.

One of the major limitations of DGGE and TGGE technology is that it can only analyze small fragments (up to 500 base pairs). Furthermore resolution is an issue. Sometimes TGGE is unable to detect or over separate small “wobbles in base pair” sequence, leading to inaccurate characterization of the gene sequence.

Application in Wine Microbiology:

 

Applications of this technology are broad from genetics and medicine, to microbial ecology, and to many industries including winemaking. The most important TGGE application in wine microbiology is the characterization of wine microbes. Wine yeasts can be characterized by TGGE. Likewise, bacterial populations can also be evaluated by TGGE. Characterization is the first step in proper identification of wine microbes. TGGE has been used to identify bacteria from wine biofilms. TGGE can also be used to screen clone libraries i.e. to identify redundant yeast strains. Microbiologists are hopeful that advances in TGGE will enable them to go beyond characterization of genetic structure to a better understanding of protein function and metabolism. This will enable winemakers to better understand microbial communities found in vineyards and wineries in order to promote desired microbes and exclude unwanted microbes and undesirable products.

References:

  • Gomez S. J Espinosa. J Ubeda. 2000. Characterization of wnie yeasts by temperature gradient gel electrophoresis. FEMS Microbiology Letters 193. pp 45-50.
  • Lopez et al. 2003. Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis. Applied and Environmental Microbiology. Vol 69: 11: pp 6801-6807.
  • Cocolin et al. 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Applied and Environmental Microbiology. Vol 67:11: pp5113-5121.