DGGE

To exploit this principle and detect the differences in sequence a vertical temperature gradient is applied alongside a “normal” acrylamide gel. AT rich sequences will melt earlier in the gel, whereas GC dominated sequences will remain double stranded until higher temperatures. To prevent single stranded “melted” DNA from just running out of the gel a GC-clamp is attached at one end of the fragment. Once separated the fragment forms a Y shaped structure due to the GC clamp and mobility in the gel is drastically reduced.

Whereas TGGE solely relies on temperature in DGGE this temperature gradient is performed not with temperature alone but with a chemical denaturans (formamide and urea). 0.4% (v/v) formamide and 0.42% (w/v) results in a 100% denaturing gel solution. 3 % in the gradient equals 1oC in temperature.

click image to enlarge




	Denaturing Gradient Gel Electrophoresis (DGGE) is a very common DNA profiling technique. It is based on the principle of electrophoresis, separating charged molecules according to their size along a gel matrix. However with DGGE double stranded DNA fragments of the same size are separated according to their base pair sequence.
	Underlying principle DNA is double stranded due to hydrogen bondings between the bases. According to their structure Adenin (A) builds two hydrogen bonds to Thymin (T) and Cytosin (C) and Guanin (G) form three hydrogen bonds. The energy which is required to separate (“melt”) these hydrogen bonds is different and hence for a double stranded DNA fragment the melting (denaturing) temperature is dependant on the sequence.
	To exploit this principle and detect the differences in sequence a vertical temperature gradient is applied alongside a “normal” acrylamide gel. AT rich sequences will melt earlier in the gel, whereas GC dominated sequences will remain double stranded until higher temperatures. To prevent single stranded “melted” DNA from just running out of the gel a GC-clamp is attached at one end of the fragment. Once separated the fragment forms a Y shaped structure due to the GC clamp and mobility in the gel is drastically reduced.
	Whereas TGGE solely relies on temperature in DGGE this temperature gradient is performed not with temperature alone but with a chemical denaturans (formamide and urea). 0.4% (v/v) formamide and 0.42% (w/v) results in a 100% denaturing gel solution. 3 % in the gradient equals 1oC in temperature. With its high resolution (up to single base transitions can be detected) this method can be used for comparative microbial community profiling based on 16S rDNA but also mutation analysis based on single genes can be performed.	click image to enlarge
	Applying a temperature gradient Standard electrophoresis along a temperature gradient of fragments of the same size is able to detect these differences in sequences. double stranded DNA fragments of different sequence will require different amount of and finally double stranded DNA is accordingly dependant on the sequence of the DNA. A GC rich sequence will require more energy to be sperated than a AT rich For more information see: Biorad product page
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