Denaturing Gradient Gel Electrophoresis

                        Denaturing gradient gel electrophoresis (DGGE) is a robust method for point mutation detection that has been widely used for many years. It is a polymerase chain reaction (PCR)-based method, the principle being the altered denaturing temperature of a PCR product with a mutation compared to the wild-type product. PCR performed on DNA of an individual with a point mutation in one of two genes will lead to a mixture of different products. 

                        PCR products from both the wild-type gene and the mutated gene will be formed. These are known as the homoduplex products. The difference in melting temperature between these two products, however, is subtle. Another type of product, heteroduplexes, consisting of a wild-type strand combined with a mutant strand of DNA, will also be formed during the last cycles of the reaction. The real strength of DGGE lies in the fact that the heteroduplex PCR products will have much lower melting temperatures compared to the homoduplex PCR products, because the heteroduplexes have a mismatch. 

                        To visualize the different melting temperatures of these homoduplexes and heteroduplexes, the products should be run on an acrylamide gel with a gradient of denaturing agents: urea and formamide. These denaturing agents alone are not sufficient. In addition, the gel should be run at a high temperature, usually 60°C. During electrophoresis, the PCR products will run through the gel as double-stranded DNA until they reach the point where they start to denature. Once denatured, the PCR products could continue running through the gel as single-stranded DNA, but the fragments have to remain precisely where they denatured. To achieve this, a so-called GC clamp is attached, to prevent complete denaturing. 

                     This GC clamp is a string of 40–60 nucleotides composed only of guanine and cytosine and is attached to one of the PCR primers. PCR with a GC clamp results in a product with one end having a very high denaturing temperature. A PCR product running through a DGGE gel will, therefore, denature partially. The GC clamp remains double stranded. The fragment will form a Y-shaped piece of DNA that will stick firmly at its position on the gel.