Denaturing+Gradient+Gel+Electrophoresis


 * Denaturing Gradient Gel Electrophoresis **
 * Introduction**

Denaturing gradient gel electrophoresis (DGGE) is a molecular fingerprinting method that allows the separation of DNA synthesized strands from another molecular technique called polymerase chain reaction (PCR). PCR works by producing (amplifying) multiple copies of a specific DNA strand. An outline of specific DNA sequences can be produced, outlining microbial communities. When PCR is amplified there can be a problem with the DNA strands in how familiar they are in size and sequence. This can make it very difficult to determine a specific band on an analysis gel. This can make the DNA content of your community very difficult to distinguish (1).

DGGE allows for a better distinguishable observation based upon PCR products by denaturing the strands by different characteristics of the DNA. As the DNA strands move through the gel they will encounter gradients of either heat or chemicals that will denature the strands on the gel. When the strand reach a point where denaturation can occur, the strands of the DNA will begin to melt (come apart) and this has the ability to slow the DNA products down. DNA sequences from prokaryotes to eukaryotes have differing DNA strands and will melt or denature at different concentrations of denaturant resulting in patterns of bands (strands). When a specific strand is amplified a G-C clamp attached to the primer will prevent the strand from coming completely apart (5).

Typically on a DGGE, a band will loosely represent an entire population in a sampled community. The desired band can then be removed (cut out) from the gel and analyzed. The resulting analysis can be uploaded into a computer's database that hosts the sequence of the desired DNA pattern which can be utilized to determine your organism. If a match is found then a general conclusion about the specific species of the microbe can decided upon. As DGGE has become one of the main molecular technique in the analysis of microbial research, there have been increasingly more PCR primers and denaturants available(5). It is now possible to analyze communities of organisms up to the phylogeny level, and even locate specific objective organisms (2). 
 * Application**

DGGE was highly utilized in the analysis of 16S rDNA fragments in a microbial mat and a bio-film created by bacteria (4). For this analysis genomic DNA was extracted and the 16S rRNA genes were placed into a PCR sequencer and amplified (6). This gave bands of dissimilar bacteria in the sample. However it was difficult to determine the specific bands and so was later divided on DGGE. The outcome a distinguishable pattern of bands in which the number of bands was proportional to the estimated number of individual species in the community. The bands were further analyzed using blots and isotope labeling to isolate the specific type of microbe. It was concluded that the species were mostly related to sulfur reducing bacteria (4). As the DNA moves through the gel the denaturant will cause the amplified strands to melt. This melting along with the G-C clamp will prevent the DNA to move further through the gel. The melting of the strand is really species specific based on sequence, this is why the technique works so well. This allows for sequences of similar size, but have different nucleic acids to be separated (3).  DGGE gels are very similar to a gel that is used for DNA electrophoresis. There are varying differences in the amount of denaturants (chemicals used to melt the DNA strand). The gels usually contain an acrylamide (a structural component) and denaturants (typically urea or formamide, sometime both are used together)(7). These two components allow for a denaturing gradient to denature the specific segments of DNA and produce the individual bands at listed above. These bands are the patterns that will be used later for species, and or population analysis, or simply re-amplified (PCR) for further molecular studies or cloning purposes (5).
 * Methods and Protocol**

The first step the process is to assemble a gel “sandwich” by setting a small glass plate on to a larger glass plate. **Components of the gel sandwich; gel clamps and spacers between two glass plates.** Once you have the glass sandwich it is time to prepare the gel. There are many commercial made solutions available and will depend greatly on the type of microbes you want to analyze. Once you have the gel you will need to fill the apparatus. The components of the apparatus are now the glass clamps the glass sandwich and the electrical units. You will now load the gel into the apparatus.
 * There are many instruction manuals on the entire assembly and techniques of DGGE. This is only a brief overview of the protocol (5).**

After the gel has set, it will be time to load any other specific denaturat and solution into the chambers. This technique is very similar to loading a normal DNA electrophoresis gel. A given amount of solution can be added if desired for further manipulation of the gel. You will let the gel sit for about an hour to allow diffusion of chemicals and polymerize with the gel.
 * Housing unit with glass sandwich and spacers attached.**


 * Loading denaturant into specific lanes**

A gel and apparatus housing will now be attached to the gel and glass apparatus. This allows for the loading of the samples and attachment of the electodes to migrate the samples through the gel. The final steps are to load your samples into the lanes and attach the electrodes and turn the machine on. After the samples have migrated through the gel you can stain the gel to determine where the specific band presides. These bands can be further analyzed or sequenced to determine the organism at such sites as: National Center for Biotechnology Information ([|http://www.ncbi.nlm.nih.gov).] (7). However, as this site is contains a majority medically of oriented microbes finding an exact match might prove more difficult to do (8).
 * Constructing and attaching the housing unit**.

**References:**

1. Amann RI, Stromley J, Devereux R, Key R&Stahl DA(1992) Molecular and microscopic identification of sulfate reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol. 58: 614–623. 2. Donner G, Schwarz K, Hoppe HG & Muyzer G (1996) Profiling the succession of bacterial populations in pelagic chemoclines. Arch. Hydrobiol. Spec. Issues Advanc. Limnol. 48: 7–14. 3. Fischer SG & Lerman LS (1983) DNA fragments differing by single base pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80: 1579–1583. 4. Muyzer G, de Waal EC & Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes encoding for 16S rRNA. Appl. Environ. Microbiol. 59: 695–700. 5. Muyzer, G. and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek //7//3:127-141. 6. Saiki RK, Gelfand DH, Stoffel SJ, Scharf SJ, Higuchi R, Horn GT, Mullis KB & Erlich HA (1988) Primer directed enzymatic amplifcation of DNA with thermostable DNA polymerase.Science 239: 487–491. 7. Lieven W., Veraeren H., Verstrate W., Boon N., Quantifying Community Dynamics of Nitrifiers in Functionally Stable Reactors. Appl. Environ. Microbiol. 74:  286–293. 8. Ercolini, D. 2004. PCR-DGGE fingerprinting: Novel strategies for detection of microbes in food. J. Microbiol. Meth//.// 56:297-314.