Temperature gradient gel electrophoresis

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Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) are forms of electrophoresis where there is a temperature or chemical gradient across the gel. TGGE and DGGE are useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins.

TGGE is one of a family of electrophoretic methods for separation of nucleic acids like DNA or RNA that rely on temperature dependent changes in structure; the original method was DGGE, which is almost identical. DGGE was invented by Leonard Lerman, while he was a professor at SUNY Albany ^[1][2][3].
While the same equipment can be used for analysis of proteins, to generate similar looking patterns, the fundamental principles are quite different for proteins and nucleic acids (Thomas E. Creighton of the MRC Laboratory of Molecular Biology, Cambridge, England was the first person to do this ^[4]).
Since a gradient of denaturant and a gradient of temperature are linearly related, the two techniques are, from a theoretical standpoint, almost identical. Thus, it stands to reason that understanding TGGE would best be accomplished by first considering the principles underlying DGGE. TGGE was first developed by Lerman and Andersen (unpublished, communication to the author), using a beryllium Oxide plate as a thermal diffuser (BeO has a very high thermal conductivity) and by Roger Wartell of Georgia Tech. Extensive work was done by the group of Riesner in Germany. Commercial equipment for DGGE is available from Bio-Rad, INGENY and CBS Scientific; a system for TGGE is available from Biometra.

To understand T/DGGE, there are two fundamental points. The first is how the structure of DNA changes with temperature; the second is how these changes in structure affect the movement of DNA through a gel. We start with a double stranded DNA molecule of a few hundred basepairs in length. At room temperature, in the presence of at least a mM of salt, the double stranded form is quite stable, and we can consider the molecule to be two strings tightly wrapped about each other so that there are effectively two ends. DNA is a negatively charged molecule (anion) and in the presence of an electric field, will move to the positive electrode. A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string. In the presence of the electric field, the DNA will attempt to move through the mesh, and for a given set of conditions, the speed of movement is roughly proportional to the length of the DNA molecule — this is the basis for size dependent separation in standard electrophoresis. As one raises the temperature, the two strands of the DNA start to come apart; this is melting. At some high temperature, the two strands will completely separate. However, at some intermediate temperature, the two strands will be partly separated,with part of the molecule still double stranded and part single stranded, just as if one took a piece of string and partially unravelled some of the strands; one could do this from one end, to make a y shaped structure with 3 ends, from both ends to make a structure with 4 ends, or in the middle to make a bubble. What makes D/TGGE useful is that the mobility of the DNA molecule through the gel decreases drastically when these partially melted structures are formed, and, most important, the exact temperature at which this occurs depends on sequence; thus D/TGGE offers a "sequence dependent, size independent method" for separating DNA molecules. A very simple, but realistic analogy is to consider a person moving through a crowded room; when you extend your arms out, your movement through the room slows drastically, even though your mass has not changed.

While the details of D/TGGE may be of interest only to specialists, a good way to see what scientists are doing is to make use of the free online search provided at this url http://www.ncbi.nlm.nih.gov/entrez/query.fcgi. Enter "pubmed" in the search menu and "DGGE" in the for menu (no quotes).

Denaturing gradient gel electrophoresis (DGGE) works by applying a small sample of DNA (or RNA) to an electrophoresis gel that contains a denaturing agent. Researchers have found that certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-700 base pairs.

Further explicating how this technique works one author notes that what is unique about the DGGE technique is that as the DNA is subjected to increasingly extreme denaturing conditions, the melted strands fragment completely into single strands. This process is unique because it stands in direct contrast to how DNA denatures in vivo. "Rather than partially melting in a continuous zipper-like manner, most fragments melt in a step-wise process. Discrete portions or domains of the fragment suddenly become single-stranded within a very narrow range of denaturing conditions" (Helms, 1990). Because of this distinctive quality of DNA when placed in denaturing gel, it is possible for researchers to discern differences in DNA sequences or mutations of various genes:

Sequence differences in otherwise identical fragments often cause them to partially melt at different positions in the gradient and therefore "stop" at different positions in the gel. By comparing the melting behavior of the polymorphic DNA fragments side-by side on denaturing gradient gels, it is possible to detect fragments that have mutations in the first melting domain (Helms, 1990). Placing two samples side-by-side on the gel and allowing them to denature together, researchers can easily see even the smallest differences in two samples or fragments of DNA.

The principles outlined above provide a rudimentary understanding of how DGGE serves to differentiate between various fragments of DNA. Although the technique for procuring finished gels that can be utilized for investigative research requires several more steps (such as amplification of the samples from the gel), one can easily understand how denaturing gels work to fragment DNA and divide components based on the amount of denaturing that has taken place.

DGGE was invented by^[5] Leonard Lerman and Stuart Fisher while at the State University of New York, Albany.

Despite the fact that DGGE produced results that were more accurate and reliable than previous gel electrophoretic techniques, the reality is that there are a number of problems inherent in this technique. "Chemical gradients such as those used in DGGE are not as reproducible, are difficult to establish and often do not completely resolve heteroduplexes" (Westburg, 2001).

Given the problem associated with DGGE, researchers began looking for new techniques capable of minimizing some of the problems encountered with DGGE. As a result of this inquiry, TGGE was developed as a suitable, more reliable technique. Much in the same way that DGGE utilizes the melting behavior of the molecule as a primary method for separating fragments on the gel, so too does TGGE. The primary difference, however, is that TGGE “provides a temperature gradient instead of a chemical gradient” (Spanevello, 1997).

The temperature at which the DNA melts is directly proportional to the GC content of DNA since GC bases have triple bonds and AT bases have double bonds connecting the strands together.

With the information provided above, it is clear that the TGGE method utilizes heat as the primary mechanism for unraveling and denaturing DNA. What is not as obvious however, is how this process occurs and how the DNA can be analyzed utilizing this technique. What is perhaps most interesting when considering the process of denaturing in the TGGE method is that it occurs in such a systematic process, that it is possible to reconstruct the fragments once they have been dissociated. Explaining the process one author reports the following:

Working with PCR fragments... electrophoresis starts with double stranded molecules. At a certain temperature, the DNA start to melt, resulting in a fork-like structure. In this conformation the migration is slowed down compared to a completely double-stranded DNA fragment. Since the melting temperature strongly depends on the base sequence, DNA fragments of the same size but different sequence can be separated. […] Thus TGGE not only separates molecules, but gives additional information about melting behavior and stability (Biometra, 2000).

The information provided above serves as the foundation for understanding the theoretical framework behind TGGE. When it comes to creating a TGGE, it is clear that the methodology employed is almost as straightforward as the principles that underlie the technique. Summarizing the steps involved in producing TGGE samples, the following are required:

• Casting the Gels - This step requires the individual to prepare the cuvettes for the machine, prepare the gel for the machine and pour the gel for electrophoresis. Of all of these steps, preparing the gel seems to pose the most significant challenge to the researcher. Because a buffered system must be chosen, it is important that the system remain stable within the context of increasing temperature. Thus, urea is typically utilized for gel preparation; however, researchers need to be aware that the amount of urea used will have an impact on the overall temperature required to separate the DNA (Biometra, 2000). Depending on which type of TGGE is to be run, either perpendicular or parallel, varying amounts of sample need to be prepared and loaded. A larger amount of one sample is used with perpendicular, while a smaller amount of many samples are used with parallel TGGE.
• Electrophoresis - This step is self-explanatory. The gel is loaded, the sample is placed on the gel according to the type of gel that is being run—i.e. parallel or perpendicular—the voltage is adjusted and the sample can be left to run (Biometra, 2000).
• Staining – Once the gel has been run, to keep the results stable and further to be able to read them, the gel must be stained. While there are a number of stains that can be used for this purpose, silver staining has proven to be the most effective tool (Biometra, 2000).
• Elution of DNA – In this step the DNA can be eluted from the silver stain for further analysis through PCR amplification (Biometra, 2000).

Considering the application of this technology within the larger framework of medical science, it is clear that TGGE and DGGE have a broad scope of utilities in scientific research. To illustrate this point, one only needs to consider current research on the subject. By considering how these methods are applied in practical research it is possible to understand the benefits that this technology has for the advancement of science and medical care.

According to a recent investigation by Wong, Liang, Kwon, Bai, Alper and Gropman, TGGE can be utilized to examine the mitochondrial DNA of an individual. According to these authors, TGGE was utilized to determine two novel mutations in the mitochondrial genome: "A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for unknown mutations in the entire mitochondrial genome by temperature gradient gel electrophoresis" (Wong et al., 2002).

Lohr and coworkers (2001) report that in a comprehensive study of pancreatic juices of individuals without pancreatic carcinoma, p53 mutations could be found in the pancreatic juices of a small percentage of participants. Because mutations of p53 has been extensively found in pancreatic carcinomas, the researchers for this investigation were attempting to determine if the mutation itself can be linked to the development of pancreatic cancer. While Lohr was able to find p53 mutations via TGGE in a few subjects, none subsequently developed pancreatic carcinoma. Thus, the researchers conclude by noting that the p53 mutation may not be the sole indicator of pancreatic carcinoma oncogenesis.

DGGE of small ribosomal subunit coding genes was first described by Gerard Muyzer^[6], while he was Post-doc at Leiden University, and has become a widely used technique in microbial ecology. PCR amplification of DNA extracted from mixed microbial communities with PCR primers specific for 16S rRNA gene fragments of Bacteria and Archaea, and 18S rRNA gene fragments of Eukaryotes results in mixtures of PCR products. Because these amplicons all have the same length, they cannot be separated from each other by agarose gel electrophoresis. However, sequence variations (i.e. differences in GC content and distribution) between different microbial rRNAs result in different denaturation properties of these DNA molecules. Hence, DGGE banding patterns can be used to visualize variations in microbial genetic diversity and provide a rough estimate of the richness and abundance of predominant microbial community members. Recently, several studies have shown that DGGE of functional genes (e.g. genes involved in sulfur reduction, nitrogen fixation, and ammonium oxidation) can provide information about microbial function and phylogeny simultaneously.

• Charles J. Sailey, M.D., M.S. Parts taken from a summary paper entitled "TGGE." 2003. The University of the Sciences in Philadelphia.

7- Meisam Tabatabaei, Mohd Rafein Zakaria, Raha Abdul Rahim, André-Denis G. Wright, Yoshihito Shirai, Norhani Abdullah, Kenji Sakai, Shinya Ikeno, Masatsugu Mori, Nakamura Kazunori, Alawi Sulaiman and Mohd Ali Hassan. PCR-Based DGGE and FISH Analysis of Methanogens in Anaerobic Closed Digester Tank Treating Palm Oil Mill Effluent (POME). (Electronic Journal of Biotechnology)