Temperature gradient gel electrophoresis

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Temperature gradient gel electrophoresis (TGGE) is a form of electrophoresis where there is a temperature gradient across the gel. TGGE is useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins. TGGE is a highly specialized technique practiced by a relatively small number of scientists[1].

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[edit] Definition

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 [2][3][4].
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 [5]).
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 TGGE was first developed by Lerman and Andersen (unpublished, communication to the author), using a beryllium Oxide plate as a thermal diffuser (BeO has a very high thermal conductivity) and by Roger Wartell of Georgia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; an expensive system for TGGE is available from Biometra.

[edit] Denaturing gradient gel electrophoresis

To understand T/DGGE, there are two fundamental points. The first is how the structure of DNA changes with temperature, and 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[6] Leonard Lerman and Stuart Fisher while at the State University of New York, Albany.

[edit] Temperature gradient gel electrophoresis

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.

[edit] Method of TGGE

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).

[edit] Applications

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

[edit] Mutations in mtDNA

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). Through TGGE it became apparent that this patient did have a mutation, however did not yet develop cystic fibrosis. This information not only provides some insight into the progression of cystic fibrosis in individuals that have these gene mutations, but also demonstrates that cystic fibrosis is a disease of the entire mitochondrial genome.

[edit] p53 mutation in pancreatic juices

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.

[edit] Mycobacterial diversity

While a number of studies have utilized TGGE for the purpose of studying internal mechanisms of mutation and disparities of DNA in the human body, the technique has also been widely applied to the field of cell biology and microbiology. Cheung and Kinkle (2001) consider the isolation of Mycobacterium sp. through the process of TGGE. According to the researchers, little is known about Mycobacterium populations because the species grows and mutates so quickly. Thus, the researchers isolated a strand on the bacteria and compared it with four contaminated soils to delineate differences in the molecular structure. The researchers were able to show that inoculation of certain chemicals into soils containing the Mycobacterium could significant improve the degradation of the soil due to the bacterium.

[edit] Source

[edit] References

  1. ^ A query on pubmed with "TGGE" pulls up only 228 citations
  2. ^ Cell. 1979 Jan;16(1):191-200. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Fischer SG, Lerman LS
  3. ^ Fischer S. G. and Lerman L. S. "Separation of random fragments of DNA according to properties of their sequences" Proc. Natl. Acad. Sci. USA, 1980, 77, 4420-4424.
  4. ^ Fischer S. G. and Lerman L. S. "DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory" Proc. Natl. Acad. Sci. USA, 1983, 80, 1579-1583.
  5. ^ J Mol Biol. 1994 Oct 7;242(5):670-82. Electrophoretic characterization of the denatured states of staphylococcal nuclease. Creighton TE, Shortle D.
  6. ^ Cell. 1979 Jan, Vol 16, pages 191-200; Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis.
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