Temperature gradient gel electrophoresis

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.

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 [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 TGGE was first developed by Lerman and Andersen (unpublished,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.

Temperature gradient gel electrophoresis

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

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.

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. (Tabatabaei et
al., 2009[1])[6] 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).

Applications

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.

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).

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.

Microbial ecology

DGGE of small ribosomal subunit coding genes was first described by Gerard Muyzer[7],
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. In addition,
many environments which carry complex microbial communities has been studied using
DGGE such as soil, sewerage, industrial effluents like Palm Oil mill Effluent (POME).
Recently, a study by DGGE in 2009 revealed that Methanosaeta concilii dominates when
POME is anaerobically treated in order to produce methane. (Tabatabaei et al.,
2009[2])[6]

DGGE

From BioMineWiki

Denaturing gradient gel electrophoresis (DGGE) is a technique used for separating DNA
fragments according to their mobilities under increasingly denaturing conditions (usually
increasing formamide/ urea concentrations).

Principle

Small samples of DNA (or RNA) are added to an electrophoresis gel that contains a
denaturing agent. The denaturing gel induces melting of the DNA at various stages. As a
result of this melting, the DNA spreads through the gel and can be analyzed for single
components.

DGGE (Muyzer et al. 1993) analyses are employed for the separation of double-stranded
DNA fragments that are identical in length, but differ in sequence.

In practice, the DNA fragments are usually produced via PCR amplification. The DGGE
technique exploits (among other factors) the difference in the stability of G-C pairing (3
hydrogen bonds per pairing) as opposed to A-T pairing (2 hydrogen bonds). A mixture of
DNA fragments of different sequence is separated by electrophoresis on an acrylamide
gel containing a linearly increasing gradient of DNA denaturants (usually urea and
formamide). In general, DNA fragments richer in GC will be more stable and remain
double-stranded until reaching higher denaturant concentrations. Double-stranded DNA
fragments migrate better in the acrylamide gel, while denatured DNA molecules slow
down or stop in the gel. In this manner, DNA fragments of differing sequence can be
separated in an acrylamide gel. DGGE is commonly performed for partial 16S rRNA
gene, but also functional genes may be used. A GC (guanine plus cytosine) rich sequence
can be incorporated into one of the primers used in the PCR to modify the melting
behaviour of the fragment of interest and to improve the separation of the fragments. The
DGGE gels can be stained with DNA binding fluorescent dyes, such as SYBR Green and
visualized under UV light. Known standards may be used for comparing the samples on
different gels. Ideally one band on the gel corresponds to one species, and therefore the
number of bands gives an idea of the diversity of the sample. The gene fragments can be
excised from the gel, eluted e.g. into sterile water and amplified for sequencing. The
relative abundance of various microorganisms can be estimated by measuring the
intensity of their bands relative to the intensity of all bands in the corresponding sample.

Advantages

• Very sensitive to variations in DNA sequence;

• Allows simultaneous analysis of multiple samples;
• Useful method for monitoring shifts in community structure over time;
• Community profiles can be analyzed with cluster analysis;
• The use of universal primers allows the analyses of microbial communities
without any prior knowledge of the species;
• The fragments separated by DGGE can be excised, cloned and sequenced for
identification;
• It is possible to identify constituents that represent only 1 % of the total
community;
• DGGE can be applied to phylogenetic and functional genes.

Disadvantages

• DGGE analysis is rather time consuming;

• DGGE analysis suffers from the same drawbacks as all PCR-based community
analysis techniques, including biases from DNA extraction and amplification;
 • The variation in 16S rRNA gene copy number in different microbes makes this
technique only “semi-quantitative”;
• Microheterogeneity in rRNA encoding genes present in some species may result
in multiple bands for a single species and subsequently to an overestimation of
community diversity;
• Heteroduplexes can cause biases to the observed diversity;
• No method for automated analyses currently available;
• Works well only with short fragments (<600 bp), thus limiting phylogenetic
characterization;
• Gels of complex communities may look smeared due to the large number of
bands;
• Band position does not provide reproducible taxonomic information;
• Results difficult to reproduce between gels and laboratories.

DNA-based DGGE as a monitoring tool

It is especially suitable for samples with few species and gives complementary
information on species diversity when combined with other methods. It is also well suited
for analysing community changes over time.

• Very suitable technique for the identification of novel or unknown organisms. The
detection sensitivity is limited due to PCR and primer artefacts, usually the most
abundant species are detected.
• Easy to perform
• The most abundant species can be readily detected.

• Limited information about the abundance of detected species and no information

about the activity of the detected species.
• Long analysis time (several days) that makes it difficult to use it as an “on-line”
tool.

Current use in bioleaching studies

DGGE has been used to elucidate and characterize microbial populations in many
bioleaching environments, including bioreactors (Kinnunen and Puhakka 2004), acidic
mining-impacted environments (González-Toril et al. 2003), and bioleaching heaps
(Hawkes et al. 2004).