Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are forms of
electrophoresis
Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions with a positive or negative net ch ...
which use either a temperature or chemical gradient to denature the sample as it moves across an
acrylamide
Acrylamide (or acrylic amide) is an organic compound with the chemical formula CH2=CHC(O)NH2. It is a white odorless solid, soluble in water and several organic solvents. From the chemistry perspective, acrylamide is a vinyl-substituted primary ...
gel. TGGE and DGGE can be applied to nucleic acids such as
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
and
RNA
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
, and (less commonly) proteins. TGGE relies on temperature dependent changes in structure to separate
nucleic acids
Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic a ...
. DGGE separates genes of the same size based on their different denaturing ability which is determined by their base pair sequence. DGGE was the original technique, and TGGE a refinement of it.
History
DGGE was invented by
Leonard Lerman, while he was a professor at SUNY Albany.
The same equipment can be used for analysis of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
, which was first done by Thomas E. Creighton of the MRC
Laboratory of Molecular Biology
The Medical Research Council (MRC) Laboratory of Molecular Biology (LMB) is a research institute in Cambridge, England, involved in the revolution in molecular biology which occurred in the 1950–60s. Since then it has remained a major medical r ...
, Cambridge, England. Similar looking patterns are produced by proteins and nucleic acids, but the fundamental principles are quite different.
TGGE was first described by Thatcher and Hodson 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
DNA has a negative charge and so will move to the positive electrode in an electric field. A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string. When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly inversely proportional to the length of the DNA molecule (shorter lengths of DNA travel faster) — this is the basis for size dependent separation in standard
electrophoresis
Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions with a positive or negative net ch ...
.
In TGGE there is also a temperature gradient across the gel. At room temperature, the DNA will exist stably in a double-stranded form. As the temperature is increased, the strands begin to separate (
melting
Melting, or fusion, is a physical process that results in the phase transition of a substance from a solid to a liquid. This occurs when the internal energy of the solid increases, typically by the application of heat or pressure, which inc ...
), and the speed at which they move through the gel decreases drastically. Critically, the temperature at which melting occurs depends on the sequence (GC basepairs are more stable than AT due to stacking interactions, not due to the difference in hydrogen bonds (there are three hydrogen bonds between a
cytosine
Cytosine () (symbol C or Cyt) is one of the four nucleotide bases found in DNA and RNA, along with adenine, guanine, and thymine ( uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attac ...
and
guanine
Guanine () (symbol G or Gua) is one of the four main nucleotide bases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine ( uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside ...
base pair, but only two between
adenine
Adenine (, ) (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a purine nucleotide base that is found in DNA, RNA, and Adenosine triphosphate, ATP. Usually a white crystalline subtance. The shape of adenine is ...
and
thymine
Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...
), so TGGE provides a ''"sequence dependent, size independent method"'' for separating DNA molecules. TGGE separates molecules and gives additional information about melting behavior and stability (Biometra, 2000).
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
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
.
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. The process of denaturation on a denaturing gel is very sharp: "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). This makes it possible to discern differences in DNA sequences or mutations of various genes: sequence differences in fragments of the same length 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.
There are a number of disadvantages to this technique: "Chemical gradients such as those used in DGGE are not as reproducible, are difficult to establish and often do not completely resolve
heteroduplex
A heteroduplex is a double-stranded ( duplex) molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from ''different'' sources, such as from different homologous chromosomes or even from diff ...
es" (Westburg, 2001). These problems are addressed by TGGE, which uses a temperature, rather than chemical, gradient to denature the sample.
Method
To separate nucleic acids by TGGE, the following steps must be performed: preparing and pouring the gels, electrophoresis, staining, and
elution
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent: washing of loaded ion-exchange resins to remove captured ions, or eluting proteins or other biopolymers from an el ...
of DNA. Because a
buffered system must be chosen, it is important that the system remain stable within the context of increasing temperature. Thus,
urea
Urea, also called carbamide (because it is a diamide of carbonic acid), is an organic compound with chemical formula . This amide has two Amine, amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest am ...
is typically utilized for gel preparation; however, researchers need to be aware that the amount of urea used will affect the overall temperature required to separate the DNA.
[(Biometra, 2000).] 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.
Depending on which type of TGGE is to be run, either
perpendicular
In geometry, two geometric objects are perpendicular if they intersect at right angles, i.e. at an angle of 90 degrees or π/2 radians. The condition of perpendicularity may be represented graphically using the '' perpendicular symbol'', � ...
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. Once the gel has been run, the gel must be stained to visualize the results. While there are a number of stains that can be used for this purpose,
silver staining has proven to be the most effective tool.
The DNA can be eluted from the silver stain for further analysis through
PCR amplification.
Applications
TGGE and DGGE are broadly useful in biomedical and ecological research; selected applications are described below.
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
Mitochondrial DNA (mtDNA and mDNA) is the DNA located in the mitochondrion, mitochondria organelles in a eukaryotic cell that converts chemical energy from food into adenosine triphosphate (ATP). Mitochondrial DNA is a small portion of the D ...
of an individual. According to these authors, TGGE was utilized to determine two novel
mutations
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosi ...
in the mitochondrial
genome
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
: "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 biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosi ...
in the entire mitochondrial genome by temperature gradient gel electrophoresis".
p53 mutation in pancreatic secretions
Lohr and coworkers (2001) report that in a comprehensive study of
pancreatic secretions of individuals without pancreatic
carcinoma
Carcinoma is a malignancy that develops from epithelial cells. Specifically, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that arises from cells originating in the endodermal, mesoder ...
,
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
Ribosomes () are macromolecular machines, found within all cells, that perform biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA molecules to fo ...
subunit coding genes was first described by
Gerard Muyzer, while he was Post-doc at
Leiden University
Leiden University (abbreviated as ''LEI''; ) is a Public university, public research university in Leiden, Netherlands. Established in 1575 by William the Silent, William, Prince of Orange as a Protestantism, Protestant institution, it holds the d ...
, 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
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
and
archaea
Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
, and 18S rRNA gene fragments of
eukaryotes
The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...
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 of abundance of predominant microbial community members. This method is often referred to as
community fingerprinting. 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. For instance, Tabatabaei et al. (2009) applied DGGE and managed to reveal the microbial pattern during the anaerobic fermentation of
palm oil
Palm oil is an edible vegetable oil derived from the mesocarp (reddish pulp) of the fruit of oil palms. The oil is used in food manufacturing, in beauty products, and as biofuel. Palm oil accounted for about 36% of global oils produced from o ...
mill effluent (POME) for the first time.
References
*
Charles J. Sailey, M.D., M.S. Parts taken from a summary paper entitled "TGGE." 2003. The University of the Sciences in Philadelphia.
{{DEFAULTSORT:Temperature Gradient Gel Electrophoresis
Biochemistry methods
Electrophoresis
Gene tests