Temperature gradient gel electrophoresis
Encyclopedia
Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) are forms of electrophoresis
which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide
gel. TGGE and DGGE can be applied to nucleic acids such as DNA
and RNA
, and (less commonly) proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE was the original technique, and TGGE a refinement of it.
, while he was a professor at SUNY Albany.
The same equipment can be used for analysis of protein
, which was first done by Thomas E. Creighton of the MRC Laboratory of Molecular Biology
, Cambridge, England. Similar looking patterns are produced by proteins and nucleic acids, but the fundamental principles are quite different.
TGGE was first developed by Lerman and Andersen (unpublished, communication to the author), using a beryllium oxide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.
.
However, 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), 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 then AT due to stacking interactions, not, as commonly thought, due to the difference in hydrogen bonds (there are three hydrogen bonds between a cytosine
and guanine
base pair, but only two between adenine
and thymine
)), so TGGE provides a "sequence dependent, size independent method" for separating DNA molecules. TGGE not only separates molecules, but gives additional information about melting behavior and stability (Biometra, 2000).
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.
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
es" (Westburg, 2001). These problems are addressed by TGGE, which uses a temperature, rather than chemical, gradient to denature the sample.
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
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.
, 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. For instance, Tabatabaei et al. (2009) applied DGGE and managed to reveal the microbial pattern during the anaerobic fermentation of palm oil mill effluent (POME) for the first time.
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide
Acrylamide
Acrylamide is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid, soluble in water, ethanol, ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts...
gel. TGGE and DGGE can be applied to nucleic acids such as DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
and RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, and (less commonly) proteins. TGGE relies on temperature dependent changes in structure to separate nucleic acids. DGGE was the original technique, and TGGE a refinement of it.
History
DGGE was invented by Leonard LermanLeonard Lerman
Leonard Lerman is an American scientist most noted for his work on DNA.As a graduate student with Linus Pauling at the California Institute of Technology, Lerman discovered that antibodies have two binding sites. Later, perhaps his most important discovery was that certain molecules bind to DNA by...
, while he was a professor at SUNY Albany.
The same equipment can be used for analysis of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
, which was first done by Thomas E. Creighton of the MRC Laboratory of Molecular Biology
Laboratory of Molecular Biology
The Laboratory of Molecular Biology is a research institute in Cambridge, England, which was at the forefront of the revolution in molecular biology which occurred in the 1950–60s, since then it remains a major medical research laboratory with a much broader focus.-Early beginnings: 1947-61:Max...
, Cambridge, England. Similar looking patterns are produced by proteins and nucleic acids, but the fundamental principles are quite different.
TGGE was first developed by Lerman and Andersen (unpublished, communication to the author), using a beryllium oxide
Beryllium oxide
Beryllium oxide , also known as beryllia, is an inorganic compound with the formula BeO. This colourless solid is a notable electrical insulator with a higher thermal conductivity than any other non-metal except diamond, and actually exceeds that of some metals. As an amorphous solid, beryllium...
plate as a thermal diffuser (BeO has a very high thermal conductivity) and by Roger Wartell
Roger Wartell
Roger Wartell is the former Chair of the School of Biology at the Georgia Institute of Technology.-Early life:Roger Wartell was born in New York, New York. He received his B.S. degree in Physics from Stevens Institute of Technology in 1966...
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 proportional to the length of the DNA molecule — this is the basis for size dependent separation in standard electrophoresisElectrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
.
However, 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), 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 then AT due to stacking interactions, not, as commonly thought, due to the difference in hydrogen bonds (there are three hydrogen bonds between a cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
and guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
base pair, but only two between adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
and thymine
Thymine
Thymine is one of the four nucleobases 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 nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
)), so TGGE provides a "sequence dependent, size independent method" for separating DNA molecules. TGGE not only separates molecules, but 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 denaturingDenaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
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.
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
Polymorphism (biology)
Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...
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
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
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
Heteroduplex
A heteroduplex is a double-stranded 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 different organisms....
es" (Westburg, 2001). These problems are addressed by TGGE, which uses a temperature, rather than chemical, gradient to denature the sample.
Method of TGGE
To separate nucleic acids by by TGGE, the following steps must be performed:- Preparing and pouring the Gels - 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).
- Electrophoresis - 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). Depending on which type of TGGE is to be run, either perpendicularPerpendicularIn geometry, two lines or planes are considered perpendicular to each other if they form congruent adjacent angles . The term may be used as a noun or adjective...
or parallelParallel (geometry)Parallelism is a term in geometry and in everyday life that refers to a property in Euclidean space of two or more lines or planes, or a combination of these. The assumed existence and properties of parallel lines are the basis of Euclid's parallel postulate. Two lines in a plane that do not...
, 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. - Staining – 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 (Biometra, 2000).
- ElutionElutionElution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....
of DNA – the DNA can be eluted from the silver stain for further analysis through PCR amplification (Biometra, 2000).
Applications
TGGE and DGGE are broadly useful in biomedical 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 DNAMitochondrial DNA
Mitochondrial DNA is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate...
of an individual. According to these authors, TGGE was utilized to determine two novel mutations in the mitochondrial genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
: "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 carcinomaCarcinoma
Carcinoma is the medical term for the most common type of cancer occurring in humans. Put simply, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during...
, p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
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, while he was Post-doc at Leiden UniversityLeiden University
Leiden University , located in the city of Leiden, is the oldest university in the Netherlands. The university was founded in 1575 by William, Prince of Orange, leader of the Dutch Revolt in the Eighty Years' War. The royal Dutch House of Orange-Nassau and Leiden University still have a close...
, 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
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and Archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
, 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. For instance, Tabatabaei et al. (2009) applied DGGE and managed to reveal the microbial pattern during the anaerobic fermentation of palm oil mill effluent (POME) for the first time.