Pulsed Field Gel Electrophoresis (PFGE)
DNA, or deoxyribonucleic acid, is the molecule that carries the genetic information of all living organisms. DNA is composed of four types of nucleotides, which are the building blocks of DNA. Each nucleotide consists of a nitrogenous base (adenine, guanine, cytosine, or thymine), a sugar (deoxyribose), and a phosphate group. The nucleotides are linked together by covalent bonds between the sugar and phosphate groups, forming a long chain called a strand. Two strands of DNA are twisted around each other to form a double helix, which is stabilized by hydrogen bonds between the complementary bases on opposite strands. The sequence of bases on one strand determines the sequence of bases on the other strand, and thus the sequence of nucleotides on both strands encodes the genetic information.
DNA is organized into structures called chromosomes, which are located in the nucleus of eukaryotic cells or in the cytoplasm of prokaryotic cells. Each chromosome consists of a single, long molecule of DNA that is tightly coiled and wrapped around proteins called histones. The human genome contains 23 pairs of chromosomes, which collectively carry about 3 billion base pairs of DNA. Each chromosome has a specific number and shape that can be distinguished by staining and microscopy techniques.
DNA is replicated before cell division, so that each daughter cell inherits an identical copy of the genetic material from the parent cell. The process of DNA replication involves unwinding the double helix and separating the two strands by breaking the hydrogen bonds between them. Then, each strand serves as a template for synthesizing a new complementary strand by adding nucleotides according to the base-pairing rules. The result is two identical copies of the original DNA molecule, each consisting of one old and one new strand.
DNA is also transcribed into RNA, which is another type of nucleic acid that plays various roles in gene expression. RNA differs from DNA in having a ribose sugar instead of a deoxyribose sugar, and having uracil instead of thymine as one of the bases. RNA can be single-stranded or double-stranded, depending on its function and structure. The most common type of RNA is messenger RNA (mRNA), which carries the genetic information from DNA to the ribosomes, where it is translated into proteins. Proteins are the molecules that perform most of the biological functions in cells, such as catalyzing reactions, transporting substances, signaling pathways, and regulating gene expression.
Pulsed Field Gel Electrophoresis (PFGE) is a technique that allows the separation of very large DNA fragments, such as whole bacterial chromosomes, by applying an electric field that changes direction periodically. The principle of PFGE is based on the fact that DNA molecules are negatively charged and will move towards the positive electrode when an electric field is applied. However, the movement of DNA molecules in a gel matrix depends not only on their charge, but also on their size and shape. Smaller and more compact DNA molecules can migrate faster and farther than larger and more linear ones, because they encounter less resistance from the gel pores.
In conventional gel electrophoresis, the electric field is constant and oriented in one direction. This means that all DNA molecules will eventually reach a limit of migration, where they cannot move any further because they are too large to pass through the gel pores. This limit is called the cliff effect, and it occurs at around 30-50 kb (kilobase pairs) for most agarose gels. Therefore, conventional gel electrophoresis cannot separate DNA fragments larger than this size.
In PFGE, the electric field is not constant, but rather changes direction at regular intervals. This means that the DNA molecules have to reorient themselves every time the field changes, which takes some time depending on their size and shape. Larger and more linear DNA molecules take longer to reorient than smaller and more compact ones, because they have more inertia and friction. Therefore, larger DNA molecules will lag behind smaller ones in each cycle of field change. Over time, this will result in a separation of DNA fragments based on their size.
The direction and frequency of the field change can be adjusted to optimize the separation of different size ranges of DNA fragments. For example, if the field changes frequently and at a low angle, smaller DNA fragments will be separated better. If the field changes less frequently and at a high angle, larger DNA fragments will be separated better. The optimal conditions for PFGE depend on the type of gel, the voltage, the buffer, the temperature, and the DNA sample.
PFGE can separate DNA fragments up to 10 Mb (megabase pairs), which is much larger than what conventional gel electrophoresis can achieve. This makes PFGE a powerful tool for analyzing whole bacterial genomes and comparing their genetic diversity.
The method of PFGE involves four main steps: lysis, digestion, electrophoresis, and analysis. Each step is briefly described below.
The first step is to prepare the DNA sample for electrophoresis. This is done by embedding the bacterial cells in agarose gel plugs, which protect the DNA from mechanical damage and prevent its degradation by nucleases. The agarose gel plugs are then treated with detergents and enzymes to lyse the cells and release the DNA. The resulting agarose-DNA plugs are also called plug molds.
The next step is to cut the DNA into fragments using restriction enzymes. Restriction enzymes are proteins that recognize specific sequences of DNA and cleave them at those sites. For PFGE, rare-cutting restriction enzymes are used, which cut the DNA infrequently and generate large fragments ranging from 10 kb to several Mb. The choice of restriction enzyme depends on the organism and the purpose of the analysis.
The third step is to separate the DNA fragments by size using an electric field. Unlike conventional gel electrophoresis, which applies a constant voltage in one direction, PFGE uses a pulsed electric field that alternates among three directions: one along the axis of the gel and two at 60 degrees on either side. The pulse times are equal for each direction, resulting in a net forward migration of the DNA. The pulsed field causes the DNA fragments to reorient themselves according to their size and shape, with larger and more linear fragments being slower than smaller and more compact ones. This allows the resolution of very large DNA fragments that would otherwise migrate as a single band in conventional gel electrophoresis.
The final step is to visualize and compare the DNA patterns generated by PFGE. The DNA fragments are stained with a fluorescent dye such as ethidium bromide or SYBR Green and detected under UV light. The resulting patterns are called fingerprints or profiles and can be compared manually or by computer software such as BioNumerics. The fingerprints can be used to identify, classify, or subtype different strains or isolates of bacteria based on their genetic similarity or diversity.
Pulsed-field gel electrophoresis (PFGE) is a technique that allows the separation of large DNA fragments by applying an electric field that periodically changes its direction. The following are the major steps involved in PFGE:
- Lysis: The first step is to prepare the DNA sample for electrophoresis. This is done by embedding the bacterial cells in agarose gel plugs, which protect the DNA from mechanical damage and prevent its diffusion. The cells are then lysed with detergents and enzymes to release the DNA into the agarose matrix.
- Digestion: The next step is to cut the DNA into smaller fragments using restriction enzymes. These are enzymes that recognize specific sequences of DNA and cleave them at those sites. The choice of restriction enzyme depends on the organism and the desired resolution of the DNA fragments. For PFGE, rare-cutting enzymes that produce large fragments are preferred over frequent-cutting enzymes that produce small fragments.
- Electrophoresis: The third step is to separate the DNA fragments by size using an electric field. Unlike conventional gel electrophoresis, where the electric field is constant and applied in one direction, PFGE uses a pulsed field that alternates among three directions: one along the axis of the gel and two at 60 degrees on either side. This causes the DNA fragments to reorient themselves according to their size and shape, and migrate at different rates through the gel matrix. The pulse time and angle can be adjusted to optimize the separation of different size ranges of DNA fragments.
- Analysis: The final step is to visualize and compare the DNA patterns generated by PFGE. This can be done by staining the gel with a fluorescent dye that binds to DNA, such as ethidium bromide, and exposing it to ultraviolet light. The resulting image shows bands of different intensities corresponding to different amounts of DNA. The bands can be compared manually or by using computer software that can measure their size and similarity. The PFGE patterns can be used for genotyping or genetic fingerprinting of bacterial strains, as well as for constructing physical maps of bacterial chromosomes.
Pulsed Field Gel Electrophoresis (PFGE) is a powerful technique that can separate large DNA molecules (up to 10 Mb) by applying an electric field that periodically changes direction. This allows the analysis of DNA fragments that are too large to be resolved by conventional gel electrophoresis. PFGE has many applications in various fields of research and practice, such as:
- Genetic mapping: PFGE can be used to construct physical maps of chromosomes from different organisms, such as bacteria, yeast, plants, and animals. Physical maps show the location and size of DNA fragments on a chromosome, which can help identify genes and their functions. PFGE can also be used to compare the genomes of different strains or species of organisms and to detect chromosomal rearrangements, such as deletions, insertions, inversions, and translocations.
- Genetic fingerprinting: PFGE can be used to generate DNA profiles or fingerprints of organisms based on their unique patterns of DNA fragments. This can help identify and differentiate individuals or groups of organisms based on their genetic similarity or diversity. PFGE is commonly used for typing bacteria, especially pathogenic bacteria that cause infectious diseases. PFGE can help track the source and transmission of outbreaks, monitor the spread and evolution of antibiotic resistance, and evaluate the effectiveness of infection control measures.
- Vaccine development: PFGE can be used to characterize the antigenic diversity of pathogens, such as viruses and bacteria, and to select the most suitable strains for vaccine production. PFGE can also be used to monitor the stability and purity of vaccine strains and to detect any contamination or mutation that may affect their safety or efficacy.
PFGE is considered a gold standard method for molecular typing of bacteria, as it has high discriminatory power, reproducibility, and concordance with epidemiological data. PFGE is also a universal method that can be applied to any organism with a suitable restriction enzyme and electrophoresis conditions. However, PFGE also has some limitations, such as being time-consuming, labor-intensive, technically demanding, and expensive. Moreover, PFGE may not be able to discriminate between some closely related isolates or may produce ambiguous or complex patterns that are difficult to interpret. Therefore, PFGE may need to be combined with other molecular methods, such as PCR-based techniques or whole-genome sequencing, for more accurate and comprehensive analysis of DNA.
Pulsed Field Gel Electrophoresis (PFGE) is a powerful technique for separating and analyzing large DNA fragments. Compared to conventional gel electrophoresis, PFGE has several advantages that make it suitable for various applications in molecular biology and microbiology. Some of the advantages of PFGE are:
- High resolution: PFGE can separate DNA fragments from a few kilobases (kb) to over 10 megabases (Mb), which is much larger than the range of conventional gel electrophoresis. This allows the analysis of whole genomes or large genomic regions of interest, such as bacterial chromosomes, plasmids, or viral genomes.
- High discrimination: PFGE can distinguish between closely related strains or isolates of bacteria based on their DNA restriction patterns. PFGE subtyping has been shown to have high concordance with epidemiological relatedness and can reveal genetic diversity and evolutionary relationships among bacterial populations. PFGE is often considered the gold standard for genotyping or genetic fingerprinting of pathogenic bacteria, such as Salmonella, Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni.
- Universal applicability: PFGE can be applied to any organism that has DNA, regardless of its size, shape, or complexity. The only requirement is to choose an appropriate restriction enzyme and optimize the conditions for electrophoresis for each species. PFGE can also be combined with other molecular techniques, such as hybridization, PCR, or sequencing, to enhance its specificity and sensitivity.
- Stability and reproducibility: The DNA restriction patterns generated by PFGE are stable and consistent over time and across laboratories. The DNA fragments are immobilized in agarose plugs during lysis and digestion, which prevents mechanical damage and degradation. The electrophoresis parameters are controlled by a computer program that ensures uniformity and accuracy. The results can be easily compared and interpreted by visual inspection or by computer software.
These advantages make PFGE a valuable tool for studying the structure, function, and evolution of DNA molecules and for investigating the molecular epidemiology and diversity of bacterial pathogens.
Although PFGE is a powerful and widely used technique for DNA separation and subtyping, it also has some limitations that need to be considered. Some of the main limitations are:
- Time-consuming: PFGE is not a quick method, as it involves several steps such as preparing the agarose plugs, digesting the DNA, running the gel, staining, and analyzing the results. The whole process can take up to two or three days, depending on the protocol and the number of samples. This can limit the applicability of PFGE for rapid identification and outbreak investigation.
- Requires trained and skilled technicians: PFGE is not a simple technique, as it requires careful handling of the samples, precise adjustment of the electrophoresis parameters, and accurate interpretation of the band patterns. A high level of technical expertise and experience is needed to perform PFGE successfully and consistently. Moreover, PFGE is sensitive to variations in the protocol, equipment, reagents, and environmental conditions, which can affect the reproducibility and comparability of the results. Therefore, standardization and quality control are essential for PFGE analysis.
- Does not discriminate between all unrelated isolates: PFGE is a highly discriminatory technique, but it is not infallible. Sometimes, PFGE may fail to distinguish between unrelated isolates that share the same or similar restriction patterns by chance or due to convergent evolution. Conversely, PFGE may also overestimate the genetic diversity of closely related isolates that have undergone minor changes in their restriction sites due to mutation or recombination. Therefore, PFGE results should be interpreted with caution and in combination with other epidemiological and molecular data.
- Pattern results vary slightly between technicians: PFGE generates complex band patterns that are not always easy to compare and interpret. Different technicians may have different criteria and methods for scoring and matching the bands, which can introduce some subjectivity and variability in the analysis. Moreover, different software tools may have different algorithms and parameters for analyzing the band patterns, which can also affect the outcome of the comparison. Therefore, it is important to use standardized and validated methods and software for PFGE analysis and to ensure inter-laboratory agreement and consistency.
- Can’t optimize separation in every part of the gel at the same time: PFGE relies on changing the direction and duration of the electric field to separate large DNA fragments according to their size and shape. However, this also means that different parts of the gel may have different optimal conditions for separation at different times. For example, smaller fragments may be better separated at shorter pulse times, while larger fragments may require longer pulse times. Therefore, it is not possible to achieve optimal separation for all fragments in all parts of the gel at the same time. This can limit the resolution and accuracy of PFGE analysis.
- Don’t really know if bands of the same size are the same pieces of DNA: PFGE separates DNA fragments based on their size and shape, but it does not provide any information about their sequence or content. Therefore, it is possible that two bands of the same size may represent different pieces of DNA that have been cut by the same restriction enzyme at different locations. Alternatively, two bands of different sizes may represent the same piece of DNA that has been cut by different restriction enzymes at different locations. Therefore, PFGE does not provide a definitive identification or characterization of the DNA fragments.
- Bands are not independent: PFGE generates band patterns that reflect the overall structure and organization of the DNA molecules. However, this also means that each band is not independent from the others, as they are derived from the same molecule. Therefore, changes in one band may affect or be correlated with changes in other bands. For example, loss or gain of one restriction site may result in more than one band change in the pattern. This can complicate the interpretation and comparison of PFGE results.
- “Relatedness” should be used as a guide, not true phylogenetic measure: PFGE can provide useful information about the genetic relatedness or similarity of different isolates based on their band patterns. However, this does not necessarily reflect their true phylogenetic or evolutionary relationships, as PFGE does not account for factors such as gene flow, recombination, horizontal gene transfer, or selection pressure that may affect the genetic diversity and divergence of bacterial populations. Therefore, PFGE should be used as a guide or a tool for subtyping rather than as a definitive measure of phylogeny.
- Some strains cannot be typed by PFGE: Finally, some bacterial strains may not be suitable for PFGE analysis due to various reasons. For example, some strains may have very large or very small genomes that are difficult to separate by PFGE. Some strains may have very few or very many restriction sites that result in too few or too many bands to be resolved by PFGE. Some strains may have unstable or variable genomes that result in inconsistent or unpredictable band patterns by PFGE. Therefore, alternative or complementary methods may be needed for typing such strains.
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