Pulsed-field gel electrophoresis (PFGE) is a technique used for the separation of large DNA molecules by applying an electric field that periodically changes direction to a gel matrix. [1] [2] Unlike standard agarose gel electrophoresis, which can separate DNA fragments of up to 50 kb, PGFE resolves fragments up to 10 Mb. [1] This allows for the direct analysis of genomic DNA. [2]
In 1984, David C. Schwartz and Charles Cantor published the first successful application of alternating electric fields for the separation of large DNA molecules. [3] [4] This technique, which they named PFGE, resulted in the development of several variations, including Orthogonal Field Alternation Gel Electrophoresis (OFAGE), Transverse Alternating Field Electrophoresis (TAFE), Field-Inversion Gel Electrophoresis (FIGE), and Clamped Homogeneous Electric Fields (CHEF), among others. [3]
The procedure for PFGE is similar to that of standard agarose gel electrophoresis, with the main exception being the application of the electric current. Generally, in PFGE electrophoresis chambers, the voltage periodically switches between three directions: one along the central axis, and two at a 60 degree angle along each side. [5] The application of the voltage can change depending on the variation of PFGE used. [6] [7]
PFGE may be used for genotyping or genetic fingerprinting. It has commonly been considered a gold standard in epidemiological studies of pathogenic organisms for several decades. For instance, subtyping bacterial isolates with this method has made it easier to discriminate among strains of Listeria monocytogenes , Lactococcus garvieae [8] and some clinical isolates of Bacillus cereus [9] group isolated from diseases aquatic organisms and thus to link environmental or food isolates with clinical infections. It is now in the process of being superseded by next generation sequencing methods. [10]
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
Agarose is a heteropolysaccharide, generally extracted from certain red algae. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.
Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
Gel electrophoresis of nucleic acids is an analytical technique to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules are placed on a gel, where an electric field induces the nucleic acids to migrate toward the positively charged anode. The molecules separate as they travel through the gel based on the each molecule's size and shape. Longer molecules move more slowly because they the gel resists their movement more forcefully than it resists shorter molecules. After some time, the electricity is turned off and the positions of the different molecules are analyzed.
Lactococcus is a genus of lactic acid bacteria that were formerly included in the genus Streptococcus Group N1. They are known as homofermenters meaning that they produce a single product, lactic acid in this case, as the major or only product of glucose fermentation. Their homofermentative character can be altered by adjusting environmental conditions such as pH, glucose concentration, and nutrient limitation. They are gram-positive, catalase-negative, non-motile cocci that are found singly, in pairs, or in chains. The genus contains strains known to grow at or below 7˚C.
Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.
The bacterial capsule is a large structure common to many bacteria. It is a polysaccharide layer that lies outside the cell envelope, and is thus deemed part of the outer envelope of a bacterial cell. It is a well-organized layer, not easily washed off, and it can be the cause of various diseases.
Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci, using DNA sequences of internal fragments of multiple housekeeping genes to characterize isolates of microbial species.
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance network.
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.
In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering.
An electropherogram is a record or chart produced when electrophoresis is used in an analytical technique, primarily in the fields of forensic biology, molecular biology, and biochemistry. The method plots data points that represent a specific time and fluorescence intensity at various wavelengths of light to represent a DNA profile.
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies, reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis, crossed immunoelectrophoresis, rocket-immunoelectrophoresis, fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen.
The single cell gel electrophoresis assay is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point and by continuous elution from a gel column.
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.
BioNumerics is a bioinformatics desktop software application that manages microbiological data. It is developed by Applied Maths NV, a bioMérieux company.
Multiple loci VNTR analysis (MLVA) is a method employed for the genetic analysis of particular microorganisms, such as pathogenic bacteria, that takes advantage of the polymorphism of tandemly repeated DNA sequences. A "VNTR" is a "variable-number tandem repeat". This method is well known in forensic science since it is the basis of DNA fingerprinting in humans. When applied to bacteria, it contributes to forensic microbiology through which the source of a particular strain might eventually be traced back, making it a useful technique for outbreak surveillance.
Long-range restriction mapping is an alternative genomic mapping technique to short-range, also called fine-scale mapping. Both forms utilize restriction enzymes in order to decipher the previously unknown order of DNA segments; the main difference between the two being the amount of DNA that comprises the final map. The unknown DNA is broken into many smaller fragments by these restriction enzymes at specific sites on the molecule, and then the fragments can later be analyzed by their individual sizes. A final long-range map can span hundreds to thousands of kilobytes of genetic data at many different loci.
