Pulsed Field Gel Electrophoresis (PFGE) is a method used to aid in the separation of huge deoxyribonucleic acids (DNA) molecules using the gel matrix an electric field that continuously shifts direction. Because DNA greater than 15-20kb moving through a gel is able to move in a size-independent manner. The conventional gel electrophoresis method was not able to separate massive DNA molecules effectively that led to the use of electrophoresis using pulsed fields.
In 1982 Schwartz came up with the notion that DNA molecules that are larger than 50kb could be separated using two electric fields that alternate.
What is Pulsed-field Gel Electrophoresis (PFGE)?
Pulsed-field Gel Electrophoresis (PFGE) can be described as a lab method used by scientists to create the DNA fingerprint of an isolate of a bacterial species. A bacteria is a type of isolate that is the same kind of bacteria. PulseNet analyzes bacterial strains found in sick people, food items that are contaminated as well as the locations in which food is manufactured.
How does Pulsed-field Gel Electrophoresis work?
- The scientist extracts the bacterial cells of an Agar Plate.
- Scientists mix the bacterial cells and agarose melts and pours the mixture into the mold of a plug.
- The cells of the bacterium are broken up using biochemicals or lysed so that DNA remains liberated from the plugs of agarose.
- The scientist inserts an DNA gel plug with and then places it within an electric field, which separates DNA fragments based on their dimensions.
- Gels are stained to allow DNA to be observed in the ultraviolet (UV) radiation. Digital cameras take photographs of the gel and saves the image on a computer.
The DNA fragments create an DNA fingerprint that has particular patterns. The image shows an example of an agarose-based gel in which each lane is an individual DNA fingerprint, or pattern. The PFGE method is distinct from traditional DNA electrophoresis as it is able to split very large fragments of DNA to produce a fingerprint, by continuously shifting how the field of electricity is directed.
When a DNA fingerprint is made and analyzed by the public health laboratory, it examines the fingerprint pattern with the software program called BioNumerics*. After analyzing the fingerprint, the lab uploads the design to the nation’s database which is where the database managers of PulseNet Central will examine the pattern to determine whether it’s creating an outbreak or may be an element of an ongoing epidemic. If it is, the database managers will work together with microbiologists from the public health and epidemiologists to further study the outbreak.
Types of Pulsed Field Gel Electrophoresis
- CHEF (clamped homogeneous electric field) technology can resolve DNA across an array of molecular masses in straight lane. It utilizes the concepts of contour-clamped electrophoresis in order to create homogeneous electrical fields.
- The PACE (programmable independently controlled electrodes) technology lets users determine the ideal angle for electrophoretic pulses that is optimal for the size range
- DR (dynamic regulation) is the design of electronics through which all 24 electrodes are controlled. CHEF-DR(r) systems can compensate for any changes in buffer conductivity and gel size and preventing them from impacting the reproducibility of results.
- FIGE (field inversion gel electrophoresis) is used to achieve rapid resolution of samples within the 100 bp-250 km size range. In FIGE, the electrical field is set at a single angles (180deg) and then reversed between forward and reverse directions.
- AFIGE (asymmetric field inversion of gel electrophoresis) is a further improvement of FIGE technology. AFIGE applies an opposite voltage to the electrical field than that of the back direction which improves the resolution of the sample within the FIGE size range.
Principle of Pulsed Field Gel Electrophoresis (PFGE)
In general, smaller fragments may be able to navigate into the matrix of gels much more easily than larger DNA fragments there is a threshold of 30-50 kb, where all the large fragments run at the same pace and will appear on the gel as a single massive diffuse band.
With the periodic alteration of field direction, different lengths of DNA respond to the change in different rate. In other words, the larger pieces of DNA will take longer to shift their charge once the direction of the field changes, and smaller ones are more efficient. As time passes, due to the constant changing of directions the bands will start to separate even at extremely large lengths. This is how the separation of huge DNA fragments with the PFGE technique is now possible.
Method of Pulsed Field Gel Electrophoresis (PFGE)
- The process is similar to the procedure used for the standard electrophoresis, except that instead of running the voltage in a single direction, the voltage is frequently changed between three directions: one running through the central direction of the gel’s axis and two running in a 60-degree angle to either from the other.
- The pulse times are the same for each direction , which results to a net movement of DNA.
The major steps involved in Pulsed-field gel electrophoresis are:
- Lysis: Firstly, the bacteria suspension is then transferred into an agarose suspension. This is done to shield the DNA of the chromosomes from mechanical harm by immobilizing it in blocks of agarose. The bacterial cells then are lysed to release DNA. The agarose-DNA suspension can also be called plug mold.
- Digestion of DNA: DNA of bacteria is processed using uncommon restriction enzymes that cut to produce a smaller amount of larger-sized DNA pieces (in contrast to the commonly used restriction enzymes that are used in RFLP which produce large amounts in smaller pieces).
- Electrophoresis: The bigger DNA fragments are subjected to pulse-field electrophoresis using an electric current and changing the direction of the current at regular intervals (in contrast to the traditional electrophoresis using agarose gel to separate smaller pieces in which the current is applied in one direction).
- Analysis: The fragments of diverse organisms produced using PFGE are compared with standards in either a manual or computer program such as BioNumerics.
Applications of Pulsed Field Gel Electrophoresis (PFGE)
- Field electrophoresis can allow the separation of DNA fragments that contain up 100,000 Bp (100 Kilobase Pairs, also known as kbp) The characterization of these large fragments has led to the construction maps physically of the chromosomes in a variety of bacteria species.
- PFGE could be used to determine genotypes or fingerprinting genetic.
- It is generally regarded as the gold standard for epidemiological research on pathogenic organisms.
- Subtyping is making it much easier to differentiate between varieties of Listeria monocytogenes. This allows us to connect food or environmental isolates to the presence of clinical infections.
Advantages of Pulsed Field Gel Electrophoresis (PFGE)
- The PFGE method separates DNAs from a couple of kb up to more than 10 Mb pair.
- PFGE subtyping is successful applied to subtyping many pathogenic bacteria . It is highly compatible with epidemiological similarity.
- It has been demonstrated repeatedly to be more specific than techniques like ribotyping and multi-locus sequence typing for a variety of bacteria.
- The PFGE method in the same format is able to be used as a universal method to subtyping bacteria. (Only the choice of restriction enzyme and the conditions for electrophoresis have to be adapted to the specific species.)
- DNA restriction patterns produced by PFGE are reliable and stable.
Limitations of Pulsed Field Gel Electrophoresis (PFGE)
- Needs skilled and trained technicians.
- Does not differentiate between any isolates that are not related.
- Results of pattern analysis can differ little between different technicians.
- There is no way to optimize separation in every area of the gel simultaneously.
- I don’t know if the bands with the same dimensions are the same parts of DNA.
- Bands aren’t independent.
- Changes in one restriction site could be more than one change to a band.
- “Relatedness” can be utilized as a reference but not as a true phylogenetic indicator.
- Certain strains cannot be typed using PFGE.