What is pulsed-field gel electrophoresis?
Pulsed-field gel electrophoresis (PFGE) is a DNA fingerprinting technique that allows separation of large DNA fragments (of more than thirty kilo-base pairs) in an agarose gel by applying an alternating electric field among twelve pairs of electrodes. The DNA fragments ultimately form the distinctive pattern of bands in a column, or lane, in the gel. Small DNA fragments form bands at the bottom, and large DNA fragments form bands at the top of the gel. Genetic variation among strains causes fragments of differing sizes to be produced when DNA is digested with enzymes called restriction endonucleases. PFGE can thus detect differences in DNA as minor as one base change.
Genetic variations revealed after digestion with restriction endonucleases are referred to as restriction fragment length polymorphisms. The pattern of banding is analyzed visually then digitized and archived to determine the relatedness of biological samples.
PFGE is used, for example, in quality control of the genetic identity and stability of grapes and hops fermented for wine and beer and in quality control of the microbes used in food production. Cancer researchers use the technique to analyze damage to DNA caused by chemicals and radiation. Genetics research laboratories use the technique to map genetic defects routinely. Outbreaks of infectious diseases can be monitored to determine the similarity of the bacterial strains involved. While genetic matching does not ensure that isolates from two infected persons came from the same source, epidemiologists can detect the rise in prevalence of a bacterial strain when an outbreak does occur. Microbes infecting hospitalized persons can be analyzed to determine if multiple patients are affected by the same strain, raising the possibility the hospital itself is the source of the infection.
In 1992, an outbreak of food-borne illness occurred in the western United States. The bacterium Escherichia coli O157:H7 was implicated in the outbreak by using traditional microbiological techniques. The PFGE patterns of isolates from infected persons and from hamburger patties from the restaurant where those persons ate were the same. Scientists concluded that the infected persons had acquired the E. coli from that particular restaurant.
The Centers for Disease Control and Prevention (CDC) led the effort to develop a standardized protocol for PFGE for analysis of clinical isolates of E. coli. The materials, methods, and controls are now standardized and can be used to yield reliable results at a number of labs around the world. The protocol served as a platform for the development of PFGE typing of a host of microbial pathogens.
The CDC then developed PulseNet, a national network of public health and food regulatory agency labs. The labs perform PFGE fingerprinting of food-borne bacteria. In addition to E. coli, Salmonella, Shigella, Listeria, and Campylobacter are fingerprinted, and their patterns are entered into the CDC database. Member labs can access the database in real time and can compare clinical specimens.
Clusters of food-borne disease are identified far more quickly, in as little as a week, using PFGE and PulseNet. The origin of the infection can be identified and isolated, preventing further transmission. PFGE has also been used to trace the source and spread of antibiotic resistant tuberculosis. Epidemiologists learned that the infected persons had acquired the infection in the hospital.
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