What is pulsed-field gel electrophoresis?

Definition

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.

Applications

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.

Public Health

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.

Impact

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.

Bibliography

Brachman, Philip S., and Elias Abrutyn, eds. Bacterial Infections of Humans: Epidemiology and Control. 4th ed. New York: Springer, 2009.

Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. Bailey and Scott’s Diagnostic Microbiology. 12th ed. St. Louis, Mo.: Mosby/Elsevier, 2007.

Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology: An Introduction. 10th ed. San Francisco: Benjamin Cummings, 2010.