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Pulsed-field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a highly discriminatory method for the differentiation of bacterial and fungal isolates based on differences in DNA content. Because chromosome composition is subject to mutation or alteration with each generation organisms that are close to one another in generation time will have few genetic differences i.e. are clones of one another or clonally related.

This method uses infrequent-cutting restriction enzymes to digest extracted genomic DNA into about 10 to 20 fragments. The fragments are then separated by PFGE to determine if the banding pattern of the fragments is the same for one or more isolates. This banding pattern is the DNA fingerprint. Clonal isolates will have the same sites for restriction enzymes and thus, indistinguishable fingerprints. Organisms which have few differences in fingerprint bands may be related to one another. Isolates which are unrelated will have distinct banding patterns.

Fungal yeast forms have multiple chromosomes that may be separated by PFGE without restriction enzyme digestion. For some species the number of chromosomes and differences (polymorphisms) in size are sufficient to yield unique banding patterns. Such fingerprints are termed the electrophoretic karyotype.

To perform PFGE genomic DNA is carefully extracted from pure culture. The cells to be analyzed are immobilized in agarose during the extraction process so that no unwanted breaks are made in the chromosome. Cells walls are broken with lysis enzymes and detergents. The DNA is purified, washed and may be cut with a selected restriction enzyme. For the nosocomial pathogens frequently tested with PFGE typical fingerprints are made up of restriction digestion fragments which range between 20 and 500 kilobase pairs (kb).

PFGE is a unique type of electrophoresis designed to separate DNA's too large to be separated by conventional gel electrophoresis. Fragments from about 20 kb up to several megabasepairs may be analyzed by this method. The electrodes are arranged in a hexagon. Current is then pulsed or switched between electric fields aligned at 120° angles (for most protocols) to one other on the hexagon. This angle and an increasing switching time from a few seconds to up to several minutes allows a wide range of band sizes to be separated.

Interpretation of PFGE Results

The banding pattern in each lane is compared visually to that in every other lane. Computer software may be used to assist in lane-to-lane and gel-to-gel comparisons. For band size determinations and lane comparisons a molecular weight marker is included on each end of the gel. Band differences are interpreted according to the following table taken from the consensus paper for PFGE interpretation in outbreak settings authored by Tenover, et al. in J. of Clin. Micro. 1995;33:2233-2239.

No. Band Differences	No. Genetic Differences	Interpretation	Epidemiologic Significance
0	0	Indistinguishable	Isolates are same strain type.
1-3	1	Closely Related	Isolates are probably related.
4-6	2	Possibly Related	Isolates are possibly related.
=>7	=>3	Different	Isolates are different

Isolates which are the same strain type (no band differences) are given a common strain letter or number i.e strain JHH-A. Closely related isolates will have from one to three bands different from one another. Closely related isolates are considered subtypes of one another and designated with the same letter, but with a different subtype number. For example: JHH-A1, JHH-A2, etc. An isolate which differs by more than 3 bands is given a new letter to denote its strain type i.e. JHH-B, JHH-C, etc.

Every isolate on each gel is interpreted according to these guidelines. Control isolates should be a different strain type than the outbreak strain. A type-written report is generated which includes a description of isolates, the epidemiologic question or reason or testing, the gel image and an interpretation. See Sample Report.