Pulsed-field gel electrophoresis
laboratory technique for separation of DNA
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. The mechanism driving these separations exploits the fact that very large DNA molecules unravel and "snake" through a gel matrix, and such electrophoretic trajectories are perturbed in a size-dependent manner by carefully oriented electrical pulses. Unlike standard agarose gel electrophoresis, which can separate DNA fragments of up to 50 kb, PFGE resolves fragments up to 10 Mb. This allows for the direct analysis of genomic DNA.
History
In 1984, David C. Schwartz and Charles Cantor published the first successful application of alternating electric fields for the separation of large DNA molecules. 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.
Procedure
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. The application of the voltage can change depending on the variation of PFGE used.
Applications
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.
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