Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 (DNA) molecule

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s by applying an electric field that periodically changes direction to a gel matrix.

Historical background

Standard gel electrophoresis

DNA electrophoresis

Nucleic acid electrophoresis is an analytical technique used to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to migrate toward the anode, due to the net...

 techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, many limitations existed with the standard protocol in that it was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15-20kb migrating through a gel will essentially move together in a size-independent manner. At Columbia University in 1984, Schwartz and Cantor

Charles Cantor

Charles Cantor is an American molecular geneticist who, in conjunction with David Schwartz, developed pulse field gel electrophoresis for very large DNA molecules....

 developed a variation on the standard protocol by introducing an alternating voltage gradient to better the resolution of larger molecules. This technique became known as Pulsed Field Gel Electrophoresis (PFGE). The development of PFGE expanded the range of resolution for DNA fragments by as much as 2 orders of magnitude.

Procedure

The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 120 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely large bands (up to around 2Mb), switching-interval ramps can be used that increases the pulse time for each direction over the course of a number of hours—take, for instance, increasing the pulse linearly from 10 seconds at 0 hours to 60 seconds at 18 hours.

This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel.

Theory

The theory behind why PFGE works pertains to the mobility of larger DNA fragments (though some finer mechanical aspects may be unclear). While in general small fragments can find their way through the gel matrix more easily than large DNA fragments, a threshold length exists above 30–50 kb where all large fragments will run at the same rate, and appear in a gel as a single large diffuse band. However, with periodic changing of field direction, the various lengths of DNA react to the change at differing rates. That is, larger pieces of DNA will be slower to realign their charge when field direction is changed, while smaller pieces will be quicker. Over the course of time with the consistent changing of directions, each band will begin to separate more and more even at very large lengths. Thus separation of very large DNA pieces using PFGE is made possible.

Applications

PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. Subtyping has made it easier to discriminate among strains of Listeria monocytogenes and thus to link environmental or food isolates with clinical infections.