Pulse field gel electrophoresis

Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction.Standard gel electrophoresis techniques for separation of DNA molecules provided huge advantages for molecular biology research. However, 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.

In DNA electrophoresis by the standard method however, DNA molecules larger than 20kb show essentially the same mobility in a static electric field, making differentiation between these DNA molecules impossible.The first attempts to resolve these larger fragments included using low percentage agarose gels and low voltage gradients. Even under these extreme conditions, separation of large DNA molecules was difficult .

In 1984, David Schwartz was able to offer a new technique. He suggested that periodically changing the orientation of the electric field would force DNA molecules in the gel to relax upon the removal of the first field and elongate to align with the new field. It was his assumption that this process should be size dependent. Schwartz was finally able to demonstrate the effectiveness of this technique when he successfully separated yeast chromosomes that were several hundred kilo bases in length.

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

• This method basically involves electrophoresis in agarose where two electric fields are applied alternately at different angles for defined time period (eg.6Osec)
• Activation of first electric field causes the coiled molecules to be stretched in the horizontal plane & start to move through the gel.
• Interruption of this field & application of second field force the molecule to move in the new direction .
• Since there is a length dependent relaxation behavior when long chain molecule undergo conformational change in an electrical field, the smaller a molecule , the quicker it  realigns  itself with new field and is able to moving through the gel. Larger molecules take longer to realign.
• In this way , with continual reversing of the field ,smaller molecule draw ahead and separate 

APPLICATION

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing technique that is used in epidemiological studies worldwide. 

• 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.
• Identifying the course of  bacterial foodborne illness(eg.Salmonella infections).
• The precise analysis provided by PFGE is unparalleled, but the process does require greater investment of funds and time. 

-Dixy