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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.
Contents
Historical background
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 15–20 kb migrating through a gel will essentially move together in a size-independent manner. At Columbia University in 1984, David C. Schwartz and Charles Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve 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 two 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 60 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely large molecules (up to around 2 Mb), 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
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.