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Pulsed field gel electrophoresis
Additional recommended knowledge
Standard gel electrophoresis 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 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.
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 reversed periodically to make each band of DNA run in the opposite direction for a set time. A net forward direction of the DNA is achieved by setting the voltage in the forward direction for a certain duration, followed by a shorter pulse duration in the reverse direction. For example, one can run the gel in the forward direction for .5 seconds followed by a reverse pulse for .25 seconds. For higher resolutions of even larger bands, these times can be increased, such as 3 seconds forward and 1 second reverse. Finally, for extremely large bands (up to around 2000kb), switching-interval ramps can be used that increases the pulse time for both directions over the course of a number of hours--take, for instance, increasing the pulse linearly from 9 seconds at 0 hours to 60 seconds at 18 hours with a reverse pulse equal to a third of the forward pulse (3-20 seconds).
Needless to say, this procedure takes longer than normal gel electrophoresis due to the fact that DNA backtracks up the gel periodically before proceeding onwards in the forward direction.
The theory behind why PFGE works pertains to the mobility of larger DNA fragments. While in general small fragments can wind their way through the gel matrix more easily than large DNA fragments, a threshold length exists where all large fragments will run at the same rate. But with a continuous changing of directions every few seconds or fraction of a second, the various lengths of DNA react to the change at differing rates. That is, larger pieces of DNA will be slower to begin moving in the opposite direction while smaller pieces will be quicker to change direction. 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 possible.
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 monocytgenes and thus to link environmental or food isolates with clinical infections.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Pulsed_field_gel_electrophoresis". A list of authors is available in Wikipedia.|