Pulsed field gel electrophoresis
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[edit] Historical Background
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.[1] 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.
[edit] 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 between 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 9 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.
[edit] Theory
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 align their charge to 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.
[edit] 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.
[edit] References
- ^ Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75.
