Pulsed field gel electrophoresis

ESR Scientist Dr Brent Gilpin explains how pulsed field gel electrophoresis is used to separate very large fragments of DNA. DNA from an organism is digested with an enzyme and run on a gel, which gives a barcode that is unique to that organism. This system is used by the PulseNet organisation to identify common sources of disease outbreaks.

Transcript

DR BRENT GILPIN So pulse field gel electrophoresis involves extracting the DNA from your pathogenic organism and digesting it, or cutting it up into fragments with a rare-cutting restriction enzyme. And what this does… from an organism which may have 5 million base pairs, you get 15 to 20 fragments, which if you separate out on a gel – you electrophorese them through a gel – you can get a DNA fingerprint or barcode. And organisms which have a common ancestor, so are from a common source, will have the same DNA fingerprint or barcode, whereas ones from a different source are likely to have a different barcode. So pulse field gel electrophoresis, compared to ordinary gel electrophoresis… because you are separating large fragments of DNA – normal electrophoresis looks at fragments that are 200–1,000 base pairs in size, and in normal electrophoresis, the very large fragments in the 20,000–1,000,000 base pairs wouldn't move into the gel – they just remain stuck at the top of the gel as a big large band. Pulse field gel electrophoresis was developed by alternating the current on the gel, a pulse in one direction is then alternated with a pulse from another direction. So you do a long electrophoresis and you alternate these currents of pulsing in one direction and then the other, and the DNA is forced in one direction and then reorientates itself through another and is able to wiggle its way along the gel. So it’s rather a clever way of getting large fragments to separate.