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Pulsed-Field Gel Electrophoresis

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P243-245

2026-04-27

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The agarose gel methods for DNA described above can fractionate DNA of 60 kb or less. The introduction of pulsed-fi eld gel electrophoresis (PFGE) and the further development of variations on the basic technique means that nowadays DNA fragments up to 2 × 10³ kb can be separated. Essentially, this allows the separation of whole chromosomes by electrophoresis. The method basically involves electrophoresis in agarose, where two electric fields are applied alternately at different angles for defined time periods (e.g. 60 s). Activation of the first electric field causes the coiled molecules to be stretched in the horizontal plane and start to move through the gel. Interruption of this field and application of the second field force the molecule to move in the new direction. Owing to a length-dependent relaxation behaviour when a long-chain molecule undergoes conformational change in an electric fi eld, the smaller a molecule, the quicker it realigns itself with the new field and is able to continue moving through the gel. Larger molecules take longer to realign. In this way, with continual reversing of the field, smaller molecules draw ahead of larger molecules and separate according to size. PFGE has proved particularly useful in identifying the course of outbreaks of bacterial food-borne illness (e.g. Salmonella infections). Having isolated the bacterial pathogen responsible for the illness from an individual, the DNA is isolated and cleaved into large fragments, which are separated by PFGE. For example, DNA from Salmonella species, when digested with the restriction enzyme Xba I, gives around 15 fragments ranging from 25 kb to 680 kb. This pattern of fragments, or ‘ fingerprint’, is unique to that strain. If the same fingerprint is found from bacteria from other infected people, then it can be assumed that they were all infected from a common source. Thus, by comparing their eating habits, food sources, etc. the source of the infection can be traced to a restaurant, food item, etc. Figure 1 shows the restriction patterns from different strains of Neisseria meningitidis.

Fig1. PFGE separation of the digestion pattern produced with the restriction enzyme Nhe I, of 21 strains of Neisseria meningitidis. There are two molecular-weight marker tracks at either end of the gel. (Courtesy of Dr Giovanna Morelli, Max Planck Institute for Molecular Genetics, Berlin, Germany.)