Abstract
Pulsed- field gel electrophoresis (PFGE) was first described by Schwartz and Cantor (1) It is now an umbrella term for the alternating of an electric field in more than one direction through a solid matrix to achieve the separation of DNA fragments. The method requires the preparation of unsheared DNA, digestion of the DNA using a rare-cutting restriction endonuclease, separation of fragments by PFGE, and the visualization and interpretation of banding patterns Fig. 1.Conventional agarose gel electrophoresis employs a static field and can resolve DNA fragments up to 50 kilobases (kb), although in practice, fragments larger than 20 kb co-migrate under the conditions usually applied. By introducing a pulse or change in the direction of the electric field, fragments as large as 10 megabases (Mb) can be separated. The time required by DNA fragments of different sizes to reorientate to the new electric field is proportional to their molecular weight and it is this factor that allows the separation and focusing of DNA fragments.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schwartz, D C and Cantor, C R (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis Cell 37, 67ā75
Grundman, H, Schneider, C, Hartung, D, Daschner, F D, and Pitt, T L (1995) Discriminatory power of three DNA-based typing techinques forPseudomonas aerugmosa. J Clin Microbiol 33, 528ā534
Carle, G C., Frank, M, and Olson, M V(1986) Electrophoretic separation of large DNA molecules by periodic inversion of the electric field Science 234, 65
Chu, G, Vollrath, D, and Davis, R W (1986) Separation of large DNA molecules by contour clamped homogeneous electric field. Science 234, 1582
Barton, B M, Harding, G P, and Zuccarelli, A J (1995) A general method for detecting and sizing large plasmids. Anal Blochem. 226, 235ā240
Romling, U., Grothues, D, Heuer, T., and Tummler, B (1992) Physical genome analysis of bacteria Electrophoresis 13, 62ā631.
Khattak, M. N., Burnie, J, Matthews, R. C., and Oppenheim, B.(1992) Clamped homogeneous electric field gel electrophoresis typing of Torulopsis glabrata isolates causing nosocomial infections. J. Clm Microbiol 30, 221lā2215.
Bostock, A., Khattak, M N, Matthews, R., and Bunie, J(1993) Comparison of PCR fingerprinting, by random amphfication of polymorphic DNA, with other molecular typing methods for Candida albicans. J. Gen Microbiol 139, 2179ā2184
Goering, R. V and Duensing, T. D. (1990)Raprd field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci.J Clan Microbiol 28, 426ā429
Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R., and Wemstock, G. M. (1990) Companson of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites J Clin Mtcrobiol 28, 1059ā1063
Brosch, R, Buchrieser, C., and Rocourt, J(1991) Subtyping of Listeria monocytogenes serovar 4b by use of low-frequency-cleavage restriction endonucleases and pulsed-field gel electrophoresis Res Microbiol 142, 667ā675
Zhang, Y, Mazurek, G H, Cave, M D, Eisenach, K D., Pang, Y, Murphy, D T, and Wallace, R. J(1992) DNA polymorphisms in strains of mycobacterium tuberculosrs analysed by pulsed-field gel electrophoresrs: a tool for epidemiology.J Clrn Mlcrobrol 30, 1551ā1556
Khattak, M. N., Matthews, R C., and Burnie, J. P.(1992)Is Bordetella pertussis clonal? BMJ 304, 813ā815
Tenover, F C, Arbeit, R D., Goering, R. V, Mickelsen, P A, Murray, B E, Persing, D H, and Swaminathan, B.(1995) Interpreting chromosomal DNA restrictron patterns produced by pulsed-field gel electrophoresrs. Criteria for bacterial strain typing J Clin Mlcrobiol. 33, 2233ā2239.
Goering, R V (1993) Molecular epidenirology of nosocomial infection. Analysis of chromosomal restriction fragment patterns by pulsed-field gel electrophoresis Infect Control Hospital Epldemrol 14, 595ā600
Struelens, M J, Deplano, A, Godard, C, Maes, N., and Serruys, E.(1992)Epldemiological typing and delineation of genetic relatedness of methlcillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis J Clan Mlcrobiol 30, 2599ā2605
Gibson, J R., Sutherland, K., and Owen, R. J.(1994) Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter Jejuni. Lett Appl Mlcrobiol 19, 357ā358
Forbes, K J., Bruce, D B., Jordens, Z., Ball, A., and Pennington, T. H(1991) Rapid methods in bacterial DNA fingerprinting. J Gen Mircobiol 137, 205lā2058
Linhardt, F., Zlebuhr, W., Meyer, P, Wrtte, W., and Hacker, J. (1992) Pulsedfield gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci. FEMS Mlcrobiol Lett. 95, 18lāl86
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 1998 Humana Press Inc.
About this protocol
Cite this protocol
Kaufmann, M.E. (1998). Pulsed-Field Gel Electrophoresis. In: Woodford, N., Johnson, A.P. (eds) Molecular Bacteriology. Methods in Molecular Medicineā¢, vol 15. Humana Press. https://doi.org/10.1385/0-89603-498-4:33
Download citation
DOI: https://doi.org/10.1385/0-89603-498-4:33
Publisher Name: Humana Press
Print ISBN: 978-0-89603-498-3
Online ISBN: 978-1-59259-599-0
eBook Packages: Springer Protocols