Abstract
During the past few years, there has been intense activity in physically mapping the genomes of a variety of organisms, including human, mouse, Drosophila, Caenorhabditis elegans, Arabidopsis thaliana, and yeast. The considerable advances made in physically mapping these genomes has demanded the development of a technique capable of filling the niche between conventional Southern blotting and such approaches as somatic cell genetics and fluorescence in situ hybridization (FISH). Pulsed-field gel electrophoresis (PFGE) fills this niche, and since its inception in 1983, has evolved into a robust and reliable technique. PFGE was first used for electrophoretically karyotyping yeasts and protozoa, but with the advent of commercially available rare cutter restriction enzymes, the technique was soon used for the long-range physical mapping of bacterial, yeast, and eukaryotic genomes.
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References
Poustka, A., Pohl, T., Barlow, D. P., Zehetner, G., Craig, A., Michiels, F., Ehrich, E., Frischauf, A.-M., and Lehrach, H. (1986) Molecular approaches to mammalian genetics. Cold Spring Harbor Symp. on Quant. Biol. 51, 131–139.
Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806–812.
Shibasaki, Y., Maule, J. C., Devon, R. S., Slorach, E. M., Gosden, J. R., Porteous, D. J., and Brookes, A. J. (1995) Catch-linker+PCR labeling a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes. PCR Methods Appl. 4, 209–211.
Monaco, A. P. and Larin, Z. (1994) YACs, BACs, PACs and MACs: artificial chromosomes as research tools. Trends Biotechnol. 12, 280–286.
Bickmore, W. A. and Bird, A. P. (1992) Use of restriction enzymes to detect and isolate genes from mammalian cells. Methods Enzymol. 216, 224–245.
Antequera, F., Boyes, J., and Bird, A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands. Cell 62, 503–514.
Maule, J. C. (1995) Colored microparticles for clear visualization of agarose beads and plugs. Trends Genet. 11, 127.
Jones, C. P., Janson, M., and Nordenskjold, M. (1989) Separation of yeast chromosomes in the megabase range suitable as size markers for pulsed-field gel electrophoresis. Technique 1, 90–95.
Wang, Y.-K. and Schwartz, D. C. (1993) Chopped inserts: a convenient alternative to agarose/DNA inserts or beads. Nucleic Acids Res. 21, 2528.
Orbach, M. J., Vollrath, D., Davis, W., and Yanofsky, C. (1988) An electrophoretic karyotype of Neurospora crassa. Mol. Cell Biol. 8, 1469–1473.
Chu, G., Vollrath, D., and Davis, R. W. (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582–1585.
Clark, S. M., Lai, E., Birren, B. W., and Hood, L. (1988) A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields. Science 241, 1203–1205.
Arker, W., Enquist, L., Hohn, B., Murray, N. E., and Murray, K. (1983) Experimental methods for use with lambda, in Lambda II (Hendrtx, R. W., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 433–466.
Maule, J. C. and Green, D. K. (1990) Semiconductor-controlled contour-clamped homogeneous electric field apparatus. Anal. Biochem. 191, 390–395.
Porteous, D. J. and Maule, J. C. (1990) Casting multiple aliquots of agarose embedded cells for PFGE analysis. Trends Genet. 6, 346.
Guido, E. C. and Abhay, K. (1994) Simple method to reduce background on autoradiographs. BioTechniques 17, 294.
Albertsen, H. M., Le Paslier, D., Abderrahim, H., Dausset, J., Cann, H., and Cohen, D. (1989) Improved control of partial DNA restriction enzyme digest in agarose using limiting concentrations of Mg++. Nucleic Acids Res. 17, 808.
Barlow, D. P. and Lehrach, H. (1990) Partial Not1 digests, generated by low enzyme concentration or the presence of ethidium in bromide, can be used to exten dthe range of pulsed-field gel mapping. Technique 2, 79–87.
Wilson, W. W. and Hoffman, R. M. (1990) Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed field electrophoresis. Anal. Biochem. 191, 370–375.
Topal, M. D., Thresher, R. J., Conrad, M., and Griffith, J. (1991) Nael endonuclease binding to pBR322 DNA induces looping. Biochemistry 30, 2006–2010.
Carle, G. F. and Olson, M. V. (1985) An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82, 3756–3760.
Smith, C. L., Klco, S. R., and Cantor, C. R. (1988) Pulsed-field gel electrophoresis and the technology of large DNA molecules, in Genome. Analysis (Davies, K. E., ed.), IRL, Oxford, UK, pp. 41–71.
Mathew, M. K., Smith, C. L., and Cantor, C. R. (1988) High-resolution separation and accurate size determination in pulsed-field gel electrophoresis: DNA size standards and the effect of agarose and temperature. Biochemistry 27, 9204–9210.
New England Biolabs Catalog (1995) Beverly, MA, p. 224.
White, H. W. (1992) Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects. BioTechniques 12, 574–579.
Birren, B. W., Lai, E., Clark, S. M., Hood, L., and Simon, M. I. (1988) Optimized conditions for pulsed field gel electrophoretic separations of DNA. Nucleic Acids Res. 16, 7563–7582.
Birren, B. W., Hood, L., and Lai, E. (1989) Pulsed field gel electrophoresis studies of DNA migration made with the PACE electrophoresis system. Electrophoresis 10, 302–309.
Vollrath, D. and Davis, R. W. (1987) Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 15, 7865–7876.
Gunderson, K. and Chu, G. (1991) Pulsed-field electrophoresis of megabase-sized DNA. Mol. Cell Biol. 11, 3348–3354.
Smith, D. R. (1990) Genomic long-range restriction mapping. Methods 1, 195–203.
Reed, K. C. and Mann, D. A. (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13, 7207–7221.
Chikashige, Y., Kinoshita, N., Nakaseko, Y., Matsumoto, T., Murakami, S., Niwa, O., and Yanagida, M. (1989) Composite motifs and repeat symmetry in S. pombe centromeres. Cell 57, 739–751.
Stoye, J. P., Frankel, W. N., and Coffin, J. M. (1991) DNA hybridization in dried gels with fragmented probes: an improvement over blotting techniques. Technique 3, 123–128.
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© 1997 Humana Press Inc., Totowa, NJ
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Maule, J. (1997). Physical Mapping by Pulsed-Field Gel Electrophoresis. In: Boultwood, J. (eds) Gene Isolation and Mapping Protocols. Methods in Molecular Biology™, vol 68. Humana Press. https://doi.org/10.1385/0-89603-482-8:93
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DOI: https://doi.org/10.1385/0-89603-482-8:93
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