Abstract
The aim of this chapter is to provide a very practical set of instructions that will enable workers with some experience in standard DNA preparation and electrophoresis techniques to prepare and analyze mammalian DNA by pulsed-field gel electrophoresis (PFGE; see also refs. 1 and 2 for additional PFGE protocols). Detailed methods are provided that describe: sources of mammalian DNA, preparation of DNA, restriction enzyme digests, standard PFGE running conditions, preparation of size markers, and preparation, hybridization, and analysis of blots. Notes about potential pitfalls accompany each section together with a trouble-shooting guide in Section 5.
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Smith, C. L. and Cantor, C. R. (1987) Purification, specific fragmentation and separation of large DNA molecules. Methods in Enzymology vol. 155 (Wu, R., ed.), Academic, London, p. 44.
Carle, G. and Olson, M. V. (1987) Orthogonal field alternation gel electrophoresis Meth Enzymol vol. 155 (Wu, R., ed.), Academic, London, p. 468.
Burmeister, M., Monaco, A. P., Gillard, E. F., van Ommen, G. J. B., Affara, N. A., Ferguson-Smith, M. A., Kunkel, L. M., and Lehrach, H. (1988) A 10 megabasepair physical map of human Xp21, including the Duchenne Muscular Dystrophy gene. Genomics 2, 189–202.
Sved, J. and Bird, A. P. (1990) The expected equilibrium of the CpG dinucleotide in vertebrate genomes under a mutation model. Proc. Natl.Acad. Sci. USA 87, 4692–4696.
Bird, A. P. (1986) CpG-rich islands and the function of DNA methylation. Nature 321, 209–213.
Lindsay, S. and Bird, A. P. (1987) Use of restriction enzymes to detect potential gene sequences m mammalian DNA. Nature 327, 336–338.
Bird, A. P. (1989) Two classes of observed frequency for rare cutter sites in CpG islands. Nucleic Acids Res. 17, 9485.
Antequera, F., Boyes, J., and Bird, A. P. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62, 503–514.
Helene, C., Thuong, N. T., Behmoaras, T. S., and Francois, J. C. (1989) Sequencc-specific artificial endonucleases. Trends Biotechnol. 7, 310–315.
Bućan, M., Zimmer, M., Whaley, W. L., Poustka, A., Youngman, S., Allitto, B. A., Ormondroyd, E., Smith, B., Pohl, T. M., McDonald, M., etal. (1990) physical maps of 4p16.3, the area expected to contain the Huntingtons disease mutation. Genomics 6, 1–15.
Barlow, D. P. and Lehrach, H. (1990) Partial Not I digests generated by low enzyme concentration or the presence of ethidium bromide can be used to extend the range of pulsed-field gel mapping, Technique 2, 79–87.
Hanish, J. and McClelland, M. (1989) Controlled partial restriction digestions of DNA by competition with modification methyltransferases. Analytical Biochemistry 179, 357–360.
Schwartz, D. C. and Cantor, C. R. (1984) Separation of yeast chromosome-sized DNAs by pulsed-field gradient gel electrophoresis. Cell 37, 67–75.
Chu, G., Vollrath, D., and Davies, R. W. (1986) Separation of large DNA molecules by Contour-Clamped Homogeneous electric fields. Science 234, 1582–1585.
Gardiner, K., Laas, W., and Patterson, D. (1986) Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient electrophoresis. Somatic Cell Mol. Gen. 12, 185–195.
Southern, E. M., Anand, R., Brown, W. R. A., and Fletcher, D. S. (1987) A model for dle separation of large DNA molecules by crossed-field gel electrophoresis. Nucleic Acids Res. 15, 5925–5943.
De Jonge, P., De Jongh, F. C. M., Meijers, R., Steensma, H. Y., and Scheffers, W. A. (1986) Orthogonal field alternation gel electrophoresis banding patterns of DNA from yeast. Yeast 2, 193–204.
Herrmann, B. G., Barlow, D. P., and Lehrach, H. (1987) A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion. Cell 48, 813–825.
Barlow, D. P., Bućan, M., Lehrach, H., Hogan, B. L. M., and Cough, N. (1987) Close genetic and physical linkage between the murine haematopoetic growth factor genes GM-CSF and Multi-CSF (IL-3). EMBO J. 6, 617–623.
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.
Smith, C. L., Matsumoto, T., Niwa, O., Klco, S., Fan, J. B., Yanagida, M., and Cantor, C. R. (1987) An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed-field gel electrophoresis. Nucleic Acids Res. 15, 4481–4489.
Gerring, S. L., Connelly, C., and Hieter, P. (1991) Positional mapping of genes by chromosome blotting and chromosomal fragmentation. Methods in Enzymology vol. 194 (Guthrie, C. and Fink, G. E., eds.), Academic, London, p. 57–77.
Link, A. J. and Olson, M. V. (1991) Physical map of the Saccharomyces cerevisiae genome at 110 kilobase resolution. Genetics 127, 681–698.
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P., D. (1992). Preparation, Restriction, and Hybridization Analysis of Mammalian Genomic DNA for Pulsed-Field Gel Electrophoresis. In: Burmeister, M., Ulanovsky, L. (eds) Pulsed-Field Gel Electrophoresis. Methods in Molecular Biology™, vol 12. Humana Press. https://doi.org/10.1385/0-89603-229-9:107
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DOI: https://doi.org/10.1385/0-89603-229-9:107
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