Abstract
Pulsed-field gel electrophoresis (PFGE) has been successfully used to generate physical maps of regions of human chromosomes near genes or single copy probes. However, many regions of the human genome contain gene families that often include pseudogenes. Other regions contain stretches of repeats. Conventional PFGE requires a series of single copy probes, which are sometimes difficult if not impossible to find for clusters of homologous genes or repeats. If probes detect several different genes that are dispersed on a chromosome, standard PFGE techniques will not be useful for the generation of large scale restriction maps. To overcome this problem, we have developed a method of two-dimensional DNA electrophoresis (2D-DE). The 2D-DE method consists of two DNA digestion steps and two electrophoretic separation steps (shown in Fig. 1)):
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References
Berman, J. E., Mellis, S.J., Pollock, R., Smith, C. L., Suh, H., Heinke, B., Kowal, C, Surti, U., Chess, L., Cantor, C.L., and Alt, F. W. (1988) Content and organization of the human Ig VH locus: Definition of three new VH families and linkage to the Ig CH locus. EMBOJ. 7, 727–738.
Schroeder H. W., Hillson, J.L., and Perlmutter, R. M. (1987) Early restriction of human antibody repertoire. Science 238, 791–793.
Walter, M. A. and Cox, D. W. (1989) A method for two dimensional DNA electrophoresis (2D-DE): Application to the immunoglobulin heavy chain variable region. Genomics 5, 157–159.
Church, G.M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci.USA 81, 1991–1995.
Boyum, A. (1968) Isolation of lymphocytes, granulocytes and macrophages. Scand.J. Invest. 5(Suppl.), 9–15.
Gardiner, K., Laas, W., and Patterson, D. (1986) Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Som. Cell Mol. Genet. 12, 185–195.
Walter, M. A., Surti, U., Hofker, M. H., and Cox, D. W. (1990) The physical organization of the human immunoglobulin heavy chain gene complex. EMBO J. 9, 3303–3313.
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© 1992 The Humana Press Inc.
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Walter, M.A., Cox, D.W. (1992). Two-Dimensional DNA Electrophoresis (2D-DE) for Mammalian DNA. In: Burmeister, M., Ulanovsky, L. (eds) Pulsed-Field Gel Electrophoresis. Methods in Molecular Biology™, vol 12. Humana Press. https://doi.org/10.1385/0-89603-229-9:285
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DOI: https://doi.org/10.1385/0-89603-229-9:285
Publisher Name: Humana Press
Print ISBN: 978-0-89603-229-3
Online ISBN: 978-1-59259-499-3
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