Abstract
The neutral/neutral two-dimensional (2D) agarose gel technique has proved to be a powerful tool for analyzing DNA structure. It was initially adapted and developed by Brewer and Fangman to localize and define bidirectional DNA replication origins on plasmids in yeast (1). Since then, neutral/neutral 2D gels have been used to analyze chromosomal replication origins, the efficiency of replication origin use, direction of replication fork movement, and barriers to DNA replication fork progression (reviewed in ref. 2). In recent years, this technique has also been used to analyze rolling circle replication in T4 phage (3) and topologically complex forms of intact plasmid DNA (4).
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References
Brewer, B. J. and Fangman, W. L. (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471.
Friedman, K. L. and Brewer, B. J. (1995) The analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol. 262, 613–627.
Boulanger, K. G., Mirzayan, C., Kreuzer, H. E., Alberts, B. A., and Kreuzer, K. N. (1996) Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase. Nucleic Acids Res. 24, 2166–2175.
MartÍn-Parras, L., Lucas, I., MartÍnez-Robles, M. L., Hernández, P., Krimer, D. B., Hyrien, O., et al. (1998) Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis. Nucleic Acids Res. 26, 3424–3432.
Lockshon, D., Zweifel, S. G., Freeman-Cook, L. L., Lorimer, H. E., Brewer, B. J., and Fangman, W. L., (1995) A role for recombination junctions in the segregation of mitochondrial DNA in yeast. Cell 81, 947–955.
MacAlpine, D. M., Perlman, P. S., and Butow, R. N. (2000) The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway. EMBO 19, 767–775.
Kunnimalaiyaan, M. and Neilsen, B. L. (1997) Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res. 25, 3681–3686.
Holt, I. J., Lorimer, H. E., and Jacobs, H. T. (2000) Coupled leading-and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524.
Hoffman, C. S. and Winston, F. (1987) A ten-minute DNA preparation from yeast effectively releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272.
Srutkowska, S., Caspi, R., Gabig, M., and Wegrzyn, G. (1999) Detection of DNA replication intermediates after two-dimensional agarose gel electrophoresis using a fluorescein-labeled probe. Anal. Biochem. 269, 221–222.
Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1982) Molecular cloning. A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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© 2002 Humana Press Inc.
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Lorimer, H.E. (2002). 2D Gel Electrophoresis of mtDNA. In: Copeland, W.C. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 197. Humana Press. https://doi.org/10.1385/1-59259-284-8:187
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DOI: https://doi.org/10.1385/1-59259-284-8:187
Publisher Name: Humana Press
Print ISBN: 978-0-89603-972-8
Online ISBN: 978-1-59259-284-5
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