Abstract
The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.
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Acknowledgement
Work in the Aparicio lab is supported by NIH grant GM065494.
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Villwock, S.K., Aparicio, O.M. (2014). Two-Dimensional Agarose Gel Electrophoresis for Analysis of DNA Replication. In: Smith, J., Burke, D. (eds) Yeast Genetics. Methods in Molecular Biology, vol 1205. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1363-3_19
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DOI: https://doi.org/10.1007/978-1-4939-1363-3_19
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1362-6
Online ISBN: 978-1-4939-1363-3
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