Neutral-Neutral 2-Dimensional Agarose Gel Electrophoresis for Visualization of E. coli DNA Replication Structures

Karla A Mettrick; Georgia M Weaver; Ian Grainge

doi:10.1007/978-1-0716-0323-9_5

Neutral-Neutral 2-Dimensional Agarose Gel Electrophoresis for Visualization of E. coli DNA Replication Structures

Methods Mol Biol. 2020:2119:61-72. doi: 10.1007/978-1-0716-0323-9_5.

Authors

Karla A Mettrick¹, Georgia M Weaver¹, Ian Grainge²

Affiliations

¹ School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia.
² School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia. ian.grainge@newcastle.edu.au.

PMID: 31989514
DOI: 10.1007/978-1-0716-0323-9_5

Abstract

Neutral-neutral 2-dimensional agarose gel electrophoresis enables the detection of replication intermediate structures in DNA. Here we describe how DNA from Escherichia coli cells can be purified to retain replication intermediates and then be separated by size and shape using two consecutive agarose gel electrophoresis protocols. The DNA structures present within a localized region can be visualized by a Southern blotting/radioactive hybridisation protocol.

Keywords: 2-dimensional agarose gel; DNA replication; Southern blotting; replication intermediates.

MeSH terms

Blotting, Southern*
DNA Replication*
DNA, Bacterial* / analysis
DNA, Bacterial* / genetics
DNA, Bacterial* / metabolism
Electrophoresis, Agar Gel
Electrophoresis, Gel, Two-Dimensional*
Escherichia coli* / genetics
Escherichia coli* / metabolism

Substances

DNA, Bacterial