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Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1837)

Abstract

This protocol describes the application of atomic force microscopy for structural analysis of the prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables step-wise dissection of the nucleoid. The procedure includes (1) on-substrate-lysis of cells, and (2) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural re-organization or building up of basic structures, as seen for Dps in Escherichia coli, and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.

Key words

Nucleoid structure On-substrate lysis Reconstitution of nucleoid Atomic force microscopy Data analysis 
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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of MedicineUniversity of TsukubaTsukubaJapan
  2. 2.Center for BiotechnologyNational Taiwan UniversityTaipeiTaiwan
  3. 3.Department of BacteriologyOsaka Dental UniversityHirakataJapan
  4. 4.Graduate School of BiostudiesKyoto UniversityKyotoJapan

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