DNA Electrophoresis: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Introduction and Perspectives of DNA Electrophoresis
	1 Introduction
	2 2D Gel Electrophoresis
	3 Comet Assay
	4 Analysis of DSBs by PFGE
	5 Other Applications
	References
Chapter 2: Two-Dimensional Gel Electrophoresis to Resolve DNA Topoisomers
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 3: Using Two-Dimensional Intact Mitochondrial DNA (mtDNA) Agarose Gel Electrophoresis (2D-IMAGE) to Detect Changes in ...
	1 Introduction
	2 Materials
		2.1 Cultured Cells or Tissue Collection and Storage
		2.2 DNA Isolation
		2.3 1D and 2D-IMAGE
		2.4 Southern Blotting (Transfer and Hybridization)
	3 Methods
		3.1 Collection of Tissues and Cells (see Fig. 1)
		3.2 DNA Isolation from Tissues or Cells (see Fig. 2)
		3.3 1D- and 2D-IMAGE (see Fig. 3)
			3.3.1 First Dimension (Day 1) (see Fig. 3a)
			3.3.2 Second Dimension (Day 2) (see Fig. 3b)
		3.4 Gel Transfer (Day 3) (see Fig. 4)
		3.5 Hybridization (Day 4)
			3.5.1 Prehybridization
			3.5.2 Probe Synthesis
			3.5.3 Blot Washing
	4 Notes
	References
Chapter 4: 2D Gel Electrophoresis to Detect DNA Replication and Recombination Intermediates in Budding Yeast
	1 Introduction
	2 Materials
		2.1 Samples Collection and Psoralen DNA Cross-Linking
		2.2 DNA Extraction with CTAB Method
		2.3 DNA Digestion
		2.4 2D Gel Electrophoresis
		2.5 Southern Blot
	3 Methods
		3.1 Samples Collection and UV Psoralen DNA Cross-Linking
		3.2 DNA Extraction with CTAB Method
		3.3 DNA Digestion
		3.4 2D Gel Electrophoresis
		3.5 Southern Blot
		3.6 Filter Stripping (See Note 15)
	4 Notes
	References
Chapter 5: Neutral-Neutral 2-Dimensional Agarose Gel Electrophoresis for Visualization of E. coli DNA Replication Structures
	1 Introduction
	2 Materials
		2.1 Preparation of Agarose Plugs
		2.2 2D Gel Electrophoresis
		2.3 Southern Hybridization
	3 Methods
		3.1 Sample Preparation for 2D Gel Electrophoresis
			3.1.1 Sample Collection
			3.1.2 Preparation of Agarose Plugs
		3.2 Two-Dimensional Agarose Gel Electrophoresis
		3.3 Southern Hybridization
			3.3.1 Transfer
			3.3.2 Probe Preparation
			3.3.3 Hybridization
	4 Notes
	References
Chapter 6: Alkali Comet Assay in Genotoxicity Tests
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 Alkaline Gel Electrophoresis
	3 Methods
		3.1 Sample Preparation
		3.2 Alkaline Gel Electrophoresis
	4 Notes
	References
Chapter 7: Analysis of DNA Interstrand Cross-Links and their Repair by Modified Comet Assay
	1 Introduction
	2 Materials
		2.1 Chemicals and Gel Reagents
		2.2 Equipment
	3 Methods
		3.1 Slide and Cell Preparation
		3.2 Cell Treatment and Sample Processing
		3.3 Slide Electrophoresis
		3.4 Sample Staining
		3.5 Data Acquisition and Analysis
	4 Notes
	References
Chapter 8: Analysis of Chromosomal DNA Fragmentation in Apoptosis by Pulsed-Field Gel Electrophoresis
	1 Introduction
	2 Materials
		2.1 Tissue Culture and Drug Treatment
		2.2 Preparation of Plugs for PFGE
		2.3 Running PFGE
	3 Methods
		3.1 Sample Preparation for PFGE
		3.2 PFGE Using Biometra´s Apparatus
	4 Notes
	References
Chapter 9: Detection of DNA Damage-Induced DSBs by the Contour-Clamped Homogeneous Electric Field (CHEF) System in Mammalian C...
	1 Introduction
	2 Materials
		2.1 Cell Culture and Treatment with DNA Damage Agents
		2.2 Buffers
		2.3 Pulsed-Field Gel Electrophoresis and Imaging
	3 Methods
		3.1 Cell Culture and Induction of DSBs
		3.2 Preparation of Agarose Sample Plugs
		3.3 Pulsed Field Gel Electrophoresis Using CHEF
		3.4 Scaling up the Experimental Process
	4 Notes
	References
Chapter 10: Investigation of DNA Double-Strand Breaks Induced in Host Cells Following Infection with Genotoxic Bacteria
	1 Introduction
	2 Materials
		2.1 Cell and Bacterial Culture
		2.2 PFGE
			2.2.1 Preparation of Plugs
			2.2.2 Running PFGE Gels
		2.3 Immunoblotting
	3 Methods
		3.1 Bacterial and Human Cell Culture
			3.1.1 Human Cell Culture
			3.1.2 Bacterial Cell Culture
		3.2 Sample Preparation for PFGE
		3.3 PFGE
		3.4 Immunoblotting
	4 Notes
	References
Chapter 11: Monitoring of DNA Replication and DNA Double-Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electro...
	1 Introduction
	2 Materials
		2.1 Culture Media and Supplements
		2.2 Yeast Strains
		2.3 Yeast DNA in Agarose Plugs
		2.4 CHEF Pulsed-Field Gel
	3 Methods
		3.1 Preparation of Agarose Plugs
		3.2 Preparing the Pulsed-Field Gel
		3.3 Running the Pulsed-Field Gel
		3.4 Detecting the Chromosomal Bands
		3.5 Monitoring the Replication Status in Synchronized Cells by PFGE
		3.6 Monitoring the Repair Status During DSBs by PFGE
	4 Notes
	References
Chapter 12: Pulsed-Field Gel Electrophoresis for Detecting Chromosomal DNA Breakage in Fission Yeast
	1 Introduction
	2 Materials
		2.1 Media for Fission Yeast Culture and DNA Damaging Agents
		2.2 Preparation of Agarose Plugs
		2.3 Pulsed-Field Gel Electrophoresis and Image Acquisition
	3 Methods
		3.1 Induction of DNA Damage by Drugs
		3.2 Induction of DNA Damage by Meiosis
		3.3 Preparation of Agarose Plugs
		3.4 Pulsed-Field Gel Electrophoresis
	4 Notes
	References
Chapter 13: Detection of DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis of Circular Bacterial Chromosomes
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 Electrophoresis
	3 Methods
		3.1 Preparing Plugs
		3.2 In-Plug Reactions
		3.3 Preparing Electrophoresis Gel
		3.4 Electrophoresis
	4 Notes
	References
Chapter 14: Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Us...
	1 Introduction
	2 Materials
		2.1 Bacterial Culture
		2.2 Sample Preparation (Plugs for PFGE)
		2.3 PFGE
	3 Methods
		3.1 Plugs Preparation
		3.2 PFGE
	4 Notes
	References
Chapter 15: Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells
	1 Introduction
	2 Materials
		2.1 Cell Lysis
		2.2 Column Chromatography
		2.3 Linear DNA Removal
		2.4 Quantification of DNA
		2.5 Concentrate DNA (Optional)
		2.6 Rolling-Circle Amplification & Sequencing
	3 Methods
		3.1 Cell Lysis
		3.2 Column Chromatography
		3.3 Linear DNA Removal
		3.4 Quantify DNA (Optional Step)
		3.5 Clean Up and Concentrate DNA (Recommended)
		3.6 Rolling Circle Amplification
		3.7 DNA Library Preparation and Sequencing
		3.8 Data Analysis
	4 Notes
	References
Chapter 16: Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation
	1 Introduction
	2 Materials
		2.1 TFO-Conjugated Plasmid for IDAP
		2.2 Human Nuclear Extracts for IDAP
		2.3 Nucleoprotein Isolation Via IDAP
		2.4 IDAP Under Cross-Linking Conditions
		2.5 A Specific DNA Substrate for IDAP
		2.6 Specific Capture of the Constructed Plasmids in IDAP
		2.7 Cross-Linking Conditions of a Human Cell Line for CoIFI
		2.8 Input Sample Preparation for CoIFI
		2.9 Nucleoprotein Isolation Via CoIFI
	3 Methods
		3.1 TFO-Conjugated Plasmid for IDAP
		3.2 Human Nuclear Extracts for IDAP
		3.3 Nucleoprotein Isolation Via IDAP
		3.4 IDAP Under Cross-Linking Conditions
		3.5 A Specific DNA Substrate for IDAP
		3.6 Specific Capture of the Constructed Plasmids in IDAP
		3.7 Cross-Linking Conditions of a Human Cell Line for CoIFI
		3.8 Input Sample Preparation for CoIFI
		3.9 Nucleoprotein Isolation Via CoIFI
	4 Notes
	References
Chapter 17: DNA Fragment Agarose Gel Electrophoresis for Chromatin Immunoprecipitation (ChIP)
	1 Introduction
	2 Materials
		2.1 Cross-Linking
		2.2 Sonication
		2.3 Agarose Gel Electrophoresis
		2.4 Immunoprecipitation and DeCrosslinking
		2.5 Slot Blot
	3 Methods
		3.1 Cross-Linking
		3.2 Sonication
		3.3 Agarose Gel Electrophoresis
		3.4 Immunoprecipitation and DeCrosslinking
		3.5 Slot Blot
	4 Notes
	References
Chapter 18: Postlabeling/PAGE Method for Detection of DNA Adducts
	1 Introduction
	2 Materials
		2.1 Equipment and Accessories
		2.2 In Vitro Activation of Chemicals by Recombinant Enzymes and DNA Isolation
		2.3 Nucleotide Digestion and Dephosphorylation
		2.4 Phosphorylation Labeling with 32P
		2.5 Polyacrylamide Electrophoresis
	3 Methods
		3.1 DNA Isolation from Cell Culture
		3.2 In Vitro DNA Adduct Formation by Activation of Chemicals Using Microsomes
		3.3 Nucleotide Digestion and Dephosphorylation/Enrichment of DNA Adducts by Nuclease P1
		3.4 Enrichment of DNA Adduct Using n-butanol-Water Two-Phase Solvent System
		3.5 Phosphorylation Labeling with 32P
		3.6 Preparation of 30% Polyacrylamide Gel
		3.7 Electrophoresis
		3.8 Autoradiography and Quantification of Radioactivity
	4 Notes
	References
Index