DNA Electrophoresis: Methods and Protocols 1071603221, 9781071603222

Table of contents :
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

Citation preview

Methods in Molecular Biology 2119

Katsuhiro Hanada Editor

DNA Electrophoresis Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

DNA Electrophoresis Methods and Protocols

Edited by

Katsuhiro Hanada Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Oita, Japan

Editor Katsuhiro Hanada Clinical Engineering Research Center Faculty of Medicine, Oita University Yufu, Oita, Japan

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0322-2 ISBN 978-1-0716-0323-9 (eBook) https://doi.org/10.1007/978-1-0716-0323-9 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Caption: Image courtesy of Dr. Luca Zardoni and Dr. Giordano Liberi. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface Gel electrophoresis of DNA is one of many routine laboratory methods in molecular biology and is used for the analysis and purification of DNA fragments. To date, electrophoresis has been used for the analysis of various DNA reactions in vitro, such as polymerase chain reaction (PCR), restriction enzyme digestion, characterization of enzymes involved in DNA reactions, and sequencing. There is no doubt that electrophoresis has contributed to biological studies, particularly the understanding of single gene function(s). A recent trend in molecular biology is to endeavor to understand the genome-wide functions of biochemical DNA reactions within cells including the detection of intermediate DNA structures. To address this, many new techniques have been developed, among them new applications for DNA electrophoresis. For successful genome-wide analysis, it is important that the technical aspects of electrophoresis and DNA sample preparation are considered. Therefore, I have collected step-by-step protocols covering these aspects that are applicable to various species including bacteria, yeasts, and mammalian cells. Finally, I would like to thank all the contributors that have enabled the publication of this book, especially to those scientists who have shared their hands-on expertise through their publications. Without their contribution, this book would not have been possible. Yufu, Oita, Japan

Katsuhiro Hanada

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v ix

1 Introduction and Perspectives of DNA Electrophoresis . . . . . . . . . . . . . . . . . . . . . . Katsuhiro Hanada 2 Two-Dimensional Gel Electrophoresis to Resolve DNA Topoisomers . . . . . . . . . Elizabeth G. Gibson, Alexandria A. Oviatt, and Neil Osheroff 3 Using Two-Dimensional Intact Mitochondrial DNA (mtDNA) Agarose Gel Electrophoresis (2D-IMAGE) to Detect Changes in Topology Associated with Mitochondrial Replication, Transcription, and Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jill E. Kolesar and Brett A. Kaufman 4 2D Gel Electrophoresis to Detect DNA Replication and Recombination Intermediates in Budding Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luca Zardoni, Eleonora Nardini, and Giordano Liberi 5 Neutral–Neutral 2-Dimensional Agarose Gel Electrophoresis for Visualization of E. coli DNA Replication Structures . . . . . . . . . . . . . . . . . . . . . . Karla A. Mettrick, Georgia M. Weaver, and Ian Grainge 6 Alkali Comet Assay in Genotoxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maya Ueda 7 Analysis of DNA Interstrand Cross-Links and their Repair by Modified Comet Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lonnie P. Swift, Lianne Castle, and Peter J. McHugh 8 Analysis of Chromosomal DNA Fragmentation in Apoptosis by Pulsed-Field Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takeshi Terabayashi, Asako Tokumaru, Toshimasa Ishizaki, and Katsuhiro Hanada 9 Detection of DNA Damage-Induced DSBs by the Contour-Clamped Homogeneous Electric Field (CHEF) System in Mammalian Cells . . . . . . . . . . . . Yuri Takiguchi, Ryo Kariyazono, and Kunihiro Ohta 10 Investigation of DNA Double-Strand Breaks Induced in Host Cells Following Infection with Genotoxic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . Rie Teshima 11 Monitoring of DNA Replication and DNA Double-Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis (PFGE) . . . . . . . Kenji Keyamura and Takashi Hishida 12 Pulsed-Field Gel Electrophoresis for Detecting Chromosomal DNA Breakage in Fission Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takatomi Yamada, Hiroshi Murakami, and Kunihiro Ohta

1

vii

15

25

43

61 73

79

89

101

111

123

135

viii

13

14

15

16

17

18

Contents

Detection of DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis of Circular Bacterial Chromosomes . . . . . . . . . . . . . . . . . . . . . Ichizo Kobayashi and Katsuhiro Hanada Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naomi Inoue, Hisashi Narahara, Yoshihiro Nishida, and Katsuhiro Hanada Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Henrik Devitt Møller Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asako Isogawa, Robert P. Fuchs, and Shingo Fujii DNA Fragment Agarose Gel Electrophoresis for Chromatin Immunoprecipitation (ChIP). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mayu Isono and Satoru Hashimoto Postlabeling/PAGE Method for Detection of DNA Adducts. . . . . . . . . . . . . . . . . Kazuhiro Shiizaki

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145

155

165

183

201 213 227

Contributors LIANNE CASTLE • Department of Oncology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK ROBERT P. FUCHS • Marseille Medical Genetics, Aix-Marseille University, Inserm, Marseille, France SHINGO FUJII • DNA Damage Tolerance, CNRS, Marseille, France; Inserm, CRCM, Marseille, France; Institut Paoli-Calmettes, Marseille, France; Aix-Marseille University, Marseille, France ELIZABETH G. GIBSON • Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA IAN GRAINGE • School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia KATSUHIRO HANADA • Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Oita, Japan SATORU HASHIMOTO • Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan TAKASHI HISHIDA • Department of Molecular Biology, Graduate School of Science, Gakushuin University, Tokyo, Japan NAOMI INOUE • Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Oita, Japan TOSHIMASA ISHIZAKI • Department of Pharmacology, Faculty of Medicine, Oita University, Yufu, Oita, Japan ASAKO ISOGAWA • DNA Damage Tolerance, CNRS, Marseille, France; Inserm, CRCM, Marseille, France; Institut Paoli-Calmettes, Marseille, France; Aix-Marseille University, Marseille, France MAYU ISONO • Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan RYO KARIYAZONO • Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan BRETT A. KAUFMAN • Division of Cardiology, Department of Medicine, Center for Metabolism and Mitochondrial Medicine and the Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA KENJI KEYAMURA • Department of Molecular Biology, Graduate School of Science, Gakushuin University, Tokyo, Japan ICHIZO KOBAYASHI • Kyorin University School of Medicine, Tokyo, Japan JILL E. KOLESAR • Department of Animal Biology, University of Pennsylvania, Philadelphia, PA, USA GIORDANO LIBERI • Istituto di Genetica Molecolare, CNR, Pavia, Italy; IFOM Foundation, Milan, Italy PETER J. MCHUGH • Department of Oncology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK KARLA A. METTRICK • School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia

ix

x

Contributors

HENRIK DEVITT MØLLER • Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark; Department of Biology, Institute of Biochemistry, ETH Zurich, Switzerland HIROSHI MURAKAMI • Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan HISASHI NARAHARA • Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Oita, Japan ELEONORA NARDINI • Istituto di Genetica Molecolare, CNR, Pavia, Italy YOSHIHIRO NISHIDA • Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Oita, Japan KUNIHIRO OHTA • Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan NEIL OSHEROFF • Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, TN, USA; VA Tennessee Valley Healthcare System, Nashville, TN, USA ALEXANDRIA A. OVIATT • Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA KAZUHIRO SHIIZAKI • Department of Applied Biosciences, Faculty of Life Sciences, Toyo University, Itakura, Gunma, Japan LONNIE P. SWIFT • Department of Oncology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK YURI TAKIGUCHI • Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan TAKESHI TERABAYASHI • Department of Pharmacology, Faculty of Medicine, Oita University, Yufu, Oita, Japan RIE TESHIMA • Shirokuma Dental Clinic, Beppu, Oita, Japan ASAKO TOKUMARU • Department of Pharmacology, Faculty of Medicine, Oita University, Yufu, Oita, Japan MAYA UEDA • BioSafety Research Center Inc. (BSRC), Shizuoka, Japan GEORGIA M. WEAVER • School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia TAKATOMI YAMADA • Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan; Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan LUCA ZARDONI • Istituto di Genetica Molecolare, CNR, Pavia, Italy; Scuola Universitaria Superiore, IUSS, Pavia, Italy

Chapter 1 Introduction and Perspectives of DNA Electrophoresis Katsuhiro Hanada Abstract Gel electrophoresis of DNA is one of the most frequently used techniques in molecular biology. Typically, it is used in the following: the analysis of in vitro reactions and purification of DNA fragments, analysis of PCR reactions, characterization of enzymes involved in DNA reactions, and sequencing. With some ingenuity gel electrophoresis of DNA is also used for the analysis of cellular biochemical reactions. For example, DNA breaks that accumulate in cells are analyzed by the comet assay and pulsed-field gel electrophoresis (PFGE). Furthermore, DNA replication intermediates are analyzed with two-dimensional (2D) gel electrophoresis. Moreover, several new methods for analyzing various chromosomal functions in cells have been developed. In this chapter, a brief introduction to these is given. Key words Mobility, Size and shape, Pulsed-field gel electrophoresis, 2-dimentional electrophoresis, In vivo biochemistry, Intermediate structures of DNAs

1

Introduction Gel electrophoresis is one of the methods used to separate charged molecules, such as DNAs, RNAs, and proteins, based on the difference of their migration speed in a gel matrix under an electric field. Gel electrophoresis is a classical technique and still the most frequently used for the identification and purification of DNA fragments [1, 2]. DNA fragments are separated based on their size and shape, and its resolution is favorable compared with other methods, such as a liquid chromatography and gradient centrifugation. An advantage of gel electrophoresis is that even low concentration of DNA can be detected by staining with fluorescent intercalators. For example, 25 picograms (pg) of DNA is detected by cyber-gold staining [3]. A standard application of DNA electrophoresis is to analyze double-stranded DNAs (dsDNAs) with nearly neutral (around pH 8.0) buffer, such as tris–acetate buffer with ethylenediamine tetraacetate (EDTA), often called TAE buffer, and tris– borate buffer with EDTA, called TBE [4]. For analysis of denatured single-stranded DNAs (ssDNAs), alkaline gel electrophoresis is the

Katsuhiro Hanada (ed.), DNA Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 2119, https://doi.org/10.1007/978-1-0716-0323-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

1

2

Katsuhiro Hanada

most commonly used [5, 6]. Electrophoresis is widely used for gene engineering in vitro, such as purification and cloning of DNA fragments that are used in the construction of various vectors for gene expression and gene targeting. It has also been established as a routine laboratory technique for genetic and biochemical analysis (e.g., functional analysis of enzymes involved in DNA reactions and analysis of PCR products [4]). Combined with a hybridization technique or PCR, it has been used for the analysis of gene mutations, polymorphisms, and gene expression. In particular, the most ambitious application of electrophoresis is to analyze in cells the intermediate structures of DNA involved in the various DNA reactions, such as DNA replication, DNA repair, recombination, transcription, and chromosome partition and segregation [7, 8]. DNA intermediate structures are usually unstable, especially in cells. Therefore, elaborate protocols have been developed that consider the preparation of DNA samples through to their fractionation with electrophoresis. In this book, methods of DNA electrophoresis are featured with an emphasis on genome-wide analysis of intermediate structures of DNA reactions in cells. 2D gel electrophoresis is widely used to understand the mechanism of DNA replication, recombination, and chromosome dynamics. The comet assay, a method of single cell electrophoresis, and PFGE are used to analyze DNA damage, in particular DNA breaks. Here these strategies are briefly introduced.

2

2D Gel Electrophoresis The theoretical model of mobility during electrophoresis has been discussed previously. The following section introduces the most classical and simplest model of the principle of electrophoresis. Several excellent reviews that summarize the kinetic model of DNA electrophoresis are found elsewhere [2, 9–11]. When a charged particle is placed in an electric field, it is accelerated and its speed increases until its resistance becomes equal to the electric force. When the electric force and the resistance become equal, the speed of the particle becomes constant, which is often called steady state. In this case, electric force and resistance force are equal. At this time, Electric force ¼ Electric charge ðQ Þ  Strength of the electric field ðE Þ Resistance force ¼ Speed ðvÞ  Frictional coefficient ð f Þ Therefore,

Perspective of DNA Electrophoresis

3

v ¼ QE=f Then mobility (μ) of the particle is μ ¼ v=E ¼ Q =f Frictional coefficient ( f ) is dependent on the size of the particle and the nature of the medium. Therefore, f ¼ k η. Coefficient (k) of the particle, viscosity coefficient (η). If the particle is a sphere, k ¼ 6 πr (r is the radius of the sphere) μ ¼ Q =6πr η The coefficient (k) of the particle is smallest when the particle is a sphere. The more the shape of the particle is distorted from the sphere, the larger the coefficient (k) becomes. In general, the intramolecular rotation of particles is much faster than the speed of electrophoresis, meaning that nonspherical particles also behave like spherical particles with a larger radius. This suggests that mobility is determined by the length of DNA molecules as long as the shape of DNAs are the same. Based on this, electrophoresis through polyacrylamide or agarose gels was developed to fractionate DNA and RNA molecules by their molecular size, and is now a standard method for the analysis and purification of DNA and RNA fragments. Small DNA fragments from one nucleotide to 500 bp are effectively separated by polyacrylamide gel electrophoresis (PAGE), and a wide range of DNA fragments from 100 bp to approximately 50 kb in length can be separated in agarose gels of various concentrations. The shape of molecules affects their mobility during electrophoresis. Even for DNA molecules of the same length, the mobilities of circular DNAs are different from that of the linear DNA because their potential for intramolecule rotation is different. The mobility of circular DNA is quite complex. Open circular DNA has a relatively large potential for intramolecular rotation, whereas supercoiled-circular DNA loses the potential for intramolecular rotation due to the tension of the supercoil, resulting in a reduced coefficient (k). This reduction in k is in inverse proportion to the number of supercoils, meaning that supercoiled DNAs display a higher mobility than open circular and linear DNAs (Fig. 1a, b). The elements that affect DNA mobility, their size and shape were often not treated differently. Using these two different parameters to separate DNA molecules has given rise to 2D gel electrophoresis. The combinations of those parameters depend on the need. Typical examples are as follows: 1. Two different concentrations of agarose gels. To detect small and large DNA fragments at the same time, two different concentrations of agarose can be combined. For the first dimension a low concentration of agarose gel, less than 0.7%, is used to

Katsuhiro Hanada

A Mobility

Open circular DNA, Nicked circular DNA Linear DNA

Coverntly colsed circular DNA (cccDNA)

B Mobility

Open circular DNA Lk = 0

・ ・ ・

Lk = 1 Lk = 2 Lk = 3 Supercoiled DNA Lk = N

C

2nd dimension Agarose gel electrophoresis in the presence of EtBr

4

1st dimension Native agarose gel electrophoresis Fig. 1 Schematic representation of gel electrophoresis of DNAs separated according to their shape. (a) Differences in the mobility of covalently closed circular DNA (cccDNA), linear DNA, and open circle DNA. (b) Schematic representation of the mobility of supercoiled DNAs. The linking number affects the mobility during gel electrophoresis. (c) Schematic representation of the detection of DNA replication structures by 2D gel electrophoresis. Replication intermediates, Y arc, and bubble structures of the restriction fragment can be analyzed by 2D gel electrophoresis, fractionated by native and ethidium bromide-containing gels

Perspective of DNA Electrophoresis

5

separate larger DNAs. In the second dimension, a higher concentration of agarose, typically more than 1.5%, is used for smaller fragments. 2. Native and intercalator-containing gels. To analyze different DNA shapes, native gels and intercalator-containing gels are often combined. In the first dimension, DNA is fractionated by a native gel. Here, DNA fragments are separated according to their size. In the second dimension, DNA is fractionated in the presence of an intercalator and the fragments are separated according to their shape. This results in the fractionation of various DNA structures. This method can be used to analyze various intermediate structures that accumulate in cells such as supercoiled DNAs [12], DNA replication forks, and recombination intermediates (Fig. 1b, c) [13, 14]. As an intercalator, ethidium bromide is often used, and chloroquine may be used for the detection of supercoiled DNAs. 3. Native and denaturing gels. To detect single-strand breaks (SSBs) on dsDNAs, native and denaturing gels are combined. In the first dimension, dsDNAs are separated in a neutral buffer, such as TAE and TBE, and in the second dimension, alkaline denatured ssDNAs are separated using an alkaline buffer. This method has been used for studies of DNA replication and recombination [7, 8]. The work of many researchers has led to the successful detection of various DNA structures, such as supercoiled DNAs [14, 15], intermediate structures of DNA replication and recombination [13, 16], and chromosome segregation [17].

3

Comet Assay The comet assay is a technique to quantify DNA breaks in individual cells and is used for evaluating genotoxicity, DNA repair, and genome instability. This technique was originally developed by ¨ stling and Johansson [18]. The method that they developed is O known as “neutral comet assay.” In their method, electrophoresis was performed in a neutral buffer, and DNA fragments as a result of DSBs were preferentially detected. Later, Singh and his coworkers modified this technique resulting in an alkaline comet assay, which is performed in alkaline denaturing conditions [19]. In the alkaline comet assay, both SSBs and DSBs in the cells can be detected, leading to enhanced sensitivity to detect DNA breaks compared with that of the neutral comet assay. However, a disadvantage of the alkaline comet assay is its inability to distinguish between DSBs and SSBs. In the comet assay, cells are fixed in an agarose gel on a glass microscope slide, and the cellular membrane is lysed in the gel. In

6

Katsuhiro Hanada

A

Intact DNA Normal cell Damaged cell Intact DNA

B

Broken DNA

Step 1. Plug preparation

Step 2. PFGE Intact DNA Broken DNA Chromosomal DNA fragmentation in apoptosis

Fig. 2 Schematic representation of DSB detection by the comet assay and PFGE. (a) Detection of DSBs by comet assay. After electrophoresis, intact DNA remains packed in the nucleus, but broken DNA migrates into the gel, forming a “comet” shaped tail. (b) Detection of DSBs by PFGE. To avoid shearing of chromosome DNAs by pipetting, cells are lysed in agarose plugs. Then DSBs are fractionated by PFGE. Intact DNA stays in the wells (plugs), whereas broken DNA migrates into the gel

the neutral comet assay, samples are fractionated by electrophoresis in neutral buffer, whereas in the alkaline comet assay, denatured DNAs are fractionated by alkaline electrophoresis. In both cases, damaged DNA fragments are fractionated and subsequently evaluated by microscopy. Intact DNA does not migrate into the agarose gel and stays in the nucleus due to its large molecular size. Damaged DNA migrates to anode of the agarose gel. This results in an image with a distinct head and tail, which resembles a comet (Fig. 2a), hence the name for this assay. The head is composed of intact DNA, while the tail consists of broken pieces of DNA. The standard application of the comet assay is to detect DNA breaks, such as DSBs and SSBs [20]. However, it is also capable of analyzing abasic sites, oxidative base damage [21], and DNA cross-linking [22]. The comet assay is also used to monitor DNA repair by living cells.

Perspective of DNA Electrophoresis

7

Here two distinct applications of the comet assay are described; one is to quantify DNA damage in cells in Chapter 6 [23], and the other is to evaluate DNA repair of interstrand DNA cross-links (ICLs) in Chapter 7 [22].

4

Analysis of DSBs by PFGE Normal gel electrophoresis, including the comet assay, is not suitable for evaluating large DNAs greater than 50 kb in length. However, Schwartz and coworkers succeeded in separating DNA fragments greater than 100 kb using PFGE system. Since PFGE can separate large DNAs between 10 kb and 10 Mb in length, PFGE has been used to purify and visualize large DNA constructs, such as bacterial artificial chromosomes (BACs) [24], phage artificial chromosomes (PACs) [25], and yeast artificial chromosomes (YACs) [26]. PFGE is also used to analyze chromosomal DNAs in various species, one example of which is the chromosomal typing of bacteria called “PFGE-typing” [27]. As with the comet assay, PFGE is widely used as a technique to quantify DSBs (Fig. 2b) [28, 29]. The advantage of PFGE is its ability to analyze DSBs across various species, from bacteria to humans [30–32]. In normal agarose gel electrophoresis, it is impossible to separate large DNA molecules (Fig. 3a). During electrophoresis, DNA molecules are stretched and migrate in the agarose matrix to the anode. At the beginning of electrophoresis, DNAs are stacked in the matrix and form U-shapes. Such stacked molecules are drawn in one direction, with one end of the DNA molecules taking priority. As a result, DNAs are fully stretched, and one of the DNA ends (the leading end) is drawn toward the anode and the other becomes the trailing end and is drawn toward the cathode [33]. However, if the direction of electric field is changed by more than 90 , the trailing end becomes the leading end and start moving toward the end of the gel (Fig. 3b). This phenomenon is called “reorientation.” To induce reorientation, the angle of the electric charge is important. If the angle is more than 90 , a trailing end of DNA molecule may become a leading one. If the angle is less than 90 , the initial leading end continues being a leading one [33]. Even if the direction of the electric field is changed, elongated DNA structures are still retained. This phenomenon, the reorientation of stretched DNA molecules, is crucial for the separation of large molecules because the length of stretched DNA during reorientation enhances the separation of large DNA molecules (Fig. 3b). If large DNAs are stretched, the distance from the head to the tail is also long, whereas the distance from the head to the tail of small DNAs is less. Continuous pulse switching results in the enhanced separation of large DNA molecules. A number of adaptations to PFGE have been developed; for example, pulsed-field gradient

8

Katsuhiro Hanada

A

Normal agarose gel electrophoresis Lage DNAs in the well

large DNA Small DNA large DNA Small DNA

B

E

E

Agarose gel

PFGE Lage DNAs in the well

Start PFGE

large DNA Small DNA

E1

Agarose gel Wel large DNA Small DNA

Angle change

E2

E2

E3

E3

Wel large DNA Small DNA

Angle change

Fig. 3 Schematic representation of DNA fractionation during gel electrophoresis. (a) Schematic representation of DNA fractionation in normal agarose gel electrophoresis. DNA molecules are stretched and move to the anode. (b) Schematic representation of DNA fractionation during PFGE. DNA molecules are stretched and move to the anode. When the direction of electric field is changed more than 90 , reorientation of migration occurs, resulting in enhanced separation of large DNA molecules

electrophoresis, orthogonal field-alternating gel electrophoresis, transverse alternating-field electrophoresis, and field inverse gel electrophoresis. However, only two systems, clamped homogeneous electric field (CHEF) (Bio-Rad Laboratories, USA) and rotating gel electrophoresis (RGE) (Analytik Jena AG, Germany), are now commercially available (Fig. 4). In PFGE, separation of

Perspective of DNA Electrophoresis

9

CHEF

A sis ore h p o ctr Ele

+ +

Ele ctr oph ore sis

Agarose gel

+ +

Agarose gel

+ +

+ + RGE

B

+ + + +

is res o h op ctr Ele Agarose gel

+

Ele ctr oph ore sis Agarose gel

+

+ + + +

Fig. 4 Systems of PFGE that are currently commercially available. (a) Clamped homogeneous electric field (CHEF) system (Bio-Rad Laboratories, USA). (b) Rotating gel electrophoresis (RGE) system (Analytik Jena AG, Germany)

DNA is determined by several parameters, such as the strength of the electric field (voltage), pulse time, the angles of the electric fields, gel concentration, and temperature [33]. 1. Electric field strength. PFGE is typically performed at a voltage of 6 V/cm. A stronger electric field is required for better separation of larger molecules. However, to separate extremely large molecules, up to 5 Mb in length, PFGE is performed with a moderate electric field (typically between 1 and 2 V/cm) and for a longer duration (typically more than 100 h). RGE (Analytik Jena AG) can vary the strength of the electric field using a gradient. 2. Pulse time. Longer pulse time leads to the better separation of larger molecules. CHEF DRIII and mapper XA (Bio-Rad Laboratories), and RGE can vary pulse time during electrophoresis. 3. Angle of the electric fields. Smaller angles between the two fields increase the speed of migration. The angles of CHEF and RGE are usually set between 110 and 120 . 4. Gel concentration. A lower concentration increases the speed of migration and higher gel concentrations lead to better separation.

10

Katsuhiro Hanada

5. Temperature. Higher temperatures increase the migration rate but decrease separation. Since PFGE is usually performed with a strong electric field, the temperature of the buffer easily rises. To keep temperature constant, a device to cool the buffer and gel is necessary. The theoretical model of PFGE is not widely appreciated. Researchers need to optimize the parameters through practice. In addition, preparation of the DNA sample is also critical for PFGE because large DNA molecules are easily sheared during the process; for example, by pipetting and mixing and during DNA extraction. Therefore, cells are lysed and proteins are removed from the DNA in an agarose plug (Fig. 2b). In this way, DNA preparation requires no pipetting and mixing. Some organisms have cell walls that protect cells from detergents. To analyze chromosomes of such organisms, additional steps are required to lyse the cells. These complexities certainly became an “entry barrier” for PFGE. To overcome this barrier, I have asked expert scientists to share their experiences. The procedures for DNA sample preparation are different between species, and parameter settings are different between CHEF and RGE systems. In this book, detailed protocols of both CHEF and RGE systems are presented for various species, with an emphasis on the detection of DSBs that have accumulated in cells after treatment with DNA damaging agents.

5

Other Applications Trends in molecular biology are now shifting from understanding the functions of single genes to genome-wide analysis of gene regulation and other chromosomal reactions. Therefore, cuttingedge techniques for DNA electrophoresis are frequently developed for genome-wide studies (e.g., chromatin regulation including epigenetics, dynamics of DNA replication, DNA damage and repair on chromosomes, and characterization of cis- and trans-acting elements on transcription and DNA replication [34–37]). Methods in genome-wide analysis for the identification of DNA replication origins, analysis of chromatin state, and activity of DNA repair are presented here. Technical breakthroughs in these methods have enriched the knowledge of intermediate structures in vitro. The techniques have the potential to unravel novel processes in chromosomal DNA reactions that are currently under study. The hope is that with increased interest generated by potential discoveries more novel methods will be developed in the next decade. In addition, one method to analyze DNA adducts by PAGE is also included in this book because of the importance of the genome-wide analysis of DNA adducts [38]. This particular analysis is used in risk assessments for environmental and public health

Perspective of DNA Electrophoresis

11

policies as well as studies in toxicology. At present, DNA adducts in cells are analyzed by liquid chromatography–tandem mass spectrometry, but the methodology for genome-wide analysis of these adducts has not yet established due to insufficient knowledge about them [39]. Many technical difficulties still remain. First, it is not easy to purify DNA adducts, especially rare ones. Second, it is not easy to prepare adducts in vitro because the formation of some DNA adducts requires the function of cytochrome P450. Even if the manipulation of a particular DNA adduct is successful, it is not easy to determine their structures. In many cases, the structure of the adduct is determined by nuclear magnetic resonance and requires substantial and expensive equipment. However, it remains important to accumulate knowledge about DNA adducts, and current efforts will strive for the development of new technologies to analyze DNA adducts in future.

Acknowledgments I thank Dominic James, PhD, from Edanz Group (www. edanzediting.com/ac) for editing a draft of the manuscript. References 1. Aaij C, Borst P (1972) The gel electrophoresis of DNA. Biochim Biophys Acta 269 (2):192–200 2. Roberts GA, Dryden DT (2013) DNA electrophoresis: historical and theoretical perspectives. Methods Mol Biol 1054:1–9 3. Noites IS, O’Kennedy RD, Levy MS, Abidi N, Keshavarz-Moore E (1999) Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye. Biotechnol Bioeng 66(3):195–201 4. Makovets S (2013) Basic DNA electrophoresis in molecular cloning: a comprehensive guide for beginners. Methods Mol Biol 1054:11–43 5. Sambrook J, Russell DW (2006) Alkaline agarose gel electrophoresis. CSH Protoc 2006(1) 6. Hanada K, Budzowska M, Davies SL, van Drunen E, Onizawa H, Beverloo HB, Maas A, Essers J, Hickson ID, Kanaar R (2007) The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat Struct Mol Biol 14(11):1096–1104 7. Allers T, Lichten M (2001) Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106 (1):47–57

8. Hunter N, Kleckner N (2001) The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell 106(1):59–70 9. Lerman LS, Frisch HL (1982) Why does the electrophoretic mobility of DNA in gels vary with the length of the molecule? Biopolymers 21(5):995–997 10. Zimm BH, Levene SD (1992) Problems and prospects in the theory of gel electrophoresis of DNA. Q Rev Biophys 25(2):171–204 11. Stellwagen NC (2009) Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 30(Suppl 1): S188–S195 12. Cebrian J, Kadomatsu-Hermosa MJ, Castan A, Martinez V, Parra C, Fernandez-Nestosa MJ, Schaerer C, Martinez-Robles ML, Hernandez P, Krimer DB, Stasiak A, Schvartzman JB (2015) Electrophoretic mobility of supercoiled catenated and knotted DNA molecules. Nucleic Acids Res 43(4):e24 13. Liberi G, Maffioletti G, Lucca C, Chiolo I, Baryshnikova A, Cotta-Ramusino C, Lopes M, Pellicioli A, Haber JE, Foiani M (2005) Rad51-dependent DNA structures

12

Katsuhiro Hanada

accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev 19 (3):339–350 14. Ashley RE, Dittmore A, McPherson SA, Turnbough CL Jr, Neuman KC, Osheroff N (2017) Activities of gyrase and topoisomerase IV on positively supercoiled DNA. Nucleic Acids Res 45(16):9611–9624 15. Tan TL, Essers J, Citterio E, Swagemakers SM, de Wit J, Benson FE, Hoeijmakers JH, Kanaar R (1999) Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation. Curr Biol 9(6):325–328 16. Alzu A, Bermejo R, Begnis M, Lucca C, Piccini D, Carotenuto W, Saponaro M, Brambati A, Cocito A, Foiani M, Liberi G (2012) Senataxin associates with replication forks to protect fork integrity across RNA-polymerase-II-transcribed genes. Cell 151 (4):835–846 17. Kolesar JE, Wang CY, Taguchi YV, Chou SH, Kaufman BA (2013) Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome. Nucleic Acids Res 41(4):e58 18. Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1):291–298 19. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191 20. Kohara A, Matsumoto M, Hirose A, Hayashi M, Honma M, Suzuki T (2018) Mutagenic properties of dimethylaniline isomers in mice as evaluated by comet, micronucleus and transgenic mutation assays. Genes Environ 40:18 21. Warpman Berglund U, Sanjiv K, Gad H, Kalderen C, Koolmeister T, Pham T, Gokturk C, Jafari R, Maddalo G, SeashoreLudlow B, Chernobrovkin A, Manoilov A, Pateras IS, Rasti A, Jemth AS, Almlof I, Loseva O, Visnes T, Einarsdottir BO, Gaugaz FZ, Saleh A, Platzack B, Wallner OA, Vallin KS, Henriksson M, Wakchaure P, Borhade S, Herr P, Kallberg Y, Baranczewski P, Homan EJ, Wiita E, Nagpal V, Meijer T, Schipper N, Rudd SG, Brautigam L, Lindqvist A, Filppula A, Lee TC, Artursson P, Nilsson JA, Gorgoulis VG, Lehtio J, Zubarev RA, Scobie M, Helleday T (2016) Validation and development of MTH1 inhibitors for treatment of cancer. Ann Oncol 27 (12):2275–2283

22. Bhagwat N, Olsen AL, Wang AT, Hanada K, Stuckert P, Kanaar R, D’Andrea A, Niedernhofer LJ, McHugh PJ (2009) XPF-ERCC1 participates in the Fanconi anemia pathway of crosslink repair. Mol Cell Biol 29(24):6427–6437 23. Kasamoto S, Masumori S, Tanaka J, Ueda M, Fukumuro M, Nagai M, Yamate J, Hayashi M (2017) Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats. Exp Toxicol Pathol 69(4):187–191 24. Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A 89(18):8794–8797 25. Sternberg N (1990) Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proc Natl Acad Sci U S A 87 (1):103–107 26. Murray AW, Szostak JW (1983) Construction of artificial chromosomes in yeast. Nature 305 (5931):189–193 27. Ferrari RG, Panzenhagen PHN, Conte-Junior CA (2017) Phenotypic and genotypic eligible methods for Salmonella Typhimurium source tracking. Front Microbiol 8:2587 28. De Silva IU, McHugh PJ, Clingen PH, Hartley JA (2000) Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol Cell Biol 20(21):7980–7990 29. Hanada K, Uchida T, Tsukamoto Y, Watada M, Yamaguchi N, Yamamoto K, Shiota S, Moriyama M, Graham DY, Yamaoka Y (2014) Helicobacter pylori infection introduces DNA double-strand breaks in host cells. Infect Immun 82(10):4182–4189 30. Fukuda E, Kaminska KH, Bujnicki JM, Kobayashi I (2008) Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases. Genome Biol 9(11):R163 31. Keyamura K, Arai K, Hishida T (2016) Srs2 and Mus81-Mms4 prevent accumulation of toxic inter-homolog recombination intermediates. PLoS Genet 12(7):e1006136 32. Kawashima Y, Yamaguchi N, Teshima R, Narahara H, Yamaoka Y, Anai H, Nishida Y, Hanada K (2017) Detection of DNA doublestrand breaks by pulsed-field gel electrophoresis. Genes Cells 22(1):84–93 33. Nassonova ES (2008) Pulsed field gel electrophoresis: theory, instruments and application. Cell Tissue Biol 2:557

Perspective of DNA Electrophoresis 34. Denker A, de Laat W (2016) The second decade of 3C technologies: detailed insights into nuclear organization. Genes Dev 30 (12):1357–1382 35. Gregoire MC, Leduc F, Morin MH, Cave T, Arguin M, Richter M, Jacques PE, Boissonneault G (2018) The DNA double-strand “breakome” of mouse spermatids. Cell Mol Life Sci 75(15):2859–2872 36. Isogawa A, Fuchs RP, Fujii S (2018) Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation. Sci Rep 8(1):5925 37. Moller HD, Mohiyuddin M, Prada-Luengo I, Sailani MR, Halling JF, Plomgaard P,

13

Maretty L, Hansen AJ, Snyder MP, Pilegaard H, Lam HYK, Regenberg B (2018) Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nat Commun 9(1):1069 38. Shiizaki K, Kawanishi M, Yagi T (2017) Modulation of benzo[a]pyrene-DNA adduct formation by CYP1 inducer and inhibitor. Genes Environ 39:14 39. Kanaly RA, Hanaoka T, Sugimura H, Toda H, Matsui S, Matsuda T (2006) Development of the adductome approach to detect DNA damage in humans. Antioxid Redox Signal 8 (5–6):993–1001

Chapter 2 Two-Dimensional Gel Electrophoresis to Resolve DNA Topoisomers Elizabeth G. Gibson, Alexandria A. Oviatt, and Neil Osheroff Abstract Agarose gel electrophoresis is one of the most straightforward techniques that can be used to differentiate between topoisomers of closed circular DNA molecules. Generally, the products of reactions that monitor the interconversion of DNA between negatively supercoiled and relaxed DNA or positively supercoiled and relaxed DNA can be resolved by one-dimensional gel electrophoresis. However, in more complex reactions that contain both positively and negatively supercoiled DNA, one-dimensional resolution is insufficient. In these cases, a second dimension of gel electrophoresis is necessary. This chapter describes the technique of two-dimensional agarose gel electrophoresis and how it can be used to resolve a spectrum of DNA topoisomers. Key words Gel electrophoresis, Two-dimensional, DNA, DNA topoisomers, DNA supercoiling, DNA intercalation, Positive supercoiling, Negative supercoiling, Relaxed DNA

1

Introduction The superhelicity of DNA can have a profound effect on a number of DNA processes including replication, transcription, and recombination [1–7]. Therefore, it is important to be able to distinguish DNA molecules that are underwound (negatively supercoiled), contain no torsional stress (relaxed), and overwound (positively supercoiled). The easiest way to resolve supercoiled and relaxed DNA topoisomers is by agarose gel electrophoresis. In order to maintain the topological state of DNA during electrophoresis, the molecules must be in a “closed” system (i.e., the ends cannot have free-rotation). Therefore, this method applies primarily to circular DNA molecules. Because the charges on DNA come from the phosphate groups in the DNA backbone, all DNA molecules have the same charge-tomass ratio. Thus, in a vacuum, DNA topoisomers would all migrate

Elizabeth G. Gibson and Alexandria A. Oviatt contributed equally to this work. Katsuhiro Hanada (ed.), DNA Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 2119, https://doi.org/10.1007/978-1-0716-0323-9_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020

15

16

Elizabeth G. Gibson et al.

Relaxation

Supercoiling

Supercoiling

Relaxation

Positively Supercoiled

Negatively Supercoiled

Fig. 1 Interconversion of DNA topoisomers. DNA that is not under torsional stress is referred to as “relaxed” (center), while over- and underwinding results in DNA that is positively (left) or negatively (right) supercoiled, respectively. The term “supercoiled” is derived from the fact that when under torsional stress, some of the stress is alleviated by the DNA forming superhelical twists about itself

at the same rate. However, when subjected to electrophoresis through a gel matrix, smaller or more compact molecules migrate more readily through the medium than a molecule that has more volume. When DNA is in a “relaxed” state, in which there is no torsional stress on the molecule, it maintains a more open structure (Fig. 1). Thus, it has a wide diameter. However, when torsional stress is applied to the molecule by increasing or decreasing the number of turns of the double helix, free DNA converts ~75% of the torsional stress to axial stress [1], which is manifested by the double helix wrapping around itself to form “superhelical twists.” As seen in Fig. 1, superhelical twisting leads to a more compact structure of DNA; the greater the superhelical twisting (or supercoiling), the more compact the structure. Therefore, the more supercoiled the DNA molecule, the faster it will migrate through an agarose gel toward the cathode. Superhelical twists occur in discrete units (i.e., molecules will have 1, 2, 3, . . . etc. DNA nodes or crossovers). Individual DNA topoisomers (i.e., plasmids with a specific number of DNA nodes or superhelical twists) will run as distinct bands. When monitoring reactions in which a DNA molecule is being converted from relaxed to negatively or positively supercoiled, or from supercoiled to relaxed, one-dimensional electrophoresis is generally sufficient to resolve topoisomers (Fig. 2). In these cases, all of the intermediate DNA topoisomers will contain a number of supercoils ranging from that of the substrate to that of the product. In the example shown in Fig. 2, the time course follows a reaction in which a relaxed plasmid is converted to a negatively supercoiled molecule by an enzyme known as DNA gyrase [5, 6, 8]. Hence, all of the individual DNA bands that are visualized contain either no supercoils (relaxed) or some number of negative supercoils. In contrast to the reaction shown in Fig. 2, some reactions are more complex. For example, Fig. 3 shows a time course for the conversion of positively to negatively supercoiled DNA by gyrase [9–11]. In this case, positive supercoils are removed to produce

Two-Dimensional Gel Electrophoresis

17

Time (min) 0

5

10

20

30

Rel

(-)SC

Fig. 2 One-dimensional resolution of relaxed and negatively supercoiled DNA topoisomers. A time course for the conversion of relaxed plasmid to negatively supercoiled molecules by Staphylococcus aureus gyrase is shown. Reaction products were resolved by one-dimensional agarose gel electrophoresis and DNA bands were visualized by mid-range ultraviolet light following staining with ethidium bromide. The positions of relaxed (Rel) and negatively supercoiled [( ) SC] DNA are indicated on the gel Time (s) 0

15

30

45

Time (min) 60

90

120

5

10

15

20

Rel (-)SC (+)SC

Fig. 3 One-dimensional resolution of negatively and positively supercoiled DNA topoisomers. A time course for the conversion of positively supercoiled plasmid to negatively supercoiled molecules by S. aureus gyrase is shown. Reaction products were resolved by one-dimensional agarose gel electrophoresis and DNA bands were visualized by mid-range ultraviolet light following staining with ethidium bromide. The positions of relaxed (Rel), positively supercoiled [(+)SC], and negatively supercoiled [( )SC] DNA are indicated on the gel

relaxed DNA followed, by the introduction of negative supercoils. Positively supercoiled DNA is slightly more compact [1] than its negatively supercoiled counterpart and therefore migrates slightly further on an agarose gel [12]. However, one-dimensional electrophoresis is insufficient to determine whether the intermediate bands are positively or negatively supercoiled. Thus, it is necessary to use two-dimensional electrophoresis to resolve this issue. The first dimension in two-dimensional gel electrophoresis separates DNA topoisomers as described above. To distinguish positive from negative intermediate topoisomers, the gel is then soaked in chloroquine, a DNA intercalative agent. DNA intercalators locally unwind the double helix. Because the number of turns of the double helix is invariant in a closed circular system [1, 2], the local underwinding (which is constrained by the presence of the intercalator) is compensated by a global overwinding [13–15].

18

Elizabeth G. Gibson et al.

Hence, negatively supercoiled molecules will tend to look less negatively supercoiled (or even fully relaxed) in the presence of an intercalator and relaxed DNA will tend to look positively supercoiled. Because molecules that already contain a high level of positive superhelical twists cannot absorb very much intercalator, positively supercoiled DNA changes very little in the presence of a DNA intercalator [12, 13]. Similarly, the migration of nicked DNA changes relatively little in the presence of an intercalator. This is because the presence of the nick opens the topological system and the absorption of an intercalator does not lead to compensatory overwinding. In the example described below, the amount of chloroquine that is added is sufficient to make negatively supercoiled DNA appear to be fully relaxed and relaxed DNA to be fully positively supercoiled. Once the gel is soaked in chloroquine, it is turned 90 clockwise and is subjected to electrophoresis once again (Fig. 4). Fully relaxed DNA, which comigrates with nicked DNA in the first dimension, migrates considerably further in the second dimension, as it appears to be positively supercoiled. Conversely, the migration of negatively supercoiled DNA (which now appears to be relaxed) in the second dimension is greatly retarded compared to positively supercoiled DNA. The addition of the second dimension allows positively and negatively supercoiled topoisomers to be readily resolved from one another; intermediate positively supercoiled molecules run in an arc between fully positively supercoiled DNA and the apex band of relaxed DNA, whereas intermediately negatively supercoiled DNA runs on an arc between relaxed and fully negatively supercoiled molecules (Fig. 4). When samples that correspond to those shown in the one-dimensional gel in Fig. 3 are subjected to two-dimensional gel electrophoresis (Fig. 5), it becomes obvious that all of the 2nd Dimension

1st Dimension

Nicked

(–)SC

Relaxed

(+)SC

Fig. 4 Schematic depicting the migration of DNA topoisomerases following two-dimensional agarose gel electrophoresis. The positions of nicked, relaxed, negatively supercoiled [( )SC], and positively supercoiled [(+)SC] DNA are shown as black bands. Gray bands represent DNA topoisomers of intermediate supercoiling. Partially negatively supercoiled molecules migrate as the arc between relaxed and ( )SC DNA and partially positively supercoiled molecules migrate as the arc between (+)SC and relaxed DNA

Two-Dimensional Gel Electrophoresis

19

2nd Dimension Nicked

Rel

(-)SC

(+)SC

Nicked

(-)SC

1st Dimension

0s

Nicked

(+)SC 90 s

(+)SC

Nicked

Nicked

(+)SC 5m

(+)SC 60 s

Rel

(-)SC

Rel

(-)SC

30 s

Rel

(-)SC

Rel

Nicked

Rel

(-)SC

(+)SC 20 m

Fig. 5 Two-dimensional resolution of negatively and positively supercoiled DNA topoisomers. A time course for the conversion of positively supercoiled plasmid to negatively supercoiled molecules by S. aureus gyrase is shown. Reaction products were resolved by two-dimensional agarose gel electrophoresis and DNA bands were visualized by mid-range ultraviolet light following staining with ethidium bromide. Samples at the reaction times shown are from the time course depicted in Fig. 3. The positions of nicked, relaxed (Rel), positively [(+)SC], and negatively supercoiled [( )SC] DNA are indicated on the gel and methods are described below

intermediate bands observed at 30 s are positively supercoiled, that a mixed population is seen at 90 s, and that all of the intermediate bands observed at 5 min are negatively supercoiled topoisomers.

2

Materials Prepare all reagents at room temperature using deionized water and analytical grade reagents. 1. Electrophoresis Buffer 10 Stock: 1 M Tris-borate, pH 8.3, and 20 mM EDTA. Add 121.1 g of Tris-base, 61.8 g of boric acid, and 5.85 g of EDTA to approximately 600 mL of H2O and stir until dissolved. Transfer the solution to a graduated cylinder, add H2O to a final volume of 1 L, and mix thoroughly (see Notes 1–3). Store at room temperature. 2. Loading Buffer: 60% sucrose, 10 mM Tris–HCl, pH 7.9, 0.5% bromophenol blue, and 0.5% xylene cyanol FF. Add 6 g of sucrose and 100 μL of 1 M Tris–HCl, pH 7.9, to 6–8 mL of H2O in a graduated cylinder and mix (see Note 4). Add 0.05 g of bromophenol blue, 0.05 g of xylene cyanol FF, and H2O to a final volume of 10 mL and mix thoroughly. Store in 1 mL

20

Elizabeth G. Gibson et al.

aliquots. For long-term storage (>1 month), store aliquots at 20  C. 3. Chloroquine: 10 mg/mL solution. Add 50 mg of chloroquine to a graduated cylinder, bring the final volume to 5 mL with H2O, and mix thoroughly. Store in a light-proof container at 4  C (see Note 5). 4. Ethidium bromide: 10 mg/mL. Add 50 mg of ethidium bromide to a graduated cylinder, bring the final volume to 5 mL with H2O, and mix thoroughly. Store in a light-proof container at 4  C (see Notes 5 and 6).

3

Methods 1. Preparation of agarose gel: mix 80 mL of Electrophoresis Buffer 10 Stock with 720 mL of H2O to yield 1 Electrophoresis Buffer. Add 1 g of molecular biology grade agarose to 100 mL of 1 Electrophoresis Buffer in a 250 mL Erlenmeyer flask. Heat the mixture in a microwave until the agarose is completely dissolved (heat for 1 min on high, swirl the mixture, and heat on high for an additional 30–45 s until the mixture has reached a low boil, see Note 7). Swirl the mixture to make sure it is fully dissolved and place it in a 50  C water bath until ready to pour. Gently pour the liquid agarose mixture into a 14  14 cm gel box (avoid trapping bubbles in the gel) and insert a comb ~1 cm from the top of the gel. For the procedure below, a 16 well comb is used. If two combs are used, insert the second comb ~7 cm from the top of the gel. Each comb can support up to three samples. Allow the gel to harden at room temperature. It will take ~10–15 min (see Note 8). Cover the solid gel with 1 Electrophoresis Buffer such that the buffer level is ~8–10 mm above the top surface of the gel and carefully remove the comb(s). This protocol will result in a gel that is ~7 mm in depth. The wells that are left following removal of the combs should be ~5–6 mm deep, ~1.5 mm high, and ~5 mm wide. 2. The example used in this chapter represents a time course for the conversion of positively supercoiled plasmid DNA (pBR322) (see Note 9) to negatively supercoiled plasmid DNA in a reaction mixture containing S. aureus gyrase [11]. This electrophoresis protocol can be used to separate supercoiled/relaxed DNA topoisomers from virtually any reaction. 3. For the purposes of the gel electrophoresis, 25 μL samples are used that contain 2 μL of loading buffer (see Note 10). Samples should contain a minimum of 0.3 μg of DNA for visibility.

Two-Dimensional Gel Electrophoresis

21

Samples (20 μL of the DNA samples described above) (see Note 11) should be loaded slowly into the wells using a 20 μL micropipette (see Note 12). If three samples are run on the gel, they should be loaded into lanes 1, 6, and 11 of a 16 well comb. If a second comb is used, a single gel can accommodate up to six samples. 4. Electrophoresis should be carried out at 150 V (~100 mA). The anode should be at the top of the gel. Electrophoresis should be carried out for ~2 h or until the bromophenol blue dye front has moved ~halfway down the length of the gel. 5. Turn off the voltage and carefully transfer the gel to a pan containing ~200 mL of 1 Electrophoresis Buffer containing 4.5 μg/mL chloroquine. The chloroquine-containing solution is prepared by adding 450 μL of 10 mg/mL chloroquine, 100 mL of Electrophoresis Buffer 10 Stock, and H2O to a final volume of 1 L. Make sure that the gel is covered with the chloroquine-containing buffer and soak it for 2 h with gentle agitation. 6. Turn the gel 90 clockwise from its original position and place it back into the electrophoresis apparatus. Add a sufficient volume of 1 Electrophoresis Buffer containing 4.5 μg/mL chloroquine to cover the gel (once again, by ~8–10 mm), and subject the gel to electrophoresis for 2 h at 120 V (~80 mA). 7. Turn off the voltage and carefully transfer the gel to a pan containing sufficient H2O to cover and soak the gel for ~15 min with gentle agitation. This step removes excess chloroquine, which will allow the ethidium bromide to intercalate more efficiently. 8. To stain the gel in order to be able to visualize the DNA bands, transfer it to a pan containing sufficient H2O containing 1 μg/ mL ethidium bromide to cover and soak for ~30 min with gentle agitation. The ethidium bromide-containing solution is prepared by adding 30 μL of 10 mg/mL ethidium bromide to 300 mL of H2O. 9. Destain the gel to remove excess ethidium bromide by transferring the gel to a pan containing sufficient H2O to cover for 10–20 min with gentle agitation (see Note 13). 10. DNA bands are observed using medium-range ultraviolet light (see Notes 14 and 15).

22

4

Elizabeth G. Gibson et al.

Notes 1. When mixed together, the Tris-borate-EDTA mixture should have a pH of ~8.3 with no further adjustment. 2. It is preferable to use EDTA, as opposed to NaEDTA, when making the Electrophoresis Buffer 10 Stock. In the presence of Na+ ions, some of the borate in the buffer can be converted to a borax complex, which tends to precipitate out of solution over time. Even when using EDTA, some precipitate may form over several weeks. 3. If the Electrophoresis Buffer 10 Stock is going to be stored for more than a month, it is desirable to filter it through a 0.22 μm sterilizing filter, which further deters precipitation. 4. If the sucrose has difficulty dissolving in the Tris–HCl, heat it at 37  C for 5–10 min. Before adding the dyes, cool the solution to room temperature. 5. Chloroquine and ethidium bromide are light-sensitive compounds. If a light-proof container is unavailable, the vessel can be wrapped in aluminum foil to protect the solution from light-induced degradation. 6. Ethidium bromide is a mild carcinogen. Therefore, appropriate caution should be used when handling the compound (including using personal protective equipment), and solutions containing ethidium bromide should be disposed of according to proper safety protocols. 7. When heating the agarose gel solution, it is critical to make sure that the container is properly vented to avoid a potential pressure explosion. 8. The clear liquid agarose solution will become opaque upon solidifying. 9. The described protocol is tailored for use with plasmid pBR322, which is 4363 bp in length. Because all DNA molecules have the same charge-to-mass ratio, electrophoretic separation is based on the size of the plasmid and its topology. Therefore, electrophoresis times or the percentage of agarose in gels may need to be modified if plasmids that are substantially longer or shorter than pBR322 are used. A slightly higher percentage of agarose can be used with shorter plasmids (which will migrate faster than pBR322), and a lower agarose percentage can be used for longer plasmids (which will migrate slower). 10. Sucrose is present in the loading buffer to increase the density of the samples so that they will sink to the bottom of the well. Therefore, the loading buffer should comprise at least 8% of

Two-Dimensional Gel Electrophoresis

23

the total sample volume. Doing so ensures that samples will stay in the wells following loading and that the DNA bands will remain tight. Two dyes, bromophenol blue and xylene cyanol FF, are included in the loading buffer to monitor the electrophoresis. The dyes run differentially when subjected to electrophoresis in the agarose gel. Bromophenol blue migrates more quickly and will move as the dye front ahead of the migrating DNA. Xylene cyanol FF migrates more slowly and follows the DNA. 11. Even though the assay samples are 25 μL in volume, we generally load only 20 μL into the wells. This keeps the wells from being overloaded and ensures that a consistent volume can be added into each well. 12. To keep DNA bands from diffusing, it is best to load wells when the power is on. As long as the gel is loaded rapidly, there is no discernible difference in electrophoretic mobility between the first and last samples that are loaded. Although the low level of current poses no danger, it is best to wear gloves while loading. 13. Even though intercalated ethidium bromide fluoresces considerably more brightly than free molecules, background fluorescence can be seen in the gel. Thus, the destaining step is recommended to decrease background levels of fluorescence. 14. Ethidium bromide fluoresces with an orange color under medium-wave ultraviolet light. Therefore, an orange filter is generally used when imaging gels. This results in DNA bands that appear as white on a black background. 15. The topological state of plasmid DNA affects the amount of ethidium bromide that can be intercalated. Nicked or linear DNA can absorb the most ethidium bromide because torsional stress on the DNA induced by intercalation is unconstrained. Other DNA topoisomers absorb ethidium bromide in the following order: negatively supercoiled > relaxed > positively supercoiled. Consequently, the same amount of nicked DNA will fluoresce more brightly than negatively supercoiled, which will fluoresce more brightly than relaxed DNA, which will fluoresce more brightly than positively supercoiled DNA. Thus, images containing different DNA topoisomers should be considered qualitative, and levels of DNA in each band cannot necessarily be compared directly with one another in a quantitative manner.

24

Elizabeth G. Gibson et al.

Acknowledgments Work in the laboratory of the senior author (N.O.) was supported by National Institutes of Health grant GM126363 and US Veterans Administration Merit Review award I01 Bx002198. E.G.G was supported by a Pharmacology Training Grant (5T32GM007628) from the National Institutes of Health and predoctoral fellowships from the PhRMA Foundation and the American Association of Pharmaceutical Scientists. A.A.O. was a trainee under National Institutes of Health grant T32-CA009582. References 1. Bates AD, Maxwell A (2005) DNA topology. Oxford University Press, New York 2. Deweese JE, Osheroff MA, Osheroff N (2008) DNA topology and topoisomerases: teaching a "knotty" subject. Biochem Mol Biol Educ 37 (1):2–10 3. Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 9(5):327–337 4. Liu Z, Deibler RW, Chan HS, Zechiedrich L (2009) The why and how of DNA unlinking. Nucleic Acids Res 37(3):661–671 5. Chen SH, Chan NL, Hsieh TS (2013) New mechanistic and functional insights into DNA topoisomerases. Annu Rev Biochem 82:139–170 6. Bush NG, Evans-Roberts K, Maxwell A (2015) DNA topoisomerases. EcoSal Plus 6(2) 7. Pommier Y, Sun Y, Huang SN, Nitiss JL (2016) Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat Rev Mol Cell Biol 17(11):703–721 8. Gellert M, Mizuuchi K, O’Dea MH, Nash HA (1976) DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A 73:3872–3876 9. Ashley RE, Dittmore A, McPherson SA, Turnbough CL Jr, Neuman KC, Osheroff N (2017) Activities of gyrase and topoisomerase IV on

positively supercoiled DNA. Nucleic Acids Res 45(16):9611–9624 10. Ashley RE, Blower TR, Berger JM, Osheroff N (2017) Recognition of DNA supercoil geometry by Mycobacterium tuberculosis gyrase. Biochemistry 56(40):5440–5448 11. Gibson EG, Bax B, Chan PF, Osheroff N (2019) Mechanistic and structural basis for the actions of the antibacterial gepotidacin against Staphylococcus aureus gyrase. ACS Infect Dis 5(4):570–581 12. McClendon AK, Rodriguez AC, Osheroff N (2005) Human topoisomerase IIα rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks. J Biol Chem 280(47):39337–39345 13. Fortune JM, Velea L, Graves DE, Osheroff N (1999) DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition. Biochemistry 38:15580–15586 14. Chaires JB (1990) Biophysical chemistry of the daunomycin-DNA interaction. Biophys Chem 35(2–3):191–202 15. Graves DE (1999) Drug-DNA interactions. In: Bjornsti M-A, Osheroff N (eds) Protocols in DNA topology and DNA topoisomerases, vol 2. Humana Press. Inc, Newark, New Jersey, pp 785–792

Chapter 3 Using Two-Dimensional Intact Mitochondrial DNA (mtDNA) Agarose Gel Electrophoresis (2D-IMAGE) to Detect Changes in Topology Associated with Mitochondrial Replication, Transcription, and Damage Jill E. Kolesar and Brett A. Kaufman Abstract The study of mitochondrial DNA (mtDNA) integrity and how replication, transcription, repair, and degradation maintain mitochondrial function has been hampered due to the inability to identify mtDNA structural forms. Here we describe the use of 2D intact mtDNA agarose gel electrophoresis, or 2D-IMAGE, to identify up to 25 major mtDNA topoisomers such as double-stranded circular mtDNA (including supercoiled molecules, nicked circles, and multiple catenated species) and various forms containing single-stranded DNA (ssDNA) structures. Using this modification of a classical 1D gel electrophoresis procedure, many of the identified mtDNA species have been associated with mitochondrial replication, damage, deletions, and possibly transcription. The increased resolution of 2D-IMAGE allows for the identification and monitoring of novel mtDNA intermediates to reveal alterations in genome replication, transcription, repair, or degradation associated with perturbations during mitochondrial stress. Key words Mitochondrial DNA (mtDNA), Topoisomers, 1D agarose gel electrophoresis, 2D agarose gel electrophoresis, mtDNA deletions, mtDNA separation, ethidium bromide, mtDNA damage, ssDNA

1

Introduction Mammalian mitochondrial DNA (mtDNA) is a small circular genome (~16 kb) that encodes transfer RNAs (tRNA), ribosomal RNAs (rRNA), and specific subunits of complexes I, III, IV, and V required for oxidative phosphorylation. Oxidative phosphorylation is essential to adenosine triphosphate (ATP) production in aerobic organisms; therefore, the maintenance of mtDNA integrity is crucial for cellular function. As such, defects in mtDNA and consequent ATP insufficiency have been identified in numerous conditions, including primary mitochondrial disorders [1], type 2 diabetes [2], cancer [3, 4], neurodegenerative diseases [5, 6],

Katsuhiro Hanada (ed.), DNA Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 2119, https://doi.org/10.1007/978-1-0716-0323-9_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020

25

26

Jill E. Kolesar and Brett A. Kaufman

autoimmunity [7, 8], musculoskeletal disorders [9], and aging [10, 11]. Hundreds to thousands of copies of the mitochondrial genome are present in cells and are organized into protein-DNA structures called nucleoids within the mitochondrial matrix. Each nucleoid may contain 1 or 2 copies of mtDNA [12]. Not all genomes are dedicated to the same task: for example, different subsets of nucleoids may be replicating or undergoing transcription at any given time [13, 14]. Thus, the partition of mtDNA-level tasks, such as transcription, replication, turnover, and quiescence could be determined at the whole genome/nucleoid level. Furthermore, how mtDNA changes in response to physiological stimuli or stress is poorly understood. One approach to investigating the dynamic mitochondrial genome is to observe mtDNA organization in association with these different processes and stressors [15]. As a biochemical approach, 1D gel electrophoresis has been used successfully to separate mtDNA according to size in several studies, including those that examined mtDNA size as a proxy for damage in isolated HeLa cell mitochondria [16, 17], ciprofloxin toxicity effects on mtDNA replication [18], and measuring changes associated with transient knockdown or induced expression of the mtDNA organizing and transcription factor TFAM [19]. These mtDNA structures can be further interrogated by enzymatic treatment or the addition of an orthogonal electrophoresis step. Indeed, separation of intact mtDNA in two dimensions resolves approximately 25 structures that include ssDNA molecules and variants. The similarity of mtDNA structural forms between human and mouse mtDNA molecules will likely facilitate better understanding of mtDNA molecules in human health and diseases.

2

Materials General instructions: All enzymes should be stored at
20  C, unless otherwise indicated by the manufacturer. Most reagents can be maintained as stock solutions. All tissue culture supplies should be sterile, and microfuge tubes should be RNase- and DNase-free. All biohazard and radioactive waste should be disposed of in accordance with appropriate local institutional policies. Deionized water (18 M Ohm) should be used. Stock solutions are prepared from reagent grade or better chemicals in advance and are either filter-sterilized or autoclaved to increase shelf life. Subsets of these stock solutions are considered reagents in this protocol. Pipettors and pipette tips are assumed to be readily available.

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

2.1 Cultured Cells or Tissue Collection and Storage

27

1. Stock solutions. (a) Phosphate buffer saline (PBS): 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, and 137 mM NaCl (Sigma). 2. Plasticware. (a) Sterile 15 mL conical tubes. (b) 1.5–1.7 mL microfuge tubes. 3. Tools or equipment. (a) Liquid nitrogen (LN2) or dry ice in the appropriate container. (b) Cryogloves. (c) Tongs or dipper for tube collection for LN2.

2.2

DNA Isolation

1. Stock solutions. (a) 1 M Tris–HCl pH 8.5 and pH 8.0. (b) 0.5 M EDTA pH 8.0. (c) 5 M NaCl. (d) 10% sodium dodecyl sulfate (SDS). (e) 100% ethanol. (f) Beta mercaptoethanol (BME; Sigma). 2. Reagents. (a) Proteinase K (ProK) buffer: 100 mM Tris–HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl made from stock components. (b) Cold 70% ethanol (store at
20  C). (c) GlycoBlue (Ambion; store at
20  C). (d) TE buffer: 10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0. (e) Dimethylurea (Sigma): 100 mM stock. (f) Proteinase K (ProK; VWR): Resuspend Proteinase K powder at 20 mg/mL in water. Aliquot and freeze at
20  C. Avoid numerous freeze–thaw cycles. (g) RNase A (Thermofisher; 20 mg/mL). Aliquot and freeze at
20  C. 3. Plasticware. (a) Sterile microfuge tubes. 4. Tools or equipment. (a) Scalpels or scissors for cutting tissues. (b) 2 mL glass tissue homogenizer (such as a Dounce homogenizer).

28

Jill E. Kolesar and Brett A. Kaufman

2.3 1D and 2D-IMAGE

1. Stock solutions or chemicals. (a) Ethidium bromide (EtBr): 10 mg/mL stock. (b) Sea Kem Gold agarose (Lonza). (c) Loading Dye (6, Fermentas). 2. Reagents and kits. (a) Running Buffer: 2 L of 0.5 TBE (without EtBr). To a 2 L graduated cylinder containing 1800 mL water, add 200 mL 5 TBE. Cover the cylinder with 2 layers of Parafilm and mix well. 3. Plasticware. (a) 10 mL pipettes. (b) Funnel. (c) 2 L graduated cylinder. (d) 1.6–2.0 mL screw-cap tubes for labeling radioactive probe. 4. Tools or equipment. (a) 20  25 cm gel tray and 20-well comb. Described here is for the Gibco-BRL Horizon 20–25. (b) Lab tape. (c) 1 L Erlenmeyer flask. (d) 9-inch  13-inch glass dishes. (e) Aluminum foil. (f) Parafilm. (g) Plastic wrap. (h) Razor blade. (i) Plastic ruler. (j) Spatula.

2.4 Southern Blotting (Transfer and Hybridization)

1. Stock solutions or chemicals. (a) 20 SSC (Thermofisher). (b) 50 Denhardt’s solution (Thermofisher). (c) Salmon sperm DNA (ssDNA; Thermofisher). (d) 1 M Na2HPO4 (Sigma): 71 g anhydrous Na2HPO4 plus 4 mL H3PO4 adjusted to 1 L with water. (e) 20% SDS (Thermofisher). 2. Reagents. (a) Nicking solution: Add 16.5 mL HCl to ~1480 mL water (1.5 L total volume). Cover and store at 22  C (make fresh daily; can be used for multiple gels).

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

29

(b) Denaturation solution: Add 30 g NaOH and 131.4 g NaCl to 1200 mL water; bring up to 1.5 L in a graduated cylinder. Cover and store at 22  C (make fresh daily; can be used for multiple gels). (c) Neutralization solution: Add 131.4 g NaCl, 90.75 g Tris, 51 mL HCl to 1200 mL water; bring up to 1.5 L in a graduated cylinder. Cover and store at 22  C (make fresh daily; can be used for multiple gels). (d) Transfer solution: Transfer in 10 SSC (made from a 1:1 dilution of 20 SSC). Cover and store at 22  C. (e) Church and Gilbert Hybridization (Hyb) solution: For 50 mL, add 12 mL of 1 M Na2HPO4, 8.75 mL of 20% SDS, 1 mL of ssDNA (boiled and snap-cooled), 1 mL of 50 Denhardt’s, 100 μL of 0.5 M EDTA pH 8.0 and 6 mL of water. Use the same day. (f) Church and Gilbert Wash solution: For 1 L, combine 100 mL of 1 M Na2HPO4, 100 mL of 20% SDS, and 800 mL of water. Store at room temperature. (g) MegaPrime DNA labeling kit (GE Healthcare): match the kit to radiolabel selected. (h) Radiolabel [alpha P32]-dCTP (provider varies): [alpha-32P] dCTP at 3000 Ci/mmol is commonly used. Replace often to maintain signal strength. (i) Double-stranded mtDNA template (can be generated by PCR). (j) Dimethyl Uridine (DMU; Sigma). Resuspend in water at 100 mM to use as a 50x solution. 3. Plasticware. (a) 10 mL pipette. (b) Screw-cap tubes. 4. Tools or supplies. (a) Thin gel blot paper: Cut 8 sheets of thin blot paper (Whatman 3MM or similar generic brand such as Denville Hyblot 3A) to 19.75 cm  25 cm. (b) Thick gel blot paper: Cut 4 sheets of thick blot paper (Denville Hyblot 30) to 19.75 cm  25 cm. (c) Nylon membrane: Cut 1 sheet of Hybond N+ (GE Healthcare) to 19.75 cm  25 cm. (d) Wick paper (long sheet of thin gel blot paper that drapes across the platform holding the gel; edges dip into transfer solution). Size will vary upon the equipment selected. (e) 20  25 cm support (rigid foam or thick cardboard rectangle).

30

Jill E. Kolesar and Brett A. Kaufman

(f) Paper towels and plastic wrap. (g) Hybridization oven, hybridization tubes. (h) UV cross-linker (such as Stratalinker 1800). (i) Phosphorimager screen and reader (GE healthcare) with associated software. (j) Long forceps. (k) Nanodrop spectrophotometer (ThermoFisher).

3

Methods It is not necessary to purify mitochondria for this assay, although this can be done. Thus, cells or tissues may be snap-frozen prior to preparation. General information: Carry out all procedures at room temperature unless otherwise noted.

3.1 Collection of Tissues and Cells (see Fig. 1)

Tissues 1. Tissues collected rapidly at the time of euthanasia and collected in RNase-free cryovials, immediately immersed in liquid nitrogen, and stored at
80  C for later analysis of DNA, RNA, protein, or enzyme activity. Cells 2. Adherent cultured cells require dissociation by trypsinization or scraping prior to collection. Once adherent or nonadherent cells are collected, cells are centrifuged at low speed (centrifuge and cell line dependent, 200  g for 5 min may serve as a starting point). Remove media (Fig. 1a). 3. Resuspend cells in cold PBS and transfer to microfuge tube; spin at 200  g for 5 min at 4  C. Carefully aspirate supernatant and snap-freeze in LN2 or dry ice (Fig. 1b). Samples should be stored at
80  C.

A. i. spin

ii. aspirate

iii. PBS, resuspend

iv. transfer v. spin

B.

i. aspirate, snap freeze

ii. store at -80 C

+ dry ice/ LN2

Fig. 1 Sample collection. (a) Example shown for cultured cells. Cells are pelleted in a conical tube by centrifugation and the supernatant removed by aspiration. The cell pellet is washed once by resuspending cells in PBS, transferred to a microfuge tube, and pelleted. (b) The supernatant is removed without disturbing the cell pellet. The pellet should be snap-frozen on dry ice or in LN2 and stored at
80  C

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

A. i. Add buffer + PK

B. i. Add salt, iii. transfer invert ii. 55C ON

+

31

iv. Add C. Remove GlycoBlue + supernatant, add 100% EtOH 70% EtOH, spin

D. Remove sup,

+

air dry

30' ice ii. spin at 4C

v. spin at 4C

E. Resuspend and Nanodrop

Fig. 2 DNA preparation. (a) Frozen pellets are thawed by resuspending in the Proteinase K buffer with proteinase K added. After resuspension, samples are digested at 55  C overnight with protection from light. (b) The following morning, samples are processed by adding salt, inverting, and placing samples on ice for 30 min. Following centrifugation, supernatants are transferred into a fresh microfuge tube, followed by addition of GlycoBlue and 100% EtOH, then spun at 4  C. (c) The supernatant is removed, pellet rinsed with 70% EtOH, and spun. (d) The supernatant is removed and pellets air dried while protected from light. Samples are resuspended and stored at
20  C. The DNA concentration of thawed samples is determined by OD260 in a NanoDrop 3.2 DNA Isolation from Tissues or Cells (see Fig. 2)

1. Premix Proteinase K digestion buffer with Proteinase K and beta-mercaptoethanol (BME) (250 μL for tissues, 500 μL for cells): (a) Tissues: 240.5 μL ProK buffer +7.5 μL Proteinase K + 2 μL 1:100 BME (diluted in water). Continue to step 2. (b) Cells: 481 μL ProK buffer+15 μL Proteinase K, +4 μL 1:100 BME. Continue to step 4. 2. For tissues, cut a small piece of tissue (1–5 mm3) on a precooled glass dish sitting on dry ice. Add tissue to a glass homogenizer containing 125 μL of the premixed digestion buffer that includes ProK and BME. Homogenize lightly (~10 passes) on ice, being sure to expose maximal surface area of tissue to the pestle. Transfer homogenate to a microfuge tube (see Note 1). 3. Rinse the homogenizer with an additional 125 μL digestion buffer and combine with the lysate in the microfuge tube. Continue to step 6. 4. For cells, grow until ~80% confluent, pellet trypsinized cells in a 15 mL conical tube at ~200  g for 5 min, aspirate medium. Resuspend in 500–1000 μL PBS, transfer to microfuge tube, and spin at 4  C at ~200  g for 5 min, aspirate PBS. Flashfreeze pellet on dry ice. 5. Add the premixed Proteinase K buffer + Proteinase K + BME to the cell pellet by using the point of the pipette tip to loosen the cell pellet at the bottom of the tube before adding the digestion buffer. Pipet up and down gently a few times, and flick or invert the tubes to ensure even distribution of buffer to cells (see Note 2). Lysis should be obvious and within 5 min. Do not vortex.

32

Jill E. Kolesar and Brett A. Kaufman

6. Digest overnight at 55  C, with lid on water bath closed to protect samples from light. 7. The following morning: (a) For tissues, dilute samples to a final volume of 500 μL Proteinase K buffer if currently at 250 μL. (b) For tissues and cells, supplement samples with another 10 μL Proteinase K. Flick tubes to mix (do not vortex). Incubate for 1 h at 55  C. 8. Add 170 μL of 5 M NaCl to each 500 μL sample, invert or rotate tube for 5 min to ensure complete distribution of NaCl (see Note 3). 9. Centrifuge at maximum speed for 15 min at 4  C. 10. Transfer supernatant to fresh tube (see Note 4). 11. Add 1 mL 100% EtOH and 1 μL GlycoBlue coprecipitate to the supernatant. Invert several times to mix. 12. Centrifuge at max speed for 15 min at 4  C. 13. Aspirate ethanol off manually or with vacuum suction (be careful not to disturb the pellet). 14. Wash pellet carefully in 500 μL cold 70% EtOH. 15. Spin 5 min at max speed at 4  C. 16. Aspirate ethanol off, air-dry pellet by placing the open tube in a drawer (protected from light). For same day processing, see Note 5. 17. Resuspend pellets from tissues in 48 μL TE + 1 μL RNAse A + 1 μL DMU. For pellets from cells, resuspend in 97 μL TE + 2 μL RNAse A + 2 μL DMU overnight in the dark. RNase A digests ribonucleic acids, and dimethyl urea is added as a radical scavenger. For optimization of resuspension (see Note 6). 18. Incubate at room temp in the dark overnight. 19. Freeze DNA at
20  C (confirm that samples are frozen). This step helps to break up local high concentrations of DNA. 20. Quantitate by NanoDrop. 21. Add 2–10 μg DNA to a microfuge tube and add water to 40 μL and an appropriate volume of loading dye (8 μL 6 loading dye (Fermentas). Mix by flicking, not pipetting. Store at
20  C protected from light; avoid multiple freeze–thaw cycles. 3.3 1D- and 2D-IMAGE (see Fig. 3)

Based on your application and desired resolution, the experiments may be 1D or 2D, and samples may be treated with enzymes for further investigation. This section describes the process of running the gels and will identify branch points in the procedures.

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

33

A. i. load ii. run ON 1D-IMAGE soak gel in EtBr

B.

i. cut out lanes

ii. rotate lanes 90

iii. cast gel

EtBr

gel transfer

~9.5 cm

2D-IMAGE iv. run ON

Fig. 3 1D and 2D-IMAGE. (a) For either format: (i) use a pipette to load samples into wells of the agarose gel (load every other lane for 2D-IMAGE), and (ii) run at 40 V for 16 h. If running 1D-IMAGE, proceed to gel transfer. (b) If running 2D-IMAGE, soak gel in EtBr, then: (i) cut out gel lanes, (ii) rotate 4 lanes 90 with wells to the left, (iii) cast second dimension gel (containing EtBr) around lanes, and then (iv) run overnight 3.3.1 First Dimension (Day 1) (see Fig. 3a)

1. Treatment of DNA may be desired. A list of several enzymes and expected effects is provided in Table 1. Be sure to stop the reaction but avoid denaturation-based approaches. If no treatment is desired, continue to step 2. 2. Tape the ends of a 20  25 cm gel tray with two layers of lab tape, being sure to seal the overlapping segments. Add a 20-well comb and place on level surface. A bubble level is recommended. 3. Prepare the 0.5 TBE running buffer as described in Subheading 2.3. 4. Prepare gel: 0.4% agarose in 500 mL 0.5 TBE. Confirm that the microwave is large enough for a 1 L Erlenmeyer flask. Add 250 mL 0.5 TBE to the flask. Weigh 2 g agarose, add to the TBE in flask and microwave for 3 min, or until the solution is boiling rapidly (see Note 7). Add stir bar. While mixing and still hot, add final 250 mL 0.5 TBE. 5. Cast the gel when the flask is no longer too hot to touch with gloves. Ensure no air bubbles surround the teeth of the comb. 6. Pour the remainder of the 0.5 TBE into the gel tank.

34

Jill E. Kolesar and Brett A. Kaufman

Table 1 Enzymes used to examine mtDNA topoisomers

Enzyme used to treat total genomic DNA

Manufacturer and reaction Expected result after enzyme treatment conditions

Ambion RNase A: Digests ss and ds RNA after U Removal of abundant RNA species associated with mtDNA that interfere 2 μL at 24  C for and C bases in low salt, present in DNA with interpretation preparations before 1D gel 1h electrophoresis RNase H: Digests RNA in RNA–DNA hybrid duplex

Increase in circular and linear ssDNA

Fermentas 15 U at 37  C for 30 min

BglII or SalI: Cleaves circular form mtDNA once (mouse or human, respectively)

Location of linearized product in the gel Fermentas marks the position of genome length 4 U at 37  C for linear mtDNA in the undigested 30 min profile.

When used as a comparison with an Topoisomerase 1: Cuts one of the two untreated sample, reveals position in strands of DNA, relaxes all supercoils gel of maximally supercoiled mtDNA from DNA molecule, and reanneals the molecules strand. ATP-independent and does not separate catenated molecules

Invitrogen 20 U at 37  C for 1h

Confirms location of supercoiled DNA gyrase: Increases the degree of mtDNA molecules supercoiling of closed circular DNA. Catalyzes the ATP-dependent negative supercoiling of ds closed-circular DNA to condense the chromosome

New England Biolabs 10 U at 37  C for 1h

Type II topoisomerases (topoisomerase II and IV): Make ds breaks that allow one duplex to pass through the other and reseals the break. Requires ATP. Remove supercoiling 2 twists at time, resulting in a relaxed circle

Decatenates or relaxes supercoiled molecules but leaves concatemers (head to tail dimers) intact

Topoisomerase II: USB Affymetrix 40 U at 37  C for 1h Topoisomerase IV: Inspiralis 20 U at 37  C for 1h

S1 nuclease: Digests ssDNA, dsDNA at nicks or gaps, and RNA

Identifies hybridization between single strands of DNA and/or RNA.

Promega (part #) 0.9 U at 37  C for 30 min.

E. coli exonuclease 1: Hydrolyzes ssDNA Digests ssDNA in a 30 ! 50 direction. stepwise in a 30 ! 50 direction. It does Does not digest dsDNA. not degrade dsDNA or DNA-RNA hybrids

NewEngland biolabs 20 U at 37  C for 30 min.

ds ¼ double-stranded; ss ¼ single-stranded

7. Take the tape off the ends of the gel tray by pulling the tape downward at a 90 angle, not outward (see Note 8). 8. Place the gel tray in the tank, and carefully remove the comb. Rinse wells with running buffer using 1 mL pipettor.

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

35

9. Gel loading. (a) FOR 1D-IMAGE: Load the gel with ladder (any; 1 kb + ladder is convenient) and samples using conventional DNA loading buffers. (b) FOR 2D-IMAGE: Load the gel with ladder and samples in every other odd numbered lane, as the gel will be cut in the center of the even numbered wells later in the procedure. 10. Connect the electrodes to the power supply and cover the gel tank with foil. Run the gel at 40 V for 16 h (see Note 9). 3.3.2 Second Dimension (Day 2) (see Fig. 3b)

If performing 1D-IMAGE, skip to Subheading 3.4. 1. Transfer the first dimension gel to a 9  13-inch glass dish, or other appropriate-sized dish that can hold enough buffer to submerge the gel in its tray (see Note 9). 2. Pour 1.4 L running buffer from the gel tank into a 2 L graduated cylinder in the sink using a funnel to prevent spillover. 3. Pour the TBE into the glass dish while adding 40.5 μL EtBr (10 mg/mL stock) to the stream of TBE coming out of the cylinder as you pour (see Note 10). 4. Cover the dish in foil and shake gently for 25 min. 5. Remove gel and set in a container. Collect and dispose of buffer + EtBr. Briefly rinse the gel under a slow stream of distilled water to remove as much EtBr as possible. Be sure to place a hand over each open end of the tray to avoid the gel sliding out. 6. Place the gel tray on a benchtop with the wells on the right; with a clean razor blade or scalpel, make a cut parallel to the wells ~9.5 cm away using a ruler to guide the razor blade. 7. Use the ruler to make cuts perpendicular to the wells in every other lane of the gel (samples were loaded in odd wells so cuts are made in the center of even wells; up to 4 gel lanes can be placed in the second dimension gel). 8. Transfer cut gel slices to a covered dish using the spatula; rotate gel slices so the wells are now on the left (this is how they will be placed in the gel tray for the second dimension). Wrap the dish in foil and keep at 4  C until the second dimension gel is ready to be poured. 9. Tape the ends of a clean, dry gel tray with 2 layers of tape, being sure the overlapping portions are well sealed. 10. Prepare the 0.4% agarose in 0.5 TBE in 500 mL (prepared as in Subheading 3.3.1), but add 15 μL EtBr when cool to the touch. Mix well.

36

Jill E. Kolesar and Brett A. Kaufman

11. Cast the second dimension gel around the gel slices when the flask is no longer too hot to touch with gloves; pour slowly to avoid moving gel slices. 12. If the gel slices move during pouring, use a P1000 pipette tip to reposition them. 13. Cover the gel tray with foil during polymerization to protect from light. 14. Prepare 2 L 0.5 TBE as above. 15. Pour the buffer into the tank while adding 60 μL EtBr to the stream of TBE coming out of the cylinder. 16. Once fully polymerized, take the tape off the ends of the gel tray by pulling the tape downward, not outward (see Note 11). 17. Place the gel in the tank, connect the electrodes, and cover the tank with foil. Run the gel for 16 h at 40 V. 3.4 Gel Transfer (Day 3) (see Fig. 4)

1. Prepare nicking solution, denaturation solution, neutralization solution, and transfer solution as described above. 2. Prepare 8 sheets of thin gel blot paper, 4 sheets of thick gel blot paper and 1 nylon membrane as described above. 3. Prepare gel: see Fig. 4a. (a) Rinse the gel under deionized water briefly and soak it in its gel tray in nicking solution in a glass dish for 25 min with gentle shaking. (b) Rinse the gel briefly (in tray for support), then soak in denaturation solution for 25 min with gentle shaking on platform shaker. (c) Rinse, soak in neutralization solution for 25 min with gentle shaking. 4. Place a large piece of plastic wrap on the benchtop and smooth out any bubbles. Briefly rinse the gel in its gel tray under deionized water and gently slide the gel off the tray and onto the left half of the plastic wrap (see Note 12). 5. Rinse the empty gel tray under deionized water and set aside. Fill a glass dish with 10 SSC to a depth of ~2 cm. 6. Holding the gel tray in your right hand, dip the wick paper (long blotting paper that will fit on your support) into the 10 SSC with your left hand and drape it over the gel tray, making sure the ends of the wick paper overhang the gel tray evenly. Use a 10 mL pipette to smooth away any bubbles under the wick paper (see Note 13). 7. Place 3 sheets of thin gel blot paper on top of the gel and pat gently to initiate adherence between the papers and the gel.

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

A.

37

soak i. Nicking solution ii. Denaturing solution quick rinse

B.

iii. Neutralization solution

place blotting paper with support on top

blotting paper on bottom

flip

C.

weight

blotting papers nylon membrane gel blotting paper

gel tray paper towels wick paper plastic wrap

~2cm SSC Fig. 4 Gel transfer. (a) For the nonelectrophoretic transfer of nucleic acids from 1D and/or 2D IMAGE gels to nylon membranes, the gel is soaked in nicking solution, denaturing solution, and neutralizing solution for 25 min with a brief water rinse between steps. (b) A piece of blotting paper (the same size as the gel) is placed on top of the gel and the gel then flipped using a support; this leads to the blotting paper being on the bottom. (c) Assemble the transfer stack in a glass or plastic dish. A support to lift the transfer stack out of the buffer, such as an upside-down gel tray, is required. On that support, stack (from bottom to top): wicking paper, blot paper + gel, nylon membrane, thin blotting paper, thick blotting paper, paper towels, gel tray, and weight

Fold the right half of the plastic wrap over the top of the gel + paper and pull it taut. 8. Place a 20  25 cm support (rigid packing foam, plexiglass or cardboard) on top of the plastic-wrapped gel and flip the gel + blotting paper over by lifting the two edges of plastic wrap up while supporting the top of the gel with your right hand on the support. 9. Pull the plastic wrap off the top and slide your hands between the blot paper and plastic wrap on the bottom. Support under the left and right side of the gel + blot paper (now face down). Gently lift the gel and place it onto the wick paper/gel tray assembly in the glass dish. 10. Smooth away bubbles using the 10 mL pipette. Be careful to not gouge the soft gel.

38

Jill E. Kolesar and Brett A. Kaufman

11. Rinse the Hybond membrane with deionized water and place onto the top of the gel. Smooth away bubbles with pipette. 12. Add 8 sheets of thin gel blot paper and 4 sheets of thick gel blot paper, followed by paper towels stacked about 5 cm high. 13. Cover the whole transfer assembly with plastic wrap, place a square dish or the gel tray on top, and make sure the tray is level. Finally, place a modest weight (such as a 250 mL solution) on top of the square dish to apply even pressure on the paper towels. 14. Allow to transfer overnight. 3.5 Hybridization (Day 4)

Instructions are provided for Church and Gilbert hybridization of a double stranded probe only. Other hybridization methods can be used.

3.5.1 Prehybridization

1. Disassemble the transfer stack, leaving one piece of thin blot paper on top of the membrane. Peel back the upper right corner of the paper and membrane slightly, then use a pencil to note pertinent identifying information such as the date the gel was initiated and the name of the samples. 2. Place the membrane (face up) onto a piece of dry thin gel blot paper and UV-cross-link the DNA to the membrane in the cross-linker before the membrane completely dries. The cross-linker should be automatically set for “1200” on the “energy” setting. 3. Roll the dry membrane into a tube, starting from right to left, and place it in a large, dry, glass hybridization tube. 4. Add 25 mL Church and Gilbert Hyb solution; pre-hyb at 65  C for at least 2 h with rotation. 5. Make the probe during the pre-hyb step (see Subheading 3.5.2). 6. Do not add probe directly to the hyb tube; predilute it in about 500 μL of pre-hyb solution and add the pre-hyb + probe to the center of the hyb tube. 7. Incubate overnight at 65  C with rotation.

3.5.2 Probe Synthesis

1. Thaw mtDNA probe template (5 ng/μL). Add 5 μL (for a total of 25 ng) to a screw-cap tube. 2. Add 5 μL random primer solution from the MegaPrime DNA labeling kit. 3. Place the lid on the tube and heat at 95  C for 5 min. 4. Add 23 μL water, 10 μL labeling buffer, 5 μL 2 μL Klenow enzyme (see Note 14). 5. Incubate at 37  C for 20 min.

32

P dCTP, and

Detection of Changes in mtDNA Topology using 1 and 2-D Gels

39

6. Stop the reaction by adding 5 μL 0.2 M EDTA (diluted from 0.5 M stock; can also use 1 μL of 0.5 M stock). 7. Immediately prior to adding probe to hyb tubes, denature probe by heating at 95  C for 5 min then placing on ice for 5 min to make single stranded. 3.5.3 Blot Washing

1. Pour the hyb solution into an appropriate liquid waste container (see Note 14). Be sure to dab a small paper towel strip onto the lip of the hyb tube to absorb any drops of hyb that have collected there during pouring (this will avoid spreading raw hyb solution around the outside neck of the hyb tube, which could then contaminate the threads of the plastic lid). 2. Add ~150 mL Church and Gilbert wash solution, replace the lid on the tube and rinse the membrane by gently tilting the tube left and right while rolling it back and forth. Pour the rinse into the liquid waste container. 3. Add 150 mL wash solution and wash at 65  C with rotation for 30 min. 4. Pour the first wash into the liquid waste container. 5. Wash twice more for 30 min each; the second and third washes can be disposed of in the sink with running water (if this is within an acceptable range according to institutional guidelines). 6. After the last wash, remove the membrane from the tube with long forceps. If necessary, crinkle the top edge of the membrane by pushing it downward slightly with the forceps to create a surface that the forceps can grasp. Dry the membrane on a large piece of thin gel blot paper until the surface of the membrane is no longer shiny. 7. Place the membrane on a piece of plastic wrap (DNA-side down) and fold over three sides. Use a paper towel to smooth away any bubbles in the plastic wrap and fold the fourth side of the plastic closed. 8. Spray a paper towel with 70% ethanol and wipe down both sides of the plastic-wrapped membrane to dissipate static electricity. 9. Place the membrane in a phosphorimager cassette, taping it in place, and scan 2 days later. An example of how to interpret the results is provided in Fig. 5.

4

Notes 1. Cryo-pulverizing of tissue improves the representativeness of a small sample and increases surface area during extraction. Pulverization improve detergent accessibility increases yield and

40

Jill E. Kolesar and Brett A. Kaufman

A. hybridization

interpretation o. high molecular weight DNA i. >1n mtDNA ii. relaxed circles iii. linear iv. supercoiled

B.

o

interpretation

hybridization i

i. catenated and concatenated mtDNA ii. 1n nicked circular mtDNA

ii

iia. single-stranded circular mtDNA

iii

iii. 1n linear mtDNA iva

iiib iiia. 1n single stranded linear mtDNA iv iiib. pH 13), and not only double-strand break but also single-strand break or DNA damage with alkali-sensitive sites such as abasic site (AP site) is possible to detect. The evaluation is possible regardless of species, age, and sex of animals used under the alkali comet assay. However, it is recommended to use both sexes for substances which show different metabolism in males and females. In case the target organ is a genital organ (e.g., a testis or uterus), animals of appropriate sex are used. In principle, the standard dose selection is the maximum tolerating dose, but 2000 mg/kg/day (when the administration period is less than 14 days) is recognized as the limit dose for test substances with low toxicity. The route selection of administration is based on the human exposure route, and the administrations are performed twice or more. In addition, because it is a detection system for initial damage, it is required to collect organs before the DNA damage caused by the test substance is repaired. Generally, two, three, or more administrations are performed, and organ harvesting at 2–6 h after the final administration is suitable for detecting DNA damage in the comet assay.

2

Materials

2.1 Sample Preparation

1. Mincing buffer (Homogenized buffer). The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO will be added immediately before use). This solution is refrigerated at