Mitochondrial DNA: Methods and Protocols

Preface
Contents
Contributors
Part I: Purification Methods for Mitochondrial DNA
Chapter 1: Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Consumables
2.3 Buffers and Solutions
2.3.1 Mitochondrial Isolation Buffers (MIB)
2.3.2 Sample Freezing Buffer
2.3.3 Mitochondria Lysis Buffer
3 Methods
3.1 Isolation of Mitochondria from Adult Heart
3.2 Isolation of Mitochondria from Neonatal Heart
3.3 Isolation of Mitochondria from Intestine
3.4 Isolation of Mitochondria from Liver
3.5 Isolation of Mitochondria from Brain
3.6 Isolation of Mitochondria from Skeletal Muscle
3.7 Isolation of Cytoplasmic and Peri-lipid Droplet Mitochondria from Adipocytes
3.8 Basic Protocol for mtDNA Purification from Isolated Mitochondria
4 Notes
References
Chapter 2: Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells
1 Introduction
2 Materials
2.1 Crude Mitochondrial Isolation
2.2 Sucrose Gradient Centrifugation
2.3 Mitoplast Preparation and Protease Treatment
2.4 Cell Fractionation for Protein Localization
2.5 mtDNA Extraction
3 Methods
3.1 Crude Mitochondrial Isolation
3.2 Sucrose Gradient Centrifugation
3.3 Mitoplast Preparation and Protease Treatment
3.4 Cell Fractionation for Protein Localization
3.5 mtDNA Extraction
4 Notes
References
Chapter 3: Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Puri...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Mitochondrial DNA Isolation
2.3 Column-Free Total DNA Isolation
2.4 Exonuclease V Treatment and SEC
2.5 Relative mtDNA Copy Number Determination by Droplet Digital PCR (ddPCR)
2.6 General Laboratory Equipment
3 Methods
3.1 Cell Culture and Preparation of Cell Pellets for Isolation of DNA
3.2 Column-Free Total DNA Isolation
3.3 mtDNA Isolation
3.4 Exonuclease V Treatment
3.5 Size Exclusion Chromatography
3.6 Assessment of Relative mtDNA Copy Number Using ddPCR
4 Notes
References
Chapter 4: Isolation and Quality Control of Yeast Mitochondria
1 Introduction
2 Materials
2.1 Yeast Culture
2.2 Isolation of Mitochondria
2.2.1 Working Solutions
2.3 Sucrose Gradient Purification of Isolated Mitochondria
2.4 Quality Control of Isolated Mitochondria
2.4.1 Protease Accessibility Assay
2.4.2 In organello Import of Su9-DHFR into Isolated Mitochondria
3 Methods
3.1 Yeast Culture
3.2 Isolation of Crude Mitochondria
3.3 Isolation of Highly Purified Mitochondria
3.4 Quality Control of Isolated Mitochondria
3.4.1 Protease Accessibility upon Osmotic Swelling
3.4.2 In organello Import of Su9-DHFR into Isolated Mitochondria
4 Notes
References
Chapter 5: Mitochondrial DNA Isolation from Plants
1 Introduction
2 Materials
2.1 Plant Culture
2.1.1 In Vitro Culture of Arabidopsis Seedlings
2.1.2 Culture of Arabidopsis Plants on Soil
2.1.3 Culture of Lettuce Seedlings on Blotting Paper
2.2 Extraction of Mitochondria
2.2.1 Materials and Solutions for Extraction of Mitochondria
2.2.2 Buffers for Mitochondria Isolation
2.2.3 Percoll Step Gradients for Arabidopsis Mitochondria Isolation
2.2.4 Percoll Step Gradients for Lettuce Mitochondria Isolation
2.3 Extraction and Purification of Nucleic Acids from Mitochondria
2.3.1 Common Materials and Solutions for Nucleic Acid Purification
2.3.2 DNA Purification Using CTAB
2.3.3 DNA Purification Using QIAamp DNA Micro Kit
2.3.4 RNase Treatment of Nucleic Acids
2.4 qPCR to Assess mtDNA Enrichment
3 Methods
3.1 Plant Growth Conditions
3.1.1 In Vitro Culture of Arabidopsis Plants
3.1.2 Arabidopsis Plants Grown in Soil
3.1.3 Culture of Etiolated Lettuce Seedlings on Blotting Paper
3.2 Purification of Mitochondria
3.2.1 Purification of Mitochondria from Arabidopsis Seedlings
3.2.2 Crude Partial Purification of Mitochondria from Single Arabidopsis Plants
3.2.3 Purification of Mitochondria from Etiolated Lettuce Seedlings
3.3 Extraction and Purification of Nucleic Acids from Mitochondria
3.3.1 Mitochondrial DNA Purification Using CTAB
3.3.2 Mitochondrial DNA Purification Using QIAamp DNA Micro Kit (Qiagen)
3.3.3 RNase Treatment of Nucleic Acids
3.4 Analyses of mtDNA Enrichment by qPCR
3.5 Analyses of mtDNA Enrichment from Illumina Whole Genome Sequencing Data
4 Notes
References
Part II: Visualising mtDNA
Chapter 6: Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies
1 Introduction
2 Materials
2.1 Observation of Total mtDNA
2.1.1 Map Total mtDNA Distribution in Fixed Cell
2.1.2 Track Total mtDNA Dynamics in Live Cell
2.2 Observation of mtDNA Replication
2.2.1 Map mtDNA Replication in Fixed Cell
2.2.2 Track mtDNA Replication in Live Cell
3 Methods
3.1 Observation of Total mtDNA
3.1.1 Map Total mtDNA Distribution in Fixed Cell
3.1.2 Track Total mtDNA Dynamics in Live Cells
Track Total mtDNA in Live Cells by Transfecting with Plasmids
Track Total mtDNA in Live Cells by Staining with PicoGreen dsDNA Reagent
3.2 Observation of mtDNA Replication
3.2.1 Map mtDNA Replication in Fixed Cell
Immunostaining Toward POLγ
Incorporation of BrdU, EdU, and PdG
BrdU Labeling
EdU Labeling
PdG Labeling
Dual BrdU and EdU/PdG Labeling
3.2.2 Observation of mtDNA Replication in Live Cells
4 Notes
References
Chapter 7: Visualization of mtDNA Using FISH
1 Introduction
2 Materials
2.1 Cell Culture and Seeding
2.2 mtDNA Probes
2.2.1 Oligonucleotides
2.2.2 mtDNA Probe Preparation
2.3 Chemicals and Equipment
2.4 Buffers and Solutions
3 Methods
3.1 Cell Seeding (3 Days Before Staining)
3.2 Preparing and Labeling mtDNA Probes
3.3 Fixation and Permeabilization
3.4 RNase Treatment
3.5 Prehybridization
3.6 Hybridization
3.7 Washes
3.8 Immunostaining (Optional) and Nuclear Counterstaining (see Note 11)
3.9 Mount
4 Notes
References
Chapter 8: In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 EdU Pulse Labeling
3.2 Fixation and Click Reaction
3.3 Immunolabeling
3.4 Imaging
4 Notes
References
Chapter 9: Measurement of Nucleoid Size Using STED Microscopy
1 Introduction
2 Materials
2.1 Immunocytochemistry
2.2 Analysis of Nucleoid Diameter
3 Methods
3.1 Immunocytochemistry
3.2 Nucleoid Analysis
3.2.1 Single Nucleoid Analysis
3.2.2 Bulk Analysis
4 Notes
References
Part III: Mitochondrial DNA Interacting Proteins
Chapter 10: How to Quantify DNA Compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence...
1 Introduction
2 Materials
2.1 TFAM Purification and Labeling
2.2 DNA Production and Labeling
2.2.1 Template for AFS Experiments
2.2.2 Template for TIRF Experiments
2.3 Preparation of the Flow Cells
2.3.1 For AFS Experiments
2.3.2 TIRF Experiments
2.4 Hardware
2.5 Software
3 Methods
3.1 Assessing DNA Compaction by TFAM with AFS
3.1.1 DNA Labeling
3.1.2 Flow Cell Functionalization
3.1.3 AFS on TFAM-Coated DNA
3.2 Assessing TFAM Dynamics with TIRF Microscopy
3.2.1 DNA Biotin Labeling
3.2.2 TFAM Labeling
3.2.3 Flow Cell Functionalization
3.2.4 Anchoring DNA Template to the Glass Surface
3.2.5 Sample Preparation
3.2.6 Imaging
3.2.7 Data Analysis
4 Notes
References
Chapter 11: Assessing TFAM Binding to Human Mitochondrial DNA
1 Introduction
2 Materials
2.1 General Consideration
2.2 Agarose Gel and Running Buffer
2.3 Preparation of Proteins and Plasmid DNA
2.4 DNA Detection After Electrophoresis
3 Methods
3.1 Preparation of Agarose Gel Running Buffer
3.2 Preparation of Agarose Gel
3.3 Preparation of Recombinant TFAM
3.4 Preparation of Plasmid DNA
3.5 Electrophoretic Mobility Shift Assay (EMSA)
3.5.1 Preparation of Linearized Plasmid
3.5.2 TFAM-Binding Incubation
3.5.3 Agarose Gel Electrophoresis and Detection of TFAM-Bound DNA
3.6 DNA-Unwinding Assay
3.6.1 Unwinding Assay
3.6.2 Agarose Gel Electrophoresis and Plasmid DNA Visualization
3.6.3 Confirmation of the Absence of DNA Nick Introduction by Southern Hybridization
4 Notes
References
Chapter 12: Identification of Proximity Interactors of Mammalian Nucleoid Proteins by BioID
1 Introduction
1.1 Background
1.2 Considerations for a BioID Experiment Design
2 Materials
2.1 Bait Cloning
2.2 Generation of Cells Stably Expressing the Bait
2.3 Selection of a Clone for Downstream Analysis
2.4 Pull-Down of Biotinylated Proteins
3 Methods
3.1 Bait Cloning
3.1.1 Primer Design
3.1.2 Cloning (See Note 4)
3.2 Generation of Cells Stably Expressing the Bait
3.3 Selection of a Clone for Downstream Analysis
3.4 Pull-Down of Biotinylated Proteins
3.4.1 SDS-PAGE and Western Blot
3.4.2 Biotin Pull-Down on Streptavidin Beads
3.4.3 Western Blotting of Biotinylated Proteins
4 Notes
References
Chapter 13: Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Generation of Murine Cell Lines Expressing Nucleoid-Targeted Transgene (See Note 4)
2.3 Validation of Cell Clones by Immunofluorescence
2.4 Sample Preparation for Electron Microscope
3 Methods
3.1 Generation of Ecotropic Retroviruses (See Note 6)
3.2 Generation of Cell Lines Stably Expressing Transgenes
3.3 Validation of the Cell Lines
3.3.1 mt-Nucleoid Localization of Twinkle-APEX2-V5
3.3.2 Proximity Biotinylation
3.4 DAB Staining of Cells and Validation by Light Microscopy
3.5 Ultrathin Sample Preparation for EM
4 Notes
References
Part IV: Mitochondrial DNA Replication and Repair
Chapter 14: In Vitro Assays of TWINKLE Function
1 Introduction
2 Materials
2.1 DNA Helicase Template Preparation
2.2 DNA Helicase Assay
2.3 ATPase Assay
2.4 Gel Electrophoresis and Visualization
3 Methods
3.1 Helicase Oligonucleotide Labeling
3.2 Helicase Template Preparation
3.3 Helicase Activity Reaction
3.4 Non-denaturing Polyacrylamide Gel
3.5 Visualization
3.6 ATPase Assay
3.7 Visualization and ATPase Activity Calculation
4 Notes
References
Chapter 15: Rolling Circle Replication and Bypass of Damaged Nucleotides
1 Introduction
2 Materials
2.1 Preparation of DNA Template
2.1.1 Circularization of Template and Purification
2.1.2 DNA Substrate Preparation
2.2 Purified Recombinant Proteins
2.3 In Vitro Replication Assay
2.4 Sequencing Gel Electrophoresis
3 Methods
3.1 DNA Template Preparation
3.1.1 Preparation of Minicircle Template
3.1.2 Purification of Circular Template on a Polyacrylamide Gel
3.1.3 Labeling of the Primer Oligonucleotide at the 5′-End
3.1.4 Primer Template Hybridization
3.2 Rolling Circle Replication Reaction
3.3 Visualization of Reaction Products
4 Notes
References
Chapter 16: Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria
1 Introduction
2 Materials
2.1 In Organello Replication and Transcription Assays
2.1.1 Mitochondrial Protein Content Quantification
2.1.2 Buffers
2.2 In Organello Replication Assay
2.2.1 mtDNA Radioactive Labeling
2.2.2 DNA Isolation
2.2.3 Southern Blot
2.3 In Organello Transcription Assay
2.3.1 mtRNA Radioactive Labeling
2.3.2 RNA Isolation
2.3.3 Northern Blot
3 Methods
3.1 In Organello Replication and Transcription Assays
3.2 In Organello Replication Assay
3.2.1 32P Labeling-Pulse
3.2.2 Chase
3.2.3 Washing
3.2.4 Mitochondrial Lysis and DNA Extraction
3.3 In Organello Transcription Assay
3.3.1 32P Labeling-Pulse
3.3.2 Chase
3.3.3 Washing
3.3.4 Mitochondrial Lysis and RNA Extraction
4 Notes
References
Chapter 17: Functional Assessment of Mitochondrial DNA Maintenance by Depletion and Repopulation Using 2´,3´-Dideoxycytidine i...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 DNA Isolation
2.3 Quantitative PCR
3 Methods
3.1 ddC Treatment
3.2 Harvesting Cells (Six-Well Plate Scale)
3.3 DNA Isolation and Determination of Concentration
3.4 Quantitative PCR
3.5 Data Analysis
4 Notes
References
Chapter 18: Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis
1 Introduction
2 Materials
2.1 Homogenization of Cultured Cells with Cytochalasin
2.2 Hypotonic Homogenization of Cultured Cells
2.3 Homogenization of Tissues
2.4 Mitochondrial Nucleic Acid Isolation
2.5 Total DNA Extraction for 2D-AGE
2.6 DNA Digestion and Two-Dimensional Neutral Agarose Gel Electrophoresis
2.7 Southern Blotting and Detection
2.8 Additional Treatments and Variations of 2D-AGE
3 Methods
3.1 Homogenization of Cultured Cells with Cytochalasin
3.2 Hypotonic Homogenization of Cultured Cells
3.3 Homogenization of Tissues
3.4 Mitochondrial Nucleic Acid Isolation
3.5 Total DNA Isolation
3.6 DNA Digestion and Two-Dimensional Agarose Gel Electrophoresis
3.7 Southern Blotting and Detection
3.8 Additional Treatments and Variations of 2D-AGE
3.8.1 Removal of Single-Stranded Nucleic Acids
3.8.2 Removal of RNA from RNA:DNA Hybrids
3.8.3 Identification of Holliday Junctions and Other Branched Structures
3.8.4 Topological Analysis of mtDNA
4 Notes
References
Chapter 19: Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chroma...
1 Introduction
2 Materials
2.1 Collection of Muscle Tissue Sample
2.2 Extraction of dNTPs and NTPs from Muscle Tissue Sample
2.3 Separation of dNTPs from NTPs with a Boronate Affinity Column
2.4 Two-Step Solid-Phase Extraction
2.5 Separation and Detection of dNTPs and NTPs by LC-MS/MS
3 Methods
3.1 Collection of Muscle Tissue Sample
3.2 Extraction of dNTPs and NTPs from the Muscle Tissue Sample
3.3 Separation of dNTPs from NTPs with the Boronate Affinity Column
3.4 Two-Step Solid-Phase Extraction
3.5 Separation and Detection of dNTPs and NTPs by LC-MS/MS
4 Notes
References
Chapter 20: Detection of UV-Induced Deletions in Mitochondrial DNA
1 Introduction
2 Materials
2.1 UVA Irradiation of Human Skin Fibroblasts
2.2 Isolation of mtDNA
2.3 Common Deletion Detection by qPCR
3 Methods
3.1 UVA Irradiation of Human Skin Fibroblasts
3.2 Isolation of mtDNA
3.3 Common Deletion Detection by qPCR
4 Notes
References
Chapter 21: Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels
1 Introduction
2 Materials
2.1 Alkaline Hydrolysis
2.2 Alkaline Gel Electrophoresis
2.3 Gel Treatments
2.4 DNA Transfer
2.5 Southern Hybridization
2.6 Quantification
3 Methods
3.1 Alkaline Hydrolysis
3.2 Alkaline Gel Electrophoresis
3.3 Gel Treatments
3.4 DNA Transfer
3.5 Southern Hybridization
3.6 Quantification
4 Notes
References
Chapter 22: 5′-End Mapping in Human Mitochondrial DNA
1 Introduction
2 Materials
2.1 DNA Isolation from Healthy and Patient-Derived Fibroblast Cells
2.2 Library Preparation
3 Methods
3.1 DNA Isolation from Human Fibroblast Cells
3.2 Phosphorylation of DNA 5′ Termini
3.3 Adapter Ligation to the 5′ End
3.4 Second Strand Synthesis
3.5 PCR Amplification, Library Analysis, and Pooling
3.6 Quality Check
3.7 Sequencing
3.8 Alignment and Visualization
4 Notes
References
Part V: Modifying Mitochondrial DNA
Chapter 23: Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs
1 Introduction
2 Materials
2.1 Genotyping Design by Pyrosequencing
2.2 Cloning ZF Arrays into mtZFN Expression Vectors
2.3 MEF Transformation and Sorting
2.4 DNA Analysis
2.5 Common Laboratory Equipment
2.6 Non-commercial Reagents
3 Methods
3.1 Designing Primers for the Measurement of Heteroplasmy
3.2 Cloning Zinc Finger Arrays into Expression Vectors
3.3 MEF Transformation by Electroporation and FACS Enrichment
3.4 Analysis of MEF Cells and mtZFN Qualification
3.4.1 Measuring Shifts in mtDNA Heteroplasmy
3.4.2 Measuring Recovery of mtDNA Copy Number
4 Notes
References
Chapter 24: Biolistic Transformation of Chlamydomonas reinhardtii and Saccharomyces cerevisiae Mitochondria
1 Introduction
1.1 Chlamydomonas Mitochondrial Transformation
1.2 Yeast Mitochondrial Transformation
2 Materials
2.1 Strains
2.2 Media and Cell Preparation
2.3 Preparation of Microprojectiles and DNA Precipitation
2.4 Bombardment
2.5 Identification of Mitochondrial Transformants
2.6 Mating and Isolation of Yeast Recombinant Cytoductants
3 Methods
3.1 Cell Culture
3.2 Particle Sterilization
3.3 DNA Coating
3.4 Bombardment (Video 1)
3.5 Identification of Chlamydomonas Transformants
3.6 Identification of Yeast Transformants
3.6.1 After Bombardment of a rho0 Recipient Strain (Fig. 2)
3.6.2 After Bombardment of a Rho+ Recipient Strain
3.7 Mating and Isolation of Recombinant Cytoductants Starting from Synthetic Rho- Clones
4 Notes
References
Chapter 25: A Method for Precisely Identifying Modifications to Plant Mitochondrial Genomes by mitoTALENs
1 Introduction
2 Materials
2.1 Genomic Transformation of Rice
2.2 FPNI-PCR
2.3 Southern Blot Analysis
2.4 General Laboratory Consumables and Equipment
3 Methods
3.1 Construction of mitoTALEN Vectors
3.2 Genetic Transformation of Rice
3.2.1 Preparation of Calli
3.2.2 Preparation of Agrobacterium
3.2.3 Infection
3.2.4 Sterilization, Selection, and Regeneration
3.2.5 Validation of Transgenic Plants
3.3 Selection of Plants with Modified Mitochondrial Genomes
3.4 Determining Precise Changes that Occurred During Modification
3.5 Determining How the Sequences Were Connected
3.5.1 FPNI-PCR
3.6 Validation of the Genomic Modification Via Homologous Recombination
3.6.1 Preparation of a DIG-Labeled Probe by PCR
3.6.2 Preparation of Membrane, Hybridization, and Detection
4 Notes
References
Part VI: Mitochondrial DNA in Human Disease
Chapter 26: Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing
1 Introduction
2 Materials and Software Programs
2.1 Mitochondrial DNA Amplification by PCR
2.2 Library Preparation for Illumina Sequencing
2.3 Next-Generation Sequencing
2.4 Bioinformatics Analysis
3 Methods
3.1 One Amplicon Long-Range PCR
3.2 DNA Quantification and Dilution
3.3 DNA Library Sequencing
3.4 Bionformatics Analysis of Raw Data
3.5 Output Visualization and Interpretation
4 Notes
References
Chapter 27: Genomic Strategies in Mitochondrial Diagnostics
1 Introduction
1.1 Genomic Strategies for Mitochondrial Disease Diagnostics
1.2 Sample Selection Strategies
1.3 Variant Detection and Characterization Workflow
1.4 Variant Prioritization and Clinical Reporting
2 Materials
2.1 Hardware
2.2 Software
2.3 User Expertise
2.4 Data Files
3 Methods
3.1 General Comments: Obtaining the Samples and Sequencing
3.2 Variant Detection Workflow
3.3 Variant Prioritization
3.4 Sanger Confirmation and Clinical Reporting
4 Notes
References
Chapter 28: Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing
1 Introduction
2 Materials
2.1 Genomic DNA Isolation
2.2 Mitochondrial DNA Enrichment
2.3 Real-Time PCR
2.4 Tagmentation
2.5 Library Purification and Sequencing
3 Methods
3.1 Genomic DNA Isolation
3.2 Mitochondrial DNA Enrichment
3.3 Real-Time PCR
3.4 Tagmentation and Amplification
3.5 Library Purification and Sequencing
4 Notes
References
Chapter 29: Single Cell Analysis of Mitochondrial DNA Deletions
1 Introduction
2 Materials
2.1 Laser Capture Microdissection of Single Cells from Tissues and Single Cell Lysis
2.2 MT-ND1/MT-ND4/D-Loop Real-Time PCR
2.3 Long-Range PCR
2.4 Sequencing of mtDNA Breakpoints
3 Methods
3.1 Laser Capture Micro-dissection (LCM) of Single Cells from Tissue and Single Cell Lysis
3.2 Real-Time PCR mtDNA Deletion Assay and Single Cell Copy Number
3.2.1 Real-Time PCR
3.2.2 Analysis of Real-Time PCR Data Pre-processing
3.2.3 Calculating Deletion Level
3.2.4 Calculating Copy Number from a Plasmid of Known Concentration and Size
3.3 Long-Range PCR
3.4 mtDNA Break Point Sequencing and Analysis
3.4.1 PCR Product Cleanup
3.4.2 Next-Generation Sequencing
3.4.3 Analysis of Sequencing Data to Identify Breakpoints
4 Notes
References
Index