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This volume compiles a comprehensive range of methods to study key aspects of mitochondrial DNA including nucleoid structure and packaging, replication, genome integrity, and disease. Chapters are organized into eight methodological sections that cover in vitro and in vivo methods, including for mtDNA isolation, visualization, deep sequencing, gene editing, and diagnostic aspects of mtDNA disease. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Mitochondrial DNA: Methods and Protocols aims to be useful and informative for researchers and clinicians with an interest in mitochondrial DNA.
List of contents
Isolation of functional mitochondria and pure mtDNA from murine tissues.- Isolating mitochondria, mitoplasts, and mtDNA from cultured mammalian cells.- Coupling differential centrifugation with exonuclease treatment and size exclusion chromatography (DIFSEC) for purification of mtDNA from mammalian cells.- Isolation and Quality Control of Yeast Mitochondria.- Mitochondrial DNA isolation from plants.- Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) nucleoids with Multiple Labelling Strategies.- Visualization of mtDNA using FISH.- In situ analysis of mitochondrial DNA synthesis using metabolic labeling coupled to fluorescence microscopy.- Measurement of nucleoid size using STED microscopy.- How to quantify DNA compaction by TFAM with Acoustic Force Spectroscopy and Total Internal Reflection Fluorescence microscopy.- Assessing TFAM binding to human mitochondrial DNA.- Identification of proximity interactors of mammalian nucleoid proteins by BioID.- Localization of mitochondrial nucleoids by transmission electron microscopy using the transgenic expression of the mitochondrial helicase TWINKLE and APEX2.- In vitro assays of TWINKLE function.- Rolling Circle Replication and Bypass of Damaged Nucleotides.- Studying mitochondrial nucleic acid synthesis utilizing intact isolated mitochondria.- Functional assessment of mitochondrial DNA maintenance by depletion and repopulation using 2',3'-dideoxycytidine in cultured cells.- Analysis of mitochondrial DNA replication by two-dimensional agarose gel electrophoresis.- Quantitative analysis of nucleoside triphosphate pools in mouse muscle using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry detection.- Detection of UV-Induced Deletions in Mitochondrial DNA.- Determination of the ribonucleotide content of mtDNA using alkaline gels.- 5´-end mapping in human mitochondrial DNA.- Manipulation of murine mitochondrial DNA heteroplasmy with mtZFNs.- Biolistic transformation of Chlamydomonas reinhardtii and Saccharomyces cerevisiae mitochondria.- A method for precisely identifying modifications to plant mitochondrial genomes by mitoTALENs.- Mitochondrial DNA sequencing and heteroplasmy quantification by Next Generation Sequencing.- Genomic strategies in mitochondrial diagnostics.- Mitochondrial DNA enrichment for sensitive next-generation sequencing.- Single cell analysis of mitochondrial DNA deletions.
