Mitochondrial DNA Methods and Protocols

Since the publication of the first edition, the number of unique heritable mtDNA mutations recognized as being associated with bioenergetic dysfunction, cell death and disease has grown. Likewise, our understanding of the basic biology of somatic mtDNA mutations continues to improve. In Mitochondrial DNA: Methods and Protocols, Second Edition, specialists from eight countries share their expertise, providing detailed protocols for studying many aspects of mtDNA. The volume is divided into three sections, which cover the transduction of information from mtDNA to functionally active respiratory complexes, mitochondrial reactive oxygen species (ROS) production as well as mtDNA damage and its repair, and the identification and quantification of heteroplasmic mtDNA mutations. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes sections, highlighting tips on troubleshooting and avoiding known pitfalls.

Comprehensive and authoritative, Mitrochondrial DNA: Methods and Protocols, Second Edition promises to aid researchers in further expanding our knowledge of this vital area of cell biology.

Mitochondrial DNA Replication, Transcription, Translation, and Respiratory Complex Assembly.- Biochemical Isolation of mtDNA Nucleoids from Animal Cells.- Analysis of Mitochondrial DNA by Two-Dimensional Agarose Gel Electrophoresis.- Comparative Purification Strategies for Drosophila and Human Mitochondrial DNA Replication Proteins: DNA Polymerase ? and Mitochondrial Single-Stranded DNA-Binding Protein.- Functional Analysis of Mutant Mitochondrial DNA Polymerase Proteins Involved in Human Disease.- Preparation of Human Mitochondrial Single-Stranded DNA-Binding Protein.- Methods for Assessing Binding of Mitochondrial Transcription Factor A (TFAM) to DNA.- Inducible Expression in Human Cells, Purification, and In Vitro Assays for the Mitochondrial DNA Helicase Twinkle.- Purification Strategy for Recombinant Formsof the Human Mitochondrial DNA Helicase.- Methods for Studying Mitochondrial Transcription Termination with Isolated Components.- Oxidative Phosphorylation: Synthesis of Mitochondrially Encoded Proteins and Assembly of Individual Structural Subunits into Functional Holoenzyme Complexes.- Mitochondrial DNA Damage and Repair.- Reactive Oxygen Species Production by Mitochondria.- Measuring mtDNA Damage Using a Supercoiling-Sensitive qPCR Approach.- Quantitative Analysis of Oxidized Guanine, 8-Oxoguanine, in Mitochondrial DNA by Immunofluorescence Method.- In Vitro Measurement of DNA Base Excision Repair in Isolated Mitochondria.- Targeting Repair Proteins to the Mitochondria of Mammalian Cells Through Stable Transfection, Transient Transfection, Viral Transduction, and TAT-Mediated Protein Transduction.- Construction and Characterization of a Cell Line Deficient in Repair of Mitochondrial, but Not Nuclear, Oxidative DNA Damage.- Mitochondrial DNA Mutations.-Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae.- Determination of DNA Mutation Load in Human Tissues Using Denaturing HPLC-Based Heteroduplex Analysis.- Rapid Identification of Unknown Heteroplasmic Mitochondrial DNA Mutations with Mismatch-Specific Surveyor Nuclease.- Collection of Isolated Cells for Studying Mitochondrial DNA Mutations Within Individual Cells.- Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCR.- Measuring DNA Precursor Pools in Mitochondria.- Establishment of Human Cell Lines Lacking Mitochondrial DNA.