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.
From the reviews of the second edition: "The book contains detailed descriptions both of established techniques that continue to be usefully applied and recent information that improves understanding of mtDNA biology. It is intended for graduate students, postdoctoral fellows, and established investigators. I think even teachers in this field and early career investigators will find this book quite useful." (Omer Iqbal, Doody's Review Service, January, 2010)
Part I: Mitochondrial DNA Replication, Transcription, Translation, and Respiratory Complex Assembly 1. Biochemical Isolation of mtDNA Nucleoids from Animal CellsDaniel F. Bogenhagen 2. Analysis of Mitochondrial DNA by Two Dimensional Agarose Gel ElectrophoresisAurelio Reyes, Takehiro Yasukawa, Tricia J. Gulliver, and Ian J. Holt 3. Comparative Purification Strategies for Drosophilia and Human Mitochondrial DNA Replication Proteins: DNA Polymerase y and Mitochondrial Single-Stranded DNA-Binding ProteinMarcos T. Oliveira and Laurie S. Kaguni 4. Functional Analysis of Mutant Mitochondrial DNA Polymerase Proteins Involved in Human DiseaseSherine S.L. Chan and William C. Copeland 5. Preparation of Human Mitochondrial Single-Stranded DNA Binding ProteinMatthew J. Longley, Leslie A. Smith, and William C. Copeland 6. Methods for Assessing Binding of Mitochondrial Transcription Factor A (TFAM) to DNAAtsushi Fukuoh and Dongchon Kang 7. Inducible Expression in Human Cells, Purification and in vitro Assays for the Mitochondrial DNA Helicase TwinkleSteffi Goffart and Hans Spelbrink 8. Purification Strategy for Recombinant Forms of the Human Mitochondrial DNA HelicaseTawn D. Ziebarth and Laurie S. Kaguni 9. Methods for Studying Mitochondrial Transcription Termination with Isolated Components Paolo Loguercio Polosa, Stefania Deceglie, Marina Roberti, Maria Nicola Gadaleta, and Palmiro Cantatore 10. Oxidative Phosphorylation: Synthesis of Mitochondrially-Encoded Proteins and Assembly of Individual Structural Subunits into Functional Holoenzyme ComplexesScot C. Leary and Florin Sasarman Part II: Mitochondrial DNA Damage and Repair 11. Reactive Oxygen Species Production by MitochondriaAdrian J. Lambert and Martin D. Brand 12. Measuring mtDNA Damage Using a Supercoiling-Sensitive qPCR ApproachSam W. Chan and Junjian Z. Chen 13. Quantitative Analysis of Oxidized Guanine, 8-oxoguanine, in Mitochondrial DNA by Immunofluorescence MethodMizuki Ohno, Sugako Oka, and Yusaku Nakabeppu 14. In vitro Measurement of DNA Base Excision Repair in Isolated MitochondriaMelissa M. Page and Jeffrey A. Stuart 15. Targeting Repair Proteins to the Mitochondria of Mammalian Cells through Stable Transfection, Transient Transfection, Viral Transduction, and TAT-Mediated Protein TransductionChristopher A. Koczor, Janet W. Snyder, Inna N. Shokolenko, Allison W. Dobson, Glenn L. Wilson, and Susan P. LeDoux 16. Construction and Characterization of a Cell Line Deficient in Repair of Mitochondrial, but not Nuclear, Oxidative DNA DamageSugako Oka, Mizuki Ohno, and Yusaku Nakabeppu Part III: Mitochondrial DNA Mutations 17. Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiaeLyra M. Griffiths, Nicole A. Doudican, Gerald S. Shadel, and Paul W. Doetsch 18. Determination of DNA Mutation Load in Human Tissues Using Denaturing HPLC-Based Heteroduplex AnalysisKok Seong Lim, Robert K. Naviaux, and Richard H. Haas 19. Rapid Identification of Unknown Heteroplasmic Mitochondrial DNA Mutations with Mismatch-Specific Surveyor NucleaseSylvie Bannwarth, Vincent Procaccio, and Veronique Paquis-Flucklinger 20. Collection of Isolated Cells for Studying Mitochondrial DNA Mutations within Individual CellsYevgenya Kraytsberg, Natalya Bodyak, Susan Myerow, Alexander Nicholas, Konstantin Ebralidze, and Konstantin Khrapko 21. Quantitative Analysis of Somatic Mitochondrial DNA Mutations by Single-Cell Single-Molecule PCRYevgenya Kraytsberg, Natalya Bodyak, Susan Myerow, Alexander Nicholas, Konstantin Ebralidze, and Konstantin Khrapko 22. Measuring DNA Precursor Pools in MitochondriaChristopher K. Mathews and Linda J. Wheeler 23. Establishment of Human Cell Lines Lacking Mitochondrial DNAKazunari Hashiguchi and Qiu-Mei Zhang-Akiyama
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