Includes supplementary material: sn.pub/extras
The aim of this book is to provide detailed protocols for studying the molecular biology of the pathogen Mycobacterium tuberculosis, and its int- actions with host cells. As established mycobacterial laboratories move - wards exploiting the genome, and laboratories with expertise in other fields apply them to mycobacteria, both traditional and novel methodologies need to be reviewed. Thus the chapters in Mycobacterium tuberculosis Protocols range from perspectives on storage of strains and safety issues to the application of the latest functional genomics technologies. The last few years have been remarkable ones for research into M. tuber- losis. The most important landmark by far has been the completion of the genome sequence of the widely studied H37Rv strain (1). We can now predict every protein and RNA molecule made by the pathogen. This information is or will soon be enriched by the addition of genome sequences of other strains from the M. tuberculosis complex: a second strain of M. tuberculosis, My- bacterium bovis, and the vaccine strain, M. bovis BCG. Valuable comparative data will also be provided by the genome sequences of Mycobacterium leprae, Mycobacterium avium, and Streptomyces coelicolor. Another recent milestone for M. tuberculosis has been the development of efficient mutagenesis me- odologies, the lack of which has been a major handicap in functional studies.
Plasmid Vectors Nicola Casali and Sabine Ehrt Isolation of DNA from Mycobacterium tuberculosis Paul D. van Helden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Helden Extraction of RNA from Intracellular Mycobacterium tuberculosis: Methods, Considerations, and Applications Irene M. Monahan, Joseph A. Mangan, and Philip D. Butcher Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages Stoyan S. Bardarov, Svetoslav S. Bardarov, Jr., and William R. Jacobs, Jr Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB Mary Jackson, Luis Reinaldo Camacho, Brigitte Gicquel, and Christophe Guilhot Gene Replacement Using Pretreated DNA Bhavna G. Gordhan and Tanya Parish Gene Replacement in Mycobacterium tuberculosis and Mycobacterium bovis BCG Using rpsL+ as a Dominant Negative Selectable Marker Peter Sander, Burkhard Springer, and Erik C. Böttger Tranillegalscription Start-Site Mapping Farahnaz Movahedzadeh, Jorge A. Gonzalez-Y-Merchand, and Robert A. Cox Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells Rory P. Cooney, Natalie J. Garton, and Michael R. Barer Phage Replication Technology for Diagnosis and Drug Susceptibility Testing Ruth McNerney Detection of Mutations in Mycobacterium tuberculosis by a Dot Blot Hybridization Strategy Thomas C. Victor and Paul D. van Helden Restriction Fragment Length Polymorphism Typing of Mycobacteria Dick van Soolingen, Petra E. W. de Haas, and Kristin Kremer Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis Ida Rosenkrands and Peter Andersen Analysis of the Capsule of Mycobacterium tuberculosis Mamadou Daffé and Marie-Antoinette Lanéelle Analysis of the Lipids of Mycobacterium tuberculosis Richard A. Slaydenand Clifton E. Barry, 3rd In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis Lawrence G. Wayne Macrophage Virulence Assays Pauline T. Lukey and Elizabeth U. Hooker Analysis of Mycobacterium-Infected Macrophages by Immunoelectron Microscopy and Cell Fractionation Wandy Beatty and David G. Russell Real-Time PCR Using Molecular Beacons: A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis Riccardo Manganelli, Sanjay Tyagi, and Issar Smith Electronic Access to Mycobacterium tuberculosis Sequence Data Julian Parkhill Proteomics Joanna C. Betts and Marjorie A. Smith Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays Michael Wilson, Martin Voskuil, Dirk Schnappinger, and Gary K. Schoolnik Storage of Mycobacterial Strains Kristin Kremer, Tridia van der Laan, and Dick van Soolingen Safety in the Laboratory Heidi Alderton and Debbie Smith
With one-third of the world's population infected with Mycobacterium tuberculosis, over two million people a year dying from tuberculosis, and the appearance of multidrug-resistant strains, the need to understand the biology of M. tuberculosis, and so to develop new interventions, has become acute. In Mycobacterium tuberculosis Protocols, leading investigators with extensive practical knowledge and experience describe their best methods for studying this dangerous pathogen. Packed with step-by-step instructions to ensure successful results, these methods range from basic handling techniques to the application of functional genomics. These molecular techniques are suitable for research in genetics, biochemistry, microbiology, cell biology, epidemiology, and diagnostics, and are at the forefront of biological research as a whole, as well as in focused M. tuberculosis research. Highlights include methods for the basic safety and culture of M. tuberculosis, fractionation of the bacterium (nucleic acids, lipids, culture filtrate, and capsule), the analysis of gene expression (start-site mapping, real-time PCR, microarrays, and proteomics), the growth of the bacterium in macrophages and low oxygen, cytological analysis of the bacteria, and diagnostics. Highly practical and accessible, Mycobacterium tuberculosis Protocols utilizes advanced functional genomics and mutagenesis methodologies to provide both experimental and clinical investigators all the powerful techniques needed to illuminate the molecular biology of tuberculosis and its interactions with host cells, and so drive work on the wide variety of emerging therapeutic opportunities.
...a very useful book for the mycobacteria research community. It is a specialist's cookbook, perfect if you want to establish a new method that is outside your scope of expertise. Individual chapters are clearly divided into introduction, materials, methods and an extensive list of notes. -- International Journal of Medical Microbiology
"For those engaged in mycobacterial research (and possibly other unrelated genera) which involves genetics, biochemistry and immunology, this textbook is essential and, moreover, I would recommend it as reading material for students undertaking molecular biology experimentation." -Today's Life Science
Plasmid Vectors.- Isolation of DNA from Mycobacterium tubercolosis.- Extraction of RNA from Intracellular Mycobacterium tuberculosis.- Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages.- Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB.- Gene Replacement using Pretreated DNA.- Gene Replacement in Mycobacterium tuberculosis and Mycobacterium bovis BCG Using rpsL as a Dominant Negative Selectable Marker.- Tranillegalscription Start-Site Mapping.- Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells.- Phage Replication Technology for Diagnosis and Drug Susceptibility Testing.- Detection of Mutations in Mycobacterium tuberculosis by a Dot Blot Hybridization Strategy.- Restriction Fragment Length Polymorphism Typing of Mycobacteria.- Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis.- Analysis of the Capsule of Mycobacterium tuberculosis.- Analysis of the Lipids of Mycobacterium tuberculosis.- In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis.- 17 Macrophage Virulence Assays.- Analysis of Mycobacterium-Infected Macrophages by Immunoelectron Microscopy and Cell Fractionation.- Real Time PCR Using Molecular Beacons.- Electronic Access to Mycobacterium tuberculosis Sequence Data.- Proteomics.- Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays.- Storage of Mycobacterial Strains.- Safety in the Laboratory.
...a very useful book for the mycobacteria research community. It is a specialist's cookbook, perfect if you want to establish a new method that is outside your scope of expertise. Individual chapters are clearly divided into introduction, materials, methods and an extensive list of notes. -- International Journal of Medical Microbiology
"For those engaged in mycobacterial research (and possibly other unrelated genera) which involves genetics, biochemistry and immunology, this textbook is essential and, moreover, I would recommend it as reading material for students undertaking molecular biology experimentation." -Today's Life Science
Inhaltsverzeichnis
Plasmid Vectors
Nicola Casali and Sabine Ehrt
Isolation of DNA from Mycobacterium tuberculosis
Paul D. van Helden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Helden
Extraction of RNA from Intracellular Mycobacterium tuberculosis: Methods, Considerations, and Applications
Irene M. Monahan, Joseph A. Mangan, and Philip D. Butcher
Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages
Stoyan S. Bardarov, Svetoslav S. Bardarov, Jr., and William R. Jacobs, Jr
Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB
Mary Jackson, Luis Reinaldo Camacho, Brigitte Gicquel, and Christophe Guilhot
Gene Replacement Using Pretreated DNA
Bhavna G. Gordhan and Tanya Parish
Gene Replacement in Mycobacterium tuberculosis and Mycobacterium bovis BCG Using rpsL+ as a Dominant Negative Selectable Marker
Peter Sander, Burkhard Springer, and Erik C. Böttger
Tranillegalscription Start-Site Mapping
Farahnaz Movahedzadeh, Jorge A. Gonzalez-Y-Merchand, and Robert A. Cox
Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells
Rory P. Cooney, Natalie J. Garton, and Michael R. Barer
Phage Replication Technology for Diagnosis and Drug Susceptibility Testing
Ruth McNerney
Detection of Mutations in Mycobacterium tuberculosis by a Dot Blot Hybridization Strategy
Thomas C. Victor and Paul D. van Helden
Restriction Fragment Length Polymorphism Typing of Mycobacteria
Dick van Soolingen, Petra E. W. de Haas, and Kristin Kremer
Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis
Ida Rosenkrands and Peter Andersen
Analysis of the Capsule of Mycobacterium tuberculosis
Mamadou Daffé and Marie-Antoinette Lanéelle
Analysis of the Lipids of Mycobacterium tuberculosis
Richard A. Slaydenand Clifton E. Barry, 3rd
In Vitro Model of Hypoxically Induced Nonreplicating Persistence of Mycobacterium tuberculosis
Lawrence G. Wayne
Macrophage Virulence Assays
Pauline T. Lukey and Elizabeth U. Hooker
Analysis of Mycobacterium-Infected Macrophages by Immunoelectron Microscopy and Cell Fractionation
Wandy Beatty and David G. Russell
Real-Time PCR Using Molecular Beacons: A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis
Riccardo Manganelli, Sanjay Tyagi, and Issar Smith
Electronic Access to Mycobacterium tuberculosis Sequence Data
Julian Parkhill
Proteomics
Joanna C. Betts and Marjorie A. Smith
Functional Genomics of Mycobacterium tuberculosis Using DNA Microarrays
Michael Wilson, Martin Voskuil, Dirk Schnappinger, and Gary K. Schoolnik
Storage of Mycobacterial Strains
Kristin Kremer, Tridia van der Laan, and Dick van Soolingen
Safety in the Laboratory
Heidi Alderton and Debbie Smith
Klappentext
With one-third of the world's population infected with Mycobacterium tuberculosis, over two million people a year dying from tuberculosis, and the appearance of multidrug-resistant strains, the need to understand the biology of M. tuberculosis, and so to develop new interventions, has become acute. In Mycobacterium tuberculosis Protocols, leading investigators with extensive practical knowledge and experience describe their best methods for studying this dangerous pathogen. Packed with step-by-step instructions to ensure successful results, these methods range from basic handling techniques to the application of functional genomics. These molecular techniques are suitable for research in genetics, biochemistry, microbiology, cell biology, epidemiology, and diagnostics, and are at the forefront of biological research as a whole, as well as in focused M. tuberculosis research. Highlights include methods for the basic safety and culture of M. tuberculosis, fractionation of the bacterium (nucleic acids, lipids, culture filtrate, and capsule), the analysis of gene expression (start-site mapping, real-time PCR, microarrays, and proteomics), the growth of the bacterium in macrophages and low oxygen, cytological analysis of the bacteria, and diagnostics. Highly practical and accessible, Mycobacterium tuberculosis Protocols utilizes advanced functional genomics and mutagenesis methodologies to provide both experimental and clinical investigators all the powerful techniques needed to illuminate the molecular biology of tuberculosis and its interactions with host cells, and so drive work on the wide variety of emerging therapeutic opportunities.
Includes supplementary material: sn.pub/extras
