Animal Biotechnology : Models in Discovery and Translation

__Animal Biotechnology: Models in Discovery and Translation, Second Edition,__ provides a helpful guide to anyone seeking a thorough review of animal biotechnology and its application to human disease and welfare. This updated edition covers vital fundamentals, including animal cell cultures, genome sequencing analysis, epigenetics and animal models, gene expression, and ethics and safety concerns, along with in-depth examples of implications for human health and prospects for the future. New chapters cover animal biotechnology as applied to various disease types and research areas, including in vitro fertilization, human embryonic stem cell research, biosensors, enteric diseases, biopharming, organ transplantation, tuberculosis, neurodegenerative disorders, and more. Cоver Animal Biotechnology: Models in Discovery and Translation Copyright Dedication Contents List of Contributors Preface Acknowledgments Section I: Human diseases: in vivo and in vitro models 1 Drosophila: a model for biotechnologist Summary What you can expect to know Introduction Classical aspects of Drosophila melanogaster Physical appearance Life cycle Drosophila development Embryogenesis in Drosophila Pattern formation in Drosophila Homeotic genes in Drosophila Drosophila genome History Historical perspective of Drosophila contributions to biotechnology Principle Methodology Culturing of Drosophila Preparation of Drosophila food medium Materials required Handling of flies Fly disposal Egg collection Dechorination of eggs Preparation of DNA for injection Protocols Protocol for germ-line transformation in Drosophila Materials required Procedure Ethical issues Translational significance Clinical significance Drosophila-based models for understanding human neurodegenerative diseases Drosophila as a model for understanding human metabolic disorders Drosophila as a model for understanding nephrolithiasis (kidney stones) Drosophila-based model for understanding the human immunodeficiency virus pathology Drosophila-based therapeutic peptide production Turning point World Wide Web resources Acknowledgments References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 2 Animal models of tuberculosis Summary Introduction Comparative pathology of tuberculosis in humans and animals Characteristics of a model for tuberculosis with respect to infection and pathogenesis Pathogen diversity: crossing species barriers Host diversity: fundamental processes and fine-tuning Animal models of tuberculosis: limits and lessons Animal models: contributions in tuberculosis vaccine testing Various animal models Mouse model Guinea pig model Rabbit model Nonhuman primate model Cattle model Protocols Preparing M. tuberculosis inoculum for aerosol exposure Aerosol infection of mice using the middlebrook apparatus Aerosol infection of guinea pigs using a madison chamber Bacteria loading Intravenous infection of mice with M. tuberculosis Isolation of samples for determining M. tuberculosis load by real-time-PCR Determination of bacterial loads in target organs Preparation of lungs or other tissues for histology Preparation of lung cell suspension Ethical issues Translational significance World wide web resources Safety considerations References Further reading Glossary Abbreviations Long-answer questions Short-answer questions Answers to short-answer questions Yes/no-type questions Answers for yes/no type questions 3 Animal models for neurodegenerative disorders Summary What you can expect to know History and methods Introduction Neurodegenerative diseases Amyotrophic lateral sclerosis Spinal muscular atrophy Spinal and bulbar muscular atrophy Principles Genetics of amyotrophic lateral sclerosis Superoxide dismutase 1-amyotrophic lateral sclerosis Amyotrophic lateral sclerosis: genes implicated in RNA metabolism TAR-DNA-binding protein 43 Fused-in sarcoma/translocated in liposarcoma protein C9orf72 Genetics of spinal muscular atrophy Genetics of spinal and bulbar muscular atrophy Methodology Generation of transgenic mice Preparation and purification of transgenic construct (step 1) Harvesting donor eggs (step 2) Microinjection of transgene to fertilized egg (step 3) Implantation of microinjected egg to pseudopregnant female mice (step 4) Screening of founder mice for expression of transgene (step 5) Establishing stable transgenic line (step 6) Cre-loxP technology Amyotrophic lateral sclerosis models SOD1G37R transgenic mice SOD1G93A transgenic mice SOD1WT transgenic mice Spinal muscular atrophy models Severe spinal muscular atrophy mice (mSMN−/−;SMN2+/+) Spinal muscular atrophy type II mice (mSMN−/−;SMN2+/+;SMNΔ7+/+) Spinal and bulbar muscular atrophy models AR-97Q and AR-24Q transgenic mice Examples and their applications Superoxide dismutase 1-linked amyotrophic lateral sclerosis Gain of toxicity from mutant superoxide dismutase 1 established as pathomechanisms through engineering mutant superoxide di... Evaluating phenotype and clinical course of mutant superoxide dismutase 1 transgenic mice Toxicity from misfolded mutant superoxide dismutase 1 protein Non-cell autonomous neurodegeneration demonstrated by superoxide dismutase 1 mouse models Stem cell-derived motor neurons established from mutant superoxide dismutase 1 mice Other amyotrophic lateral sclerosis Spinal muscular atrophy Human SMN2 transgenic mice Neuron-specific deletion of survival of motor neuron in mice using Cre-loxP systems Spinal and bulbar muscular atrophy AR-97Q mice as spinal and bulbar muscular atrophy Model Androgen hormone and mutant androgen receptor central to spinal and bulbar muscular atrophy pathogenesis Clinical correlations Protocols Ethical issues Translational significance World Wide Web resources Acknowledgment References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers for yes/no type questions 4 Epigenetics and animal models: applications in cancer control and treatment Summary What you expect to know Introduction History Principle Use of mouse models in the epigenetics of cancer Examples with applications Brain cancer Breast cancer Colorectal cancer Esophageal cancer Gastric cancer Head and neck cancer Lung cancer Lymphoma and leukemia Prostate cancer Liver cancer Other approaches Methodology Methylation profiling Histone profiling Nucleosome mapping Protocols Ethical issues Translation significance Clinical significance Web resources Turning point Flow chart World Wide Web resources Something interesting about this chapter References Glossary (terms used in text with examples) Abbreviations Long answer questions Answers to long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 5 Development of mouse models for cancer research Summary What you can expect to know Introduction History Principle Institutional Animal Care and Use Committee approval Institutional Animal Care and Use Committee guidelines Methodology Inbred mice Examples with applications Immunocompetent mice Spontaneous tumor models The genetically engineered mouse models The Cre/Lox system: a superior genetically engineered mouse model Immunodeficient mice Allograft transplants Xenograft transplants Humanized mice Checklist for a successful in vivo experiment Protocols An orthotopic mouse model of colorectal cancer Design and execution Interpretation of results A xenograft model of prostate cancer metastasis Design and execution Interpretation of results Humanized mouse models for tumor xenografts Design and execution Ethical issues Turning point Translational significance World Wide Web resources References Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 6 The clinico-molecular approaches for detection of human papillomavirus Summary What you can expect to know Introduction Cancer Cervical cancer Historical overview Mistaken theories of cervical cancer causation The first breakthrough zur Hausen Prevalence and epidemiology of cervical cancer Global scenario Symptoms of cervical cancer Anatomy of female pelvis Types of cervical cancer Risk factors for cervical cancer Human papillomaviruses Genomic organization of human papillomavirus Transcriptional regulation of human papillomavirus Life cycle of human papillomavirus Functions of human papillomavirus oncoproteins E6 and E7 Inactivation and degradation of p53 through the E6/E6AP complex Screening and diagnostic methodologies of cervical cancer Screening Methods used for screening/diagnosis of cervical cancer Visual methods Indications Other screening tests Reporting systems terminology Precancer classification WHO classification CIN classification (Bhambhani., 2007) Bethesda classification Cancer classification Colposcopy and biopsy New technologies DNA cytometry Human papillomavirus DNA-based screening methods (protocol) Urine-based noninvasive human papillomavirus DNA detection method Simple “paper smear” method for rapid detection of human papillomavirus infection Detection of HPV by multiplex PCR and RFLP Statistical information Hybrid Capture II Method Principle Applications Next-generation sequencing Clinical correlations Treatment Ablative techniques Excisional techniques Follow-up for excisional/ablative treatment Hysterectomy Stage-wise management of cervical cancer Treatment of microinvasive carcinoma Stage IA1 and IA2 Treatment of early invasive cancer (stage IB1 and IIA %3c 4cm) Treatment of early bulky disease (stage IB2 and IIA %3e 4cm) Treatment of extensive disease: stages IIB–IIIB Treatment of stage IVA Treatment of stage IVB or recurrent disease Human papillomavirus vaccines Prophylactic human papillomavirus vaccines Therapeutic human papillomavirus vaccine Genetic-based DNA vaccine Issues/unanswered questions associated with human papillomavirus vaccine Vaccine efficacy Vaccine protection Who should be vaccinated Ethical issues With screening implementation Risk Benefit versus cost Patient autonomy and coercion With vaccine implementation Translational significance MicroRNA expression profiles in cervical cancer siRNAs for human papillomavirus oncogenes as potential gene therapy for cervical cancer Chemotherapeutic drugs and siRNAs Conclusion World Wide Web resources The American Cancer Society Southwest Division CancerNet People Living Through Cancer American Institute for Cancer Research American Society of Plastic and Reconstructive Surgeons Asian and Pacific Islander American Health Forum Avon’s Breast Cancer Awareness Crusade Cancer Information Service Cancer Mail CancerNet Cancer Patient Education Database Cancer Research Foundation of America Cancer source.com Further information Keywords Key points to remember Key points for effective cervical cancer screening program in low-resource settings Ablative techniques Excisional techniques References Glossary Abbreviations Long answer questions Answers to long answer questions Short answer questions Answers to short answer questions “Yes/no” type questions Answers to yes/no type questions 7 Human DNA tumor viruses and oncogenesis Summary What you can expect to know History and methods Transformation and oncogenesis History of human DNA tumor viruses and cancer Epstein–Barr virus Kaposi’s sarcoma–associated herpesvirus Kaposi’s sarcoma Primary effusion lymphoma Multicentric Castleman’s disease KSHV inflammatory cytokine syndrome Human papillomavirus Hepatitis B virus Human polyomaviruses Merkel cell polyomavirus Principle Epstein–Barr virus genome structure Entry into the cell Epstein–Barr virus lytic replication Epstein–Barr virus latency Epstein–Barr virus latent genes Epstein–Barr virus nuclear antigen 1 Epstein–Barr virus nuclear antigen 2 Epstein–Barr virus nuclear antigen 3 family EBNA-LP LMP1 LMP2 EBERs BARTs Micro RNAs Clinical correlation Burkitt’s lymphoma Nasopharyngeal cancer Hodgkin’s lymphoma Infectious mononucleosis X-linked lymphoproliferative disease Research methods and protocols Turning point: modeling Epstein–Barr virus infection and pathogenesis Current research perspectives Ethical issues Translational significance World Wide Web resources References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 8 Animal models for human disease Summary Outline What you expect to know Introduction Rheumatoid arthritis Epidemiology and etiology Pathogenesis Clinical manifestations Treatment Experimental models Spontaneous models Induced models Pathology of collagen-induced arthritis Methodology and protocols Clinical assessment of collagen-induced arthritis Histological assessment Radiographic evaluation Multiple sclerosis Epidemiology and etiology Pathogenesis Clinical manifestations Treatment Experimental models Spontaneous models Induced models Methodology and protocol Protocol Clinical evaluation Histology Immunohistochemistry Enzyme-linked immunosorbent assay Real-time polymerase chain reaction Ethical issues Translational significance Clinical correlations Conclusion World Wide Web resources References Further reading Glossary Abbreviations Long-answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 9 HIV and antiretroviral drugs Summary What you can expect to know History and methods Introduction Discovery and origin of HIV History of HIV and AIDS Global disease burden Clinical stages of HIV Classification of clinical stages Stage I: primary HIV infection or seroconversion stage Stage II: asymptomatic stage Stage III: persistent generalized lymphadenopathy Stage IV: symptomatic stage Molecular biology of HIV Envelope Group-specific antigen Long-terminal repeats Negative factor Polymerase Regulator of expression of viral proteins Transactivator of transcription Viral infectivity factor Viral protein U Viral protein R Replication: steps and drug targets Antiretroviral drugs HIV resistance and antiretroviral treatment Highly active antiretroviral treatment Salvage therapy Drug holiday New types of antiretrovirals Methodology and principles Growing HIV stock Principles Crucial steps Assays for antiretroviral drugs Monitoring antiretroviral drug toxicity Principles Special note Crucial steps Evaluating anti-HIV effects of antiretroviral drugs Principles Special note Crucial steps Understanding HIV reservoir Mechanism of viral latency NeuroAIDS: an emerging health concern Bone marrow transplantation: a probable cure for HIV Ethical issues Translational significance Clinical correlation Acknowledgments World Wide Web resources References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 10 Animal models in advancement of research in enteric diseases Summary What you can expect to know Introduction Animal models for enterotoxigenic Escherichia coli Suckling mouse model Infant mouse model Adult mouse model Rat model Adult rabbit ligated ileal loop model Animal models for Vibrio cholerae Infant mouse model Suckling mouse model Infant rabbit model Rabbit ligated ileal loop model Ileal ligated mouse model of cholera Animal models for Campylobacter jejuni Nonhuman primate model Ferret model Pig model Mouse model Rabbit model Chicken model Animal models for Shigella Macaque monkey model Pig model Rabbit model Guinea pig model Mouse model Animal models for Salmonella typhi Mouse model Animal models for nontyphoidal Salmonella Opium-treated guinea pig model Calf gastroenteritis model Mouse model Coinfection model Translational significance World Wide Web resources References Further reading Glossary Long answer questions Short answer questions Yes/no types questions Answers to yes/no questions 11 Chick chorioallantoic membrane assay: a 3D animal model for cancer invasion and metastasis Summary What you can expect to know Introduction History Principle Structure and function of the CAM Methodology Examples With Applications Tumor Growth Tumor Invasion Tumor Metastasis Patient-Derived Xenografts Response to anticancer drugs Advantages and limitations of the cam assay Conclusions Ethical Issues Translational significance Clinical correlations Turning point References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Section II: Animal biotechnology: tools and techniques 12 Animal biotechnology as a tool to understand and fight aging Summary What you expect to know Introduction Theories of aging Evolutionary theories Molecular theories Cellular theories Cell senescence/telomere theory Free radical theory of aging System-based theories Neuroendocrine theory Caloric restriction theory Principle Reactive oxygen species—causative agent of aging Superoxide anion (O2−) Hydroxyl radical (OH) Hydrogen peroxide (H2O2) Singlet oxygen Peroxyl and alkoxyl radicals Nitric oxide and nitric dioxide Peroxynitrite Enzymatic formation Methodology: measurement of free radicals and methods to monitor aging Protein oxidation/protein carbonyl content Antioxidant capacity Common laboratory animal experimental models for aging research Mice Fish Polyphenols as an agent to fight aging Flavonoids Tea as antiaging compound Types of teas Tea catechins Health benefits of tea Molecular mechanisms of green tea effects Green tea in aging and neurodegenerative diseases Animal biotechnology as a tool to understand aging and fight aging Human aging: a translational perspective and significance Ethical issues related with aging research World wide web resources Clinical correlation References Further reading Glossary Abbreviations Long-answer questions Short-answer questions Answers to short-answer questions Yes/no-type questions Answers to yes/no-type questions 13 Multicellular tumor spheroids as in vitro models for studying tumor responses to anticancer therapies Summary What you can expect to know History and methods Introduction Multicellular tumor spheroids Historical facts toward the development of tissue culture technology from 2D and 3D cultures Examples where 3D culture is more beneficial over 2D culture Techniques for the generation of spheroids Hanging-drop method Liquid overlay method Microfabricated microstructures method Rotatory flask methods Surface modification-based methods Chip-based spheroid generation Emerging technologies for the generation of spheroids Protocol for tumor spheroid generation Drug treatment protocol Parameters to monitor drug efficacy in 3D cultures Radiation response of tumor cells and its modifications Response to anticancer drugs Response to photodynamic therapy Response to antiangiogenesis therapeutics Evaluation of response to immunotherapy Application of 3D cultures in other diseases Conclusions Ethical issues Translational significance World Wide Web resources References Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions 14 Animal tissue culture principles and applications Summary What you can expect to know History and methods Introduction Development of animal cell culture Basic concept of cell culture How are cell cultures obtained? Organ culture Primary explant culture Cell culture Monolayer cultures Suspension cultures Cell passage and use of trypsin Quantitation Hemocytometer Electronic counting Other quantitation Reconstruction of three-dimensional structures Histotypic culture Organotypic culture Types of cell culture Primary cell culture Advantages and disadvantages of primary cell culture Anchorage-dependent/adherent cells Anchorage-independent/suspension cells Secondary cell culture Advantages and disadvantages of secondary cell culture Cell line Finite cell lines Indefinite cell lines Commonly used cell lines Advantages of continuous cell lines Growth cycle Phases of the growth cycle Lag phase Log phase Plateau phase Monitoring cell growth Characteristics of cell cultures Temperature pH Oxygen Cell viability Cytotoxicity Hayflick’s phenomenon Culture media Basic components in culture media Natural media Artificial media Serum Advantages of serum in cell culture medium Disadvantages of serum-containing medium Serum-free media Advantages of serum-free culture media Disadvantages of serum-free media Chemically defined media Protein-free media Characterization of cell lines Identity testing Karyotyping Purity testing Stability testing Viral testing assays Advantages of animal cell culture Disadvantages of animal cell culture Ethical issues Use of fetal bovine serum in animal culture of media Translational significance Antiviral vaccines Viral particles production by cell culture Production of virus-like particles Vaccines based on virus-like particles Human papilloma virus vaccine Recombinant therapeutic proteins Main therapeutic proteins Cytokines Applications of interferons Growth factors Hormones Therapeutic enzymes Blood coagulation factors Antibodies Gene therapy Importance of cell culture in gene therapy Clinical correlation Biopesticides Baculovirus production in animal cell culture Cell lines for biopesticide production Viral mutant formation in cell culture Monoclonal antibodies Stem cells Culturing embryonic stem cells in the laboratory Microfluidics three-dimensional culture Applications Organ-on-a-chip technology Tissue models on a chip Liver-on-a-chip Tumor-on-a-chip World Wide Web resources References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 15 Concepts of tissue engineering Summary What can you expect to know? Introduction History Basic approach to tissue engineering: principles and methodology Cells Scaffolds Media Bioreactors Methodology Scaffold design Materials for scaffolds Scaffold fabrication methods Fiber bonding Solvent casting and particulate leaching Melt molding Membrane lamination Phase separation Gas foaming Polymer ceramic composite foam Solid-free form techniques Selective laser sintering Stereolithography Fused deposition modeling 3D Printing Pressure-assisted micro-syringe method Freeze-drying Examples of tissue-engineered organs Skin Pancreas Liver Kidney Bone/cartilage Nerves Blood vessels Tissue engineering using stem cells Issues and challenges Ethical issues Translational significance World wide web resources References Further reading Glossary Abbreviations Long-answer questions Short-answer questions Answers to short-answer questions Kindly state yes or no against the following statements Answers to yes/no statements 16 Nanotechnology and its applications to animal biotechnology Summary What you can expect to know History and methods Introduction Methodologies Nanotools and nanotechniques SPM techniques Raman spectroscopy and imaging AFM–Raman confocal hybrid systems Chemical modification of AFM probes Nanostructural features of animal cells and tissues Nanomechanical properties of animal cells and tissues Nanomanipulation Nanofabrication Examples of nanotechnology applications to animal biotechnology Nanoparticles in animal production Nanosurgery AFM as a diagnostic tool to identify orthopoxvirus in animals Frictional response of bovine articular cartilage Microstructure and nanomechanical properties of cortical bone osteons from baboons Use of calf thymus DNA for cancer experiments Characterization of mitochondria isolated from normal and ischemic hearts in rats Polymorphism and ultrastructural organization of prion protein Ultrastructural investigation of animal spermatozoa using AFM Multifactor analysis of living animal cells for biotechnology and medicine Ethical issues Translational significance Clinical correlations Acknowledgments World wide web resources References Further reading Glossary Abbreviations Long Answer Questions Short answer questions Answers to short answer questions Yes/no type questions Answers to “Yes/no” questions 17 Antibodies: monoclonal and polyclonal Summary What you can expect to know History and methods Introduction Tiselius and Kabat’s experiment History Elucidation of immunoglobulin structure Edelman’s experiment Porter’s experiment Nisonoff’s experiment Conclusion from papain and pepsin digestion Turning point Immunoglobulin G: a prototype for immunoglobulin Polyclonal antibody versus monoclonal antibody Polyclonal antibodies Monoclonal antibodies Naming monoclonal antibodies Prefix Infix-1 Infix-2 Additional words Antibodies as therapeutics: adverse effects Serum sickness Human antimonoclonal antibody response Human antichimeric antibody response Human antihumanized antibody response Applications of antibodies Therapeutic applications Analytical applications Preparative applications Methodology, principles, and protocols Polyclonal antibodies Principle Methodology and rationale for PoAb production Step 1: antigen preparation Step 2: immunization of animals Step 3: antibody titer Step 4: purification and identification Monoclonal antibodies Principle Methodology and rationale of MoAb production Step 1: immunization of mouse Step 2: preparation of splenocytes Step 3: fusion of cells Step 4: selection of hybrid cells Step 5: selection of clones Step 6: expansion of clone Step 7: purification Antibody titration Biochemical pathway: hybridoma selection Ethical issues Camelid nanobodies/single-domain antibodies/variable domain of camelid heavy chain only antibody Translational significance Clinical correlations World Wide Web resources Acknowledgments References Further reading Glossary Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type question Answers to yes/no type questions 18 Molecular markers: tool for genetic analysis Summary What can you expect to know Introduction Methodology Restriction fragment length polymorphism Steps involved in restriction fragment length polymorphism analysis Applications of restriction fragment length polymorphism Allele-specific oligonucleotide Applications of allele-specific oligonucleotide Allele-specific polymerase chain reaction Applications of allele-specific polymerase chain reaction Single-strand conformation polymorphism Applications of single-strand conformation polymorphism Sequence-tagged site Applications of sequence-tagged site Random amplified polymorphic DNA Steps involved in random amplified polymorphic DNA analysis Applications of random amplified polymeric DNA analysis Restriction landmark genome scanning Applications of landmark genome scanning Single nucleotide polymorphisms Applications of single-nucleotide polymorphisms Amplified fragment length polymorphism Steps involved in amplified fragment length polymorphism analysis Applications of amplified fragment length polymorphism Methylation-sensitive amplification polymorphism Applications of methylation-sensitive amplification polymorphism Miniature inverted-repeat transposable element Applications of miniature inverted-repeat transposable element Microsatellites Simple sequence repeats/Short tandem repeats/Simple sequence tandem repeats Applications of simple sequence repeats Inter simple sequence repeats Applications of inter simple sequence repeats Variable number of tandem repeat markers Applications of variable number of tandem repeat markers Sequence tagged microsatellite site Applications of sequence-tagged microsatellite site Simple sequence length polymorphisms Applications of sequence length polymorphisms Example of microsatellites Advantage of microsatellite marker Disadvantages of microsatellite marker Some general applications of microsatellites Parentage determination Determination of twin zygosity and freemartinism Identification of disease carrier Ethical issues Translational significance Clinical correlations Turning point World wide web resources References Further reading Glossary Abbreviations Long-answer questions Short-answer questions Answers to short questions Yes/no-type questions Answers to yes/No-type questions 19 Ribotyping: a tool for molecular taxonomy Summary What you can expect to know History and methods Introduction Historical developments in bacterial taxonomy Typing methods used for bacterial systematics Phenotypic typing methods Genotypic typing methods Basis of using rRNA and rRNA genes as taxonomic tools Organization of the ribosomal operon Different techniques of ribotyping Conventional ribotyping Selection of restriction endonuclease for ribotyping by sequence analysis (in silico) Automated ribotyping Polymerase chain reaction ribotyping Polymerase chain reaction ribotyping and endonuclease subtyping Polymerase chain reaction ribotyping followed by sequencing of ISR Amplified ribosomal DNA restriction analysis Terminal restriction fragment length polymorphism of 16S rRNA gene Long polymerase chain reaction ribotyping Broad-range polymerase chain reaction ribotyping Limitations of polymerase chain reaction ribotyping Ribosomal DNA sequence analysis In situ hybridization targeted to detect rRNA Clone-fluorescent in situ hybridization Catalyzed reported deposition fluorescent in situ hybridization Recognition of individual gene fluorescent in situ hybridization Stable isotope-labeled probing of rRNA and rDNA Peptide nucleic acid probes Limitations of ribotyping Other genotyping methods Multilocus sequence typing Whole genome sequencing Single cell sequencing Metagenomics Future perspectives Ethical issues Translational significance Clinical significance World Wide Web resources References Abbreviations Long answer questions Short answer questions Answers to short answer questions Yes/no type questions Answers to yes/no type questions 20 Next generation sequencing and its applications Summary What you can expect to know Introduction History of DNA sequencing Generation of sequencing technologies Principle of Sanger sequencing versus NGS NGS technologies: Initial phase Pyrosequencing technology Sequencing by ligation technology True single molecule sequencing NGS technologies: recent phase Reversible terminator technology Ion semiconductor sequencing Single molecule real time sequencing Other newer sequencing technologies Nanopore sequencing Polony-based sequencing technology DNA nanoball sequencing Downstream bioinformatics Primary analysis Secondary analysis Tertiary analysis General principles of NGS methods in va