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An Introduction to Molecular Biotechnology

E-BookPDF2 - DRM Adobe / Adobe Ebook ReaderE-Book
520 Seiten
Englisch
Wiley-VCH Verlag GmbH & Co. KGaAerschienen am03.12.20203. vollständig überarbeitete Auflage
Completely updated in line with the rapid progress made in the field, this new edition of the highly-praised textbook addresses powerful new methods and concepts in biotechnology, such as genome editing, reprogrammed stem cells, and personalized medicine.
An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including purification and analysis of biomolecules, cloning techniques, gene expression systems, genome editing methods, labeling of proteins and in situ-techniques, standard and high resolution microscopy. The third part focuses on key areas in research and application, ranging from functional genomics, proteomics and bioinformatics to drug targeting, recombinant antibodies and systems biology. The final part looks at the biotechnology industry, explaining intellectual property issues, legal frameworks for pharmaceutical products and the interplay between start-up and larger companies. The contents are beautifully illustrated throughout, with hundreds of full color diagrams and photographs.
Provides students and professionals in life sciences, pharmacy and biochemistry with everything they need to know about molecular biotechnology.


Michael Wink studied biology and chemistry in Bonn and was awarded his doctorate from TU Braunschweig in 1980. After gaining his lecturing qualification in 1984/1985, he was awarded a Heisenberg grant by the German Research Council to work at the Max Planck Institute for Breeding Research in Cologne and from then at the Gene Center of Ludwig-Maximilians University in Munich. Following a chair for Pharmaceutical Biology at Mainz University in 1988, he accepted the post of Professor for Pharmaceutical Biology at the University of Heidelberg one year later. His areas of interest include pharmaceutical research, molecular biotechnology, and medicinal plants, as well as research into natural products and evolution.mehr
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Produkt

KlappentextCompletely updated in line with the rapid progress made in the field, this new edition of the highly-praised textbook addresses powerful new methods and concepts in biotechnology, such as genome editing, reprogrammed stem cells, and personalized medicine.
An introduction to the fundamentals in molecular and cell biology is followed by a description of standard techniques, including purification and analysis of biomolecules, cloning techniques, gene expression systems, genome editing methods, labeling of proteins and in situ-techniques, standard and high resolution microscopy. The third part focuses on key areas in research and application, ranging from functional genomics, proteomics and bioinformatics to drug targeting, recombinant antibodies and systems biology. The final part looks at the biotechnology industry, explaining intellectual property issues, legal frameworks for pharmaceutical products and the interplay between start-up and larger companies. The contents are beautifully illustrated throughout, with hundreds of full color diagrams and photographs.
Provides students and professionals in life sciences, pharmacy and biochemistry with everything they need to know about molecular biotechnology.


Michael Wink studied biology and chemistry in Bonn and was awarded his doctorate from TU Braunschweig in 1980. After gaining his lecturing qualification in 1984/1985, he was awarded a Heisenberg grant by the German Research Council to work at the Max Planck Institute for Breeding Research in Cologne and from then at the Gene Center of Ludwig-Maximilians University in Munich. Following a chair for Pharmaceutical Biology at Mainz University in 1988, he accepted the post of Professor for Pharmaceutical Biology at the University of Heidelberg one year later. His areas of interest include pharmaceutical research, molecular biotechnology, and medicinal plants, as well as research into natural products and evolution.
Details
Weitere ISBN/GTIN9783527812868
ProduktartE-Book
EinbandartE-Book
FormatPDF
FormatFormat mit automatischem Seitenumbruch (reflowable)
Erscheinungsjahr2020
Erscheinungsdatum03.12.2020
Auflage3. vollständig überarbeitete Auflage
Seiten520 Seiten
SpracheEnglisch
Dateigrösse22252 Kbytes
Artikel-Nr.5503715
Rubriken
Genre9201

Inhalt/Kritik

Inhaltsverzeichnis
1;Cover;0
2;Title Page;0
3;Copyright;0
4;Contents;7
5;Abbreviations;19
6;Part I Fundamentals of Cellular and Molecular Biology;27
6.1;Chapter 1 The Cell as the Basic Unit of Life;29
6.1.1;References;34
6.1.2;Further Reading;34
6.2;Chapter 2 Structure and Function of Cellular Macromolecules;35
6.2.1;2.1 Structure and Function of Sugars;35
6.2.2;2.2 Structure of Membrane Lipids;39
6.2.3;2.3 Structure and Function of Proteins;43
6.2.4;2.4 Structure of Nucleotides and Nucleic Acids (DNA and RNA);51
6.2.5;References;58
6.2.6;Further Reading;58
6.3;Chapter 3 Structure and Functions of a Cell;59
6.3.1;3.1 Structure of a Eukaryotic Cell;59
6.3.1.1;3.1.1 Structure and Function of the Cytoplasmic Membrane;59
6.3.1.1.1;3.1.1.1 Membrane Permeability;59
6.3.1.1.2;3.1.1.2 Transport Processes Across Biomembranes;60
6.3.1.1.3;3.1.1.3 Receptors and Signal Transduction at Biomembranes;63
6.3.1.2;3.1.2 Endomembrane System in a Eukaryotic Cell;66
6.3.1.3;3.1.3 Mitochondria and Chloroplasts;71
6.3.1.4;3.1.4 Cytoplasm;75
6.3.1.5;3.1.5 Cytoskeleton;77
6.3.1.6;3.1.6 Cell Walls;79
6.3.2;3.2 Structure of Bacteria;79
6.3.3;3.3 Structure of Viruses;81
6.3.4;3.4 Differentiation of Cells;82
6.3.5;3.5 Cell Death;86
6.3.6;References;87
6.3.7;Further Reading;87
6.4;Chapter 4 Biosynthesis and Function of Macromolecules (DNA, RNA, and Proteins);89
6.4.1;4.1 Genomes, Chromosomes, and Replication;89
6.4.1.1;4.1.1 Genome Size;89
6.4.1.2;4.1.2 Composition and Function of Chromosomes;93
6.4.1.3;4.1.3 Mitosis and Meiosis;95
6.4.1.4;4.1.4 Replication;97
6.4.1.5;4.1.5 Mutations and Repair Mechanisms;98
6.4.2;4.2 Transcription: From Gene to Protein;103
6.4.3;4.3 Protein Biosynthesis (Translation);107
6.4.4;Further Reading;111
6.5;Chapter 5 Distributing Proteins in the Cell (Protein Sorting);113
6.5.1;5.1 Import and Export of Proteins via the Nuclear Pore;113
6.5.2;5.2 Import of Proteins in Mitochondria, Chloroplasts, and Peroxisomes;114
6.5.3;5.3 Protein Transport into the Endoplasmic Reticulum;115
6.5.4;5.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane;118
6.5.5;References;120
6.5.6;Further Reading;120
6.6;Chapter 6 Evolution and Diversity of Organisms;121
6.6.1;6.1 Prokaryotes;121
6.6.2;6.2 Eukaryotes;121
6.6.3;References;127
6.6.4;Further Reading;127
7;Part II Standard Methods in Molecular Biotechnology;129
7.1;Chapter 7 Isolation and Purification of Proteins;131
7.1.1;7.1 Introduction;131
7.1.2;7.2 Producing a Protein Extract;132
7.1.3;7.3 Gel Electrophoretic Separation Methods;133
7.1.3.1;7.3.1 Principles of Electrophoresis;133
7.1.3.2;7.3.2 Native Gel Electrophoresis;133
7.1.3.3;7.3.3 Discontinuous Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS?PAGE);133
7.1.3.4;7.3.4 Two?Dimensional (2D) Gel Electrophoresis and Isoelectric Focusing (IEF);134
7.1.3.5;7.3.5 Detecting Proteins in Gels;134
7.1.4;7.4 Methods of Protein Precipitation;135
7.1.5;7.5 Column Chromatography Methods;135
7.1.5.1;7.5.1 General Principles of Separation;135
7.1.5.1.1;7.5.1.1 Size Exclusion Chromatography (Gel Filtration);135
7.1.5.1.2;7.5.1.2 Hydrophobic Interaction Chromatography;137
7.1.5.1.3;7.5.1.3 Ion Exchange Chromatography;137
7.1.5.1.4;7.5.1.4 Hydroxyapatite Chromatography;138
7.1.5.2;7.5.2 Group?Specific Separation Techniques;138
7.1.5.2.1;7.5.2.1 Chromatography on Protein A or Protein G;138
7.1.5.2.2;7.5.2.2 Chromatography on Cibacron Blue (Blue Gel);138
7.1.5.2.3;7.5.2.3 Chromatography on Lectins;138
7.1.5.2.4;7.5.2.4 Chromatography on Heparin;139
7.1.5.3;7.5.3 Purification of Recombinant Fusion Proteins;139
7.1.5.3.1;7.5.3.1 Chromatography on Chelating Agents;139
7.1.5.3.2;7.5.3.2 Chromatography on Glutathione Matrices;140
7.1.6;7.6 Examples;140
7.1.6.1;7.6.1 Example 1: Purification of Nucleoside Diphosphate Kinase from the Cytosol of Bovine Retina Rod Cells;140
7.1.6.2;7.6.2 Example 2: Purification of Recombinant His6?RGS16 After Expression in E. coli;140
7.1.7;Further Reading;141
7.2;Chapter 8 Mass Spectrometry and Applications in Proteomics and Microbial Identification;143
7.2.1;8.1 Principles of ESI and MALDI Mass Spectrometry;143
7.2.2;8.2 Instrumental Setup;144
7.2.3;8.3 Intact Protein Analysis;145
7.2.3.1;8.3.1 Protein Digestion;145
7.2.3.2;8.3.2 Peptide Fragmentation;145
7.2.3.3;8.3.3 Protein Identification with MS/MS Spectra;147
7.2.4;8.4 Protein and Proteome Quantification;147
7.2.4.1;8.4.1 Label?Free Quantification;147
7.2.4.2;8.4.2 Chemical Stable Isotope Labeling;147
7.2.4.3;8.4.3 Metabolic Stable Isotope Labeling;148
7.2.5;8.5 Protein-Protein Interaction Analysis;149
7.2.6;8.6 Analysis of Posttranslational Modifications;150
7.2.7;8.7 Microbial Identification and Resistance Detection;151
7.2.8;References;152
7.3;Chapter 9 Isolation of DNA and RNA;155
7.3.1;9.1 Introduction;155
7.3.2;9.2 DNA Isolation;155
7.3.3;9.3 RNA Isolation;157
7.3.3.1;9.3.1 Enrichment of mRNA;157
7.3.4;Reference;157
7.4;Chapter 10 Chromatography and Electrophoresis of Nucleic Acids;159
7.4.1;10.1 Introduction;159
7.4.2;10.2 Chromatographic Separation of Nucleic Acids;159
7.4.3;10.3 Electrophoresis;160
7.4.3.1;10.3.1 Agarose Gel Electrophoresis: Submarine Electrophoresis;160
7.4.3.2;10.3.2 Pulsed?Field Agarose Gel Electrophoresis;160
7.4.3.3;10.3.3 Polyacrylamide Gel Electrophoresis (PAGE);161
7.4.4;Further Reading;161
7.5;Chapter 11 Hybridization of Nucleic Acids;163
7.5.1;11.1 Significance of Base Pairing;163
7.5.2;11.2 Experimental Hybridization: Kinetic and Thermodynamic Control;163
7.5.3;11.3 Analytical Techniques;164
7.5.3.1;11.3.1 Clone Detection, Southern Blotting, Northern Blotting, and Gene Diagnosis;164
7.5.3.2;11.3.2 Systematic Gene Diagnosis and Expression Screening Based on Gene Arrays;165
7.5.3.3;11.3.3 In Situ Hybridization;165
7.5.4;References;166
7.5.5;Further Reading;166
7.6;Chapter 12 Use of Enzymes in the Modification of Nucleic Acids;167
7.6.1;12.1 Restriction Enzymes (Restriction Endonucleases);167
7.6.2;12.2 Ligases;168
7.6.3;12.3 Methyltransferases;168
7.6.4;12.4 DNA Polymerases;169
7.6.5;12.5 RNA Polymerases and Reverse Transcriptase;170
7.6.6;12.6 Nucleases;170
7.6.7;12.7 T4 Polynucleotide Kinase;170
7.6.8;12.8 Phosphatases;171
7.6.9;Further Reading;171
7.7;Chapter 13 Polymerase Chain Reaction;173
7.7.1;13.1 Introduction;173
7.7.2;13.2 PCR Methods;173
7.7.2.1;13.2.1 Basic Principle;173
7.7.2.2;13.2.2 Primer Design and Hot Start PCR;174
7.7.2.3;13.2.3 Multiplex PCR;175
7.7.2.4;13.2.4 RT?PCR;175
7.7.2.5;13.2.5 Qualitative Analysis of the PCR Products;175
7.7.3;13.3 PCR as a Quantitative Method;175
7.7.3.1;13.3.1 PCR Phases and PCR Efficiency;175
7.7.3.2;13.3.2 Quantitative Real?Time PCR;176
7.7.3.3;13.3.3 Digital PCR;177
7.7.4;13.4 Areas of Application;177
7.7.4.1;13.4.1 Genome Analysis;177
7.7.4.2;13.4.2 Cloning Techniques;178
7.7.4.3;13.4.3 Gene Expression Studies;178
7.7.5;Further Reading;178
7.8;Chapter 14 DNA Sequencing;179
7.8.1;14.1 Introduction;179
7.8.2;14.2 The Sanger Method;179
7.8.3;14.3 Pyrosequencing;180
7.8.4;14.4 Second?Generation Sequencing: Illumina and Ion Torrent;181
7.8.4.1;14.4.1 Overview;181
7.8.4.2;14.4.2 The Illumina Sequencing System;181
7.8.4.3;14.4.3 The Ion Torrent Sequencing System;182
7.8.5;14.5 Third?Generation Sequencing Techniques;182
7.8.5.1;14.5.1 Overview;182
7.8.5.2;14.5.2 SMRT Sequencing;183
7.8.5.3;14.5.3 Nanopore Sequencing;183
7.8.6;14.6 The Impact of the DNA Sequencing Technology;184
7.8.7;References;184
7.8.8;Further Reading;184
7.8.9;Websites;184
7.9;Chapter 15 Cloning Procedures;185
7.9.1;15.1 Introduction;185
7.9.2;15.2 Construction of Recombinant Vectors;185
7.9.2.1;15.2.1 Insert;185
7.9.2.2;15.2.2 Vector;187
7.9.2.3;15.2.3 Essential Components of Vectors;188
7.9.2.3.1;15.2.3.1 Bacterial Origin of Replication (ori);188
7.9.2.3.2;15.2.3.2 Antibiotic Resistance;188
7.9.2.3.3;15.2.3.3 Polylinkers;188
7.9.2.4;15.2.4 Cloning Using Recombination Systems;188
7.9.2.5;15.2.5 Further Components of Vectors for Prokaryotic Expression Systems;189
7.9.2.5.1;15.2.5.1 Promoter;189
7.9.2.5.2;15.2.5.2 Ribosome?Binding Site;189
7.9.2.5.3;15.2.5.3 Termination Sequence;190
7.9.2.5.4;15.2.5.4 Fusion Sequence;190
7.9.2.6;15.2.6 Further Components of Eukaryotic Expression Vectors;190
7.9.2.6.1;15.2.6.1 Eukaryotic Expression Vectors: Yeast;190
7.9.2.6.2;15.2.6.2 Eukaryotic Expression Vectors for Mammal Cells;191
7.9.2.6.3;15.2.6.3 Viral Expression Systems for Mammalian Cells;193
7.9.2.7;15.2.7 Nonviral Introduction of Heterologous DNA to Host Organisms (Transformation, Transfection);194
7.9.2.7.1;15.2.7.1 Transformation of Prokaryotes;194
7.9.2.7.2;15.2.7.2 Transformation of Yeast Cells;195
7.9.2.7.3;15.2.7.3 Transfection of Mammal Cells;195
7.9.3;Further Reading;196
7.10;Chapter 16 Expression of Recombinant Proteins;197
7.10.1;16.1 Introduction;197
7.10.2;16.2 Expression of Recombinant Proteins in Host Organisms;197
7.10.2.1;16.2.1 Expression in E. coli;198
7.10.2.2;16.2.2 Expression in Yeasts;201
7.10.2.3;16.2.3 Expression in Insect Cells;203
7.10.2.3.1;16.2.3.1 Expression Based on Recombinant Baculoviruses;203
7.10.2.3.2;16.2.3.2 Expression of Proteins in Stably Transfected Insect Cells;204
7.10.2.4;16.2.4 Expression of Proteins in Mammalian Cells;204
7.10.3;16.3 Expression in Cell?Free Systems;205
7.10.3.1;16.3.1 Expression of Proteins in Reticulocyte Lysates;206
7.10.3.2;16.3.2 Protein Expression Using E. coli Extracts;206
7.10.4;Further Reading;206
7.11;Chapter 17 Patch Clamp Method;207
7.11.1;17.1 Ion Channels;207
7.11.2;17.2 Technical Requirements of the Patch Clamp Method;207
7.11.3;17.3 Patch Clamp Configurations;208
7.11.4;17.4 Applications of the Patch Clamp Method;209
7.11.5;Reference;211
7.11.6;Further Reading;211
7.12;Chapter 18 Cell Cycle Analysis;213
7.12.1;18.1 Introduction;213
7.12.2;18.2 Analyzing the Cell Cycle;213
7.12.3;18.3 Experimental Analysis of the Cell Cycle;215
7.12.3.1;18.3.1 Preparing Synchronized Cell Cultures of S. cerevisiae;215
7.12.3.1.1;18.3.1.1 Centrifugal Elutriation;216
7.12.3.1.2;18.3.1.2 Cell Cycle Arrest Using &rmalpha;?Factor;216
7.12.3.2;18.3.2 Identification of Cell Cycle Stages;217
7.12.3.2.1;18.3.2.1 Budding Index;217
7.12.3.2.2;18.3.2.2 Fluorescent Staining of the Nucleus;217
7.12.3.2.3;18.3.2.3 Detection of Cell Cycle Phases Using Fluorescent Proteins as Reporters;220
7.12.4;Acknowledgments;221
7.12.5;Further Reading;222
7.13;Chapter 19 Microscopic Techniques;223
7.13.1;19.1 Introduction;223
7.13.2;19.2 Electron Microscopy;223
7.13.2.1;19.2.1 Cryo?electron Microscopy;225
7.13.2.2;19.2.2 Electron Tomography;225
7.13.3;19.3 Atomic or Scanning Force Microscopy;225
7.13.3.1;19.3.1 Force Spectroscopy;226
7.13.3.2;19.3.2 Advantages and Disadvantages;227
7.13.4;19.4 Light Microscopy;227
7.13.4.1;19.4.1 Deconvolution;228
7.13.4.2;19.4.2 Confocal Microscopy;228
7.13.4.3;19.4.3 Why Fluorescence?;229
7.13.4.4;19.4.4 Nanoscopy;229
7.13.5;19.5 Microscopy in the Living Cell;230
7.13.5.1;19.5.1 Analysis of Fluorescently Labeled Proteins In Vivo;231
7.13.5.2;19.5.2 Fluorescence Recovery After Photobleaching;232
7.13.5.3;19.5.3 Fluorescence Correlation Spectroscopy;232
7.13.5.4;19.5.4 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy;233
7.13.5.5;19.5.5 Single?Molecule Fluorescence;233
7.13.6;Further Reading;233
7.14;Chapter 20 Laser Applications;235
7.14.1;20.1 Laser Development: A Historical Perspective;235
7.14.2;20.2 Types of Lasers and Setups;236
7.14.3;20.3 Properties of Laser Radiation;236
7.14.4;20.4 Applications;237
7.14.4.1;20.4.1 Laser Scanning Microscopy;237
7.14.4.2;20.4.2 Optical Tweezers;238
7.14.4.3;20.4.3 Laser Microdissection and Laser Therapy;238
7.14.4.4;20.4.4 Manufacturing of Products in Medical Technology and Biotechnology Products;239
7.14.5;Further Reading;239
8;Part III Key Topics;241
8.1;Chapter 21 Sequencing the Universe of Life;243
8.1.1;21.1 What to Sequence?;243
8.1.1.1;21.1.1 Whole?Genome Sequencing;243
8.1.1.2;21.1.2 Exome Sequencing;246
8.1.1.3;21.1.3 (Gene) Panel Sequencing;246
8.1.1.4;21.1.4 RNA Sequencing;247
8.1.1.4.1;21.1.4.1 Tag? vs. Full?Length Sequencing;247
8.1.1.4.2;21.1.4.2 Sequencing of RNA Species and Modifications;247
8.1.1.4.3;21.1.4.3 Sequencing of Single Cells;248
8.1.1.4.4;21.1.4.4 In Situ Sequencing;248
8.1.1.5;21.1.5 (Whole?Genome) Bisulfite Sequencing of DNA;249
8.1.1.6;21.1.6 Sequencing to Characterize Chromatin Structure and Beyond;249
8.1.2;21.2 Sequencing Projects: Human;250
8.1.2.1;21.2.1 Initial Sequencing of the Human Genome;250
8.1.2.2;21.2.2 The 1000 Genomes Project: Assessing Natural Variation;250
8.1.2.3;21.2.3 Screening for Genetic Disease;251
8.1.2.4;21.2.4 Sequencing of Populations;252
8.1.2.5;21.2.5 TCGA and ICGC: Screening for Cancer Driver Mutations;252
8.1.3;21.3 Sequencing Other Species, Environments, â¦;254
8.1.4;21.4 Sequencing in the Clinics: Personalizing Oncology;254
8.1.5;21.5 Sequencing in the Private Sector: Direct to Consumer Testing (DTC);257
8.1.6;21.6 The Information Content of a Genome Sequence and Ethical Consequences;257
8.1.7;References;258
8.2;Chapter 22 Cellular Systems Biology;265
8.2.1;22.1 Introduction;265
8.2.2;22.2 Analysis of Cellular Networks by Top?Down Approaches;266
8.2.2.1;22.2.1 Motivation;266
8.2.2.2;22.2.2 Definitions and Construction of the Networks;266
8.2.2.3;22.2.3 Gene Set Enrichment Tests;267
8.2.2.4;22.2.4 Inferring Gene Regulators Employing Gene Regulatory Models;268
8.2.2.5;22.2.5 Network Descriptors;269
8.2.2.5.1;22.2.5.1 Scale?Free Networks;269
8.2.2.5.2;22.2.5.2 Centrality;269
8.2.2.5.3;22.2.5.3 The Clustering Coefficient;270
8.2.2.6;22.2.6 Detecting Essential Enzymes with a Machine Learning Approach;270
8.2.2.7;22.2.7 Elementary Flux Modes;270
8.2.3;22.3 Overview over Bottom?Up Modeling of Biochemical Networks;273
8.2.3.1;22.3.1 Motivation;273
8.2.3.2;22.3.2 Choosing Model Complexity and Model Building;274
8.2.3.3;22.3.3 Model Simulation;277
8.2.3.4;22.3.4 Model Calibration;278
8.2.3.5;22.3.5 Model Verification and Analysis;280
8.2.3.6;22.3.6 Examples;280
8.2.4;Further Reading;284
8.2.5;References;285
8.3;Chapter 23 Protein-Protein and Protein-DNA Interactions;287
8.3.1;23.1 Protein-Protein Interactions;287
8.3.1.1;23.1.1 Classification and Specificity: Protein Domains;287
8.3.1.2;23.1.2 Protein Networks and Complexes;288
8.3.1.3;23.1.3 Structural Properties of Interacting Proteins;288
8.3.1.4;23.1.4 Which Forces Mediate Protein-Protein Interactions?;289
8.3.1.4.1;23.1.4.1 Thermodynamics;290
8.3.1.4.2;23.1.4.2 Energetics;290
8.3.1.5;23.1.5 Methods to Examine Protein-Protein Interactions;290
8.3.1.6;23.1.6 Theoretical Prediction of Protein-Protein Interactions;292
8.3.1.7;23.1.7 Regulation of Protein-Protein Interactions;292
8.3.1.8;23.1.8 Biotechnological and Medical Applications of Protein-Protein Interactions;294
8.3.2;23.2 Protein-DNA Interactions;295
8.3.2.1;23.2.1 Specific Protein-DNA Interaction;295
8.3.2.2;23.2.2 Thermodynamic Consideration;296
8.3.2.3;23.2.3 Methods to Study Protein-DNA Interactions;296
8.3.2.3.1;23.2.3.1 Structural Classification of Protein-DNA Complexes;296
8.3.2.4;23.2.4 Regulatory Networks and System Biology;298
8.3.2.5;23.2.5 Medical Importance of Protein-DNA Interactions;300
8.3.2.6;23.2.6 Biotechnological Applications;300
8.3.3;References;301
8.3.4;Further Reading;302
8.4;Chapter 24 Bioinformatics;303
8.4.1;24.1 Introduction;303
8.4.2;24.2 Data Sources;303
8.4.2.1;24.2.1 Primary Databases: EMBL/GenBank/DDBJ, PIR, and Swiss?Prot;303
8.4.2.2;24.2.2 Genome Databases: Ensembl and GoldenPath;304
8.4.2.3;24.2.3 Motif Databases: BLOCKS, PROSITE, Pfam, ProDom, and SMART;304
8.4.2.4;24.2.4 Molecular Structure Databases: PDB and SCOP;304
8.4.2.5;24.2.5 Transcriptome Databases: SAGE, ArrayExpress, and GEO;305
8.4.2.6;24.2.6 Reference Databases: PubMed, OMIM, and GeneCards;305
8.4.2.7;24.2.7 Pathway Databases and Gene Ontology;305
8.4.3;24.3 Sequence Analysis;306
8.4.3.1;24.3.1 Kyte-Doolittle Plot, Helical Wheel Analysis, and Signal Sequence Analysis;306
8.4.3.2;24.3.2 Pairwise Alignment;307
8.4.3.2.1;24.3.2.1 Local/Global;307
8.4.3.2.2;24.3.2.2 Optimal/Heuristic;308
8.4.3.3;24.3.3 Alignment Statistics;308
8.4.3.4;24.3.4 Multiple Alignment;308
8.4.4;24.4 Evolutionary Bioinformatics;309
8.4.4.1;24.4.1 Statistical Models of Evolution;309
8.4.4.2;24.4.2 Relation to Score Matrices;310
8.4.4.3;24.4.3 Phylogenetic Analysis;311
8.4.5;24.5 Gene Prediction;311
8.4.5.1;24.5.1 Neural Networks or HMMs Based on Hexanucleotide Composition;312
8.4.5.2;24.5.2 Comparison with Expressed Sequence Tags or Other Genomes (Fugu, Mouse);312
8.4.6;24.6 Bioinformatics in Transcriptome and Proteome Analysis;313
8.4.6.1;24.6.1 Preprocessing and Normalization;313
8.4.6.2;24.6.2 Feature Selection;314
8.4.6.3;24.6.3 Similarity Measures: Euclidean Distance, Correlation, Manhattan Distance, Mahalanobis Distance, and Entropy Measures;314
8.4.6.4;24.6.4 Unsupervised Learning Procedures: Clustering, Principal Component Analysis, Multidimensional Scaling, and Correspondence Analysis;315
8.4.6.5;24.6.5 Supervised Learning Procedures: Linear Discriminant Analysis, Decision Trees, Support Vector Machines, and ANNs;315
8.4.6.6;24.6.6 Analysis of Overrepresentation of Functional Categories;316
8.4.7;24.7 Analysis of Ultraparallel Sequencing Data;317
8.4.7.1;24.7.1 Mapping of Ultraparallel Sequencing Data;317
8.4.7.2;24.7.2 Genome (Re?)sequencing;318
8.4.7.3;24.7.3 Transcriptome Sequencing;318
8.4.7.4;24.7.4 ChIP?seq;319
8.4.7.5;24.7.5 Epigenetic Analysis;319
8.4.7.6;24.7.6 Single?Cell Analysis;320
8.4.7.7;24.7.7 Bioethics of Human Sequencing Data;320
8.4.8;24.8 Bioinformatic Software;320
8.4.9;Further Reading;321
8.5;Chapter 25 Drug Research;323
8.5.1;25.1 Introduction;323
8.5.2;25.2 Active Compounds and Their Targets;323
8.5.2.1;25.2.1 Identification of Potential Targets in the Human Genome;324
8.5.2.2;25.2.2 Comparative Genome Analysis;324
8.5.2.3;25.2.3 Experimental Target Identification: In Vitro Methods;325
8.5.2.4;25.2.4 Experimental Identification of Targets: Model Organisms;326
8.5.2.5;25.2.5 Experimental Target Identification in Humans;326
8.5.2.6;25.2.6 Difference Between Target Candidates and Genuine Targets;327
8.5.2.7;25.2.7 Biologicals;327
8.5.2.8;25.2.8 DNA and RNA in New Therapeutic Approaches;328
8.5.2.9;25.2.9 Patent Protection for Targets;329
8.5.2.10;25.2.10 Compound Libraries as a Source of Drug Discovery;330
8.5.2.11;25.2.11 High?Throughput Screening;330
8.5.2.12;25.2.12 High?Quality Paramounts in Screening Assays;330
8.5.2.13;25.2.13 Virtual Ligand Screening;332
8.5.2.14;25.2.14 Activity of Drugs Described in Terms of Efficacy and Potency;333
8.5.2.15;25.2.15 Chemical Optimization of Lead Structures;333
8.5.3;25.3 Preclinical Pharmacology and Toxicology;334
8.5.4;25.4 Clinical Development;335
8.5.5;25.5 Clinical Testing;335
8.5.6;Further Reading;336
8.6;Chapter 26 Drug Targeting and Prodrugs;337
8.6.1;26.1 Drug Targeting;337
8.6.1.1;26.1.1 Passive Targeting by Exploiting Special Physiological Properties of the Target Tissue;337
8.6.1.2;26.1.2 Physical Targeting;338
8.6.1.3;26.1.3 Active Targeting;339
8.6.1.4;26.1.4 Cellular Carrier Systems;342
8.6.2;26.2 Prodrugs;342
8.6.2.1;26.2.1 Prodrugs to Improve Drug Solubility;342
8.6.2.2;26.2.2 Prodrugs to Increase Stability;343
8.6.3;26.3 Penetration of Drugs Through Biological Membranes;343
8.6.4;26.4 Prodrugs to Extend Duration of Effect;344
8.6.5;26.5 Prodrugs for the Targeted Release of a Drug;344
8.6.6;26.6 Prodrugs to Minimize Side Effects;346
8.6.7;References;346
8.7;Chapter 27 Molecular Diagnostics in Medicine;349
8.7.1;27.1 Introduction;349
8.7.2;27.2 Uses of Molecular Diagnostics;349
8.7.2.1;27.2.1 Introduction;349
8.7.2.2;27.2.2 Monogenic and Polygenic Diseases;349
8.7.2.3;27.2.3 Individual Variability in the Genome: Forensics;351
8.7.2.4;27.2.4 Individual Variability in the Genome: HLA Typing;351
8.7.2.5;27.2.5 Individual Variability in the Genome: Pharmacogenomics;351
8.7.2.6;27.2.6 Individual Variability in the Genome: Susceptibility to Infectious Diseases;352
8.7.2.7;27.2.7 Viral Diagnosis;352
8.7.2.8;27.2.8 Microbial Diagnosis and Resistance Diagnosis;353
8.7.3;27.3 Which Molecular Variations Should be Detected;353
8.7.3.1;27.3.1 Point Mutations;353
8.7.3.2;27.3.2 Insertions and Deletions;354
8.7.3.3;27.3.3 Nucleotide Repeats;354
8.7.3.4;27.3.4 Deletion or Duplication of Genes;354
8.7.3.5;27.3.5 Recombination Between Chromosomes;355
8.7.3.6;27.3.6 Epigenetic Changes;355
8.7.4;27.4 Molecular Diagnostic Methods;356
8.7.4.1;27.4.1 DNA/RNA Purification;357
8.7.4.2;27.4.2 Detection of Target Sequence and Known Sequence Variations;357
8.7.4.2.1;27.4.2.1 Nucleic Acid Tests;357
8.7.4.2.2;27.4.2.2 Quantitative PCR;358
8.7.4.2.3;27.4.2.3 Multiplexing of Nucleic Acid Detection: Nucleic Acid Microarrays;359
8.7.4.2.4;27.4.2.4 Production and Manufacture of Microarrays;360
8.7.4.2.5;27.4.2.5 Applications of Fragment Length Analysis;361
8.7.4.2.6;27.4.2.6 Minisequencing;362
8.7.4.2.7;27.4.2.7 Determination of Unknown Mutations;362
8.7.5;27.5 Outlook;363
8.7.6;Further Reading;364
8.7.7;Historic Article: News & Views ;364
8.7.8;Reviews;364
8.7.9;Web Link;364
8.7.10;Textbooks;364
8.8;Chapter 28 Recombinant Antibodies and Phage Display;365
8.8.1;28.1 Introduction;365
8.8.2;28.2 Generation of Specific Recombinant Antibodies;366
8.8.2.1;28.2.1 Generation of Antibody Gene Libraries;367
8.8.2.2;28.2.2 Selection Systems for Recombinant Antibodies;368
8.8.2.2.1;28.2.2.1 Transgenic Mice with Human IgG Genes;368
8.8.2.2.2;28.2.2.2 In Vitro Selection Systems;368
8.8.3;28.3 Production and Purification of Recombinant Antibodies;374
8.8.4;28.4 Features and Applications of Recombinant Antibodies;375
8.8.4.1;28.4.1 Advantages of Recombinant Antibodies;375
8.8.4.2;28.4.2 Formats and Applications of Recombinant Antibodies;376
8.8.4.2.1;28.4.2.1 Camelid Antibodies and VH Domains;377
8.8.4.2.2;28.4.2.2 scFv and dsFv;377
8.8.4.2.3;28.4.2.3 scFv-Fc Fusions, Fc Engineering, and the Addition of Constant Domains;378
8.8.4.2.4;28.4.2.4 IgG, Fusion Proteins, and Derivatives for Therapy;378
8.8.4.2.5;28.4.2.5 Bispecific Antibodies;380
8.8.4.2.6;28.4.2.6 Chimeric Antigen Receptors (CARs);381
8.8.4.3;28.4.3 The Future of Therapeutic Antibodies;381
8.8.4.4;28.4.4 Research and In Vitro Diagnostics;382
8.8.4.5;28.4.5 Intracellular and Cell?Penetrating Antibodies;382
8.8.5;28.5 Outlook;383
8.8.6;Further Reading;383
8.8.7;Textbooks;383
8.8.8;References;384
8.9;Chapter 29 Genetically Modified Mice and Their Impact in Medical Research;387
8.9.1;29.1 Overview;387
8.9.2;29.2 Transgenic Mice;388
8.9.2.1;29.2.1 Retroviral Infection;388
8.9.2.2;29.2.2 Pronuclear Injection;389
8.9.3;29.3 Homologous Recombination: Knockout (Knock?In) Mice;390
8.9.4;29.4 Endonuclease?Based Knockout Mice;392
8.9.5;29.5 Endonuclease?Based Knock?In Mice;393
8.9.6;29.6 Conditionally Regulated Gene Expression;393
8.9.7;29.7 Gene Transfer to Subpopulations of Cells;394
8.9.7.1;29.7.1 Electroporation of Mouse Embryos (Plasmid DNA);394
8.9.7.2;29.7.2 Virus?Mediated Gene Transfer (Lentivirus, rAAVs);395
8.9.7.3;29.7.3 Virus?Mediated Gene Deletion (Cre/lox);396
8.9.7.4;29.7.4 Virus?Mediated Gene Knockdown (shRNA, Antagomirs);396
8.9.8;29.8 Impact of Genetically Modified Mice in Biomedicine;396
8.9.8.1;29.8.1 Alzheimer's Disease;396
8.9.8.2;29.8.2 Amyotrophic Lateral Sclerosis (ALS);396
8.9.8.3;29.8.3 Psychological and Cognitive Disorders;397
8.9.8.4;29.8.4 Autism Spectrum Disorder (ASD);397
8.9.8.5;29.8.5 Chemogenetics, Optogenetics, and Magnetogenetics;398
8.9.9;29.9 Outlook;398
8.9.10;Reference;399
8.9.11;Further Reading;399
8.10;Chapter 30 Plant Biotechnology;401
8.10.1;30.1 Introduction;401
8.10.1.1;30.1.1 Green Genetic Engineering: A New Method Toward Traditional Goals;401
8.10.1.2;30.1.2 Challenges in Plant Biotechnology;402
8.10.2;30.2 Gene Expression Control and Genome Editing;402
8.10.2.1;30.2.1 Gene Expression Control;403
8.10.2.2;30.2.2 Genome Editing;403
8.10.3;30.3 Production of Transgenic Plants;404
8.10.3.1;30.3.1 Transformation Systems;405
8.10.3.1.1;30.3.1.1 Agrobacterium as a Natural Transformation System;405
8.10.3.1.2;30.3.1.2 Biolistic Method: Gene Gun;407
8.10.3.1.3;30.3.1.3 Plastid Transformation;408
8.10.3.1.4;30.3.1.4 Viral Systems;408
8.10.4;30.4 Selection of Transformed Plant Cells;409
8.10.4.1;30.4.1 Requirements for an Optimal Selection Marker System;409
8.10.4.2;30.4.2 Negative Selection Marker Systems;410
8.10.4.3;30.4.3 Positive Selection Marker Systems;411
8.10.4.4;30.4.4 Selection Systems, Genetic Engineering Safety, and Marker?Free Plants;411
8.10.5;30.5 Regeneration of Transgenic Plants;413
8.10.5.1;30.5.1 Regeneration Procedures;413
8.10.5.2;30.5.2 Composition of Regeneration Media;413
8.10.6;30.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants;414
8.10.6.1;30.6.1 DNA and RNA Verification;414
8.10.6.2;30.6.2 Protein Analysis;415
8.10.6.3;30.6.3 Genetic and Molecular Maps;415
8.10.6.4;30.6.4 Stability of Transgenic Plants;416
8.10.7;Further Reading;416
8.11;Chapter 31 Biocatalysis in the Chemical Industry;419
8.11.1;31.1 Introduction;419
8.11.2;31.2 Bioconversion/Enzymatic Procedures;421
8.11.3;31.3 Development of an Enzyme for Industrial Biocatalysis;423
8.11.3.1;31.3.1 Identification of Novel Biocatalysts;423
8.11.3.2;31.3.2 Improvement of Biocatalysts;425
8.11.3.3;31.3.3 Production of Biocatalysts;425
8.11.3.4;31.3.4 Outlook;425
8.11.3.5;31.3.5 Case Study 1: Screening for New Nitrilases;426
8.11.3.6;31.3.6 Case Study 2: Use of Known Enzymes for New Reactions: Lipases for the Production of Optically Active Amines and Alcohols;426
8.11.3.7;31.3.7 Case Study 3: Enzyme Optimization with Rational and Evolutive Methods;427
8.11.4;31.4 Fermentative Procedures;428
8.11.4.1;31.4.1 Improvement of Fermentation Processes;428
8.11.4.2;31.4.2 Classical Strain Optimization;429
8.11.4.3;31.4.3 Metabolic Engineering;430
8.11.4.4;31.4.4 Case Study 4: Fermentative Production of n?Butanol;431
8.11.4.5;31.4.5 Case Study 5: Production of Glutamic Acid with C. glutamicum;432
8.11.4.5.1;31.4.5.1 Molecular Mechanism of Glutamate Overproduction;432
8.11.4.6;31.4.6 Case Study 6: Production of Lysine with C. glutamicum;433
8.11.4.6.1;31.4.6.1 Molecular Mechanism of Lysine Biosynthesis;433
8.11.4.6.2;31.4.6.2 Deregulation of the Key Enzyme Aspartate Kinase;434
8.11.4.7;31.4.7 Genomic Research and Functional Genomics;435
8.11.4.8;31.4.8 Case Study 7: Fermentative Penicillin Production;435
8.11.4.9;31.4.9 Case Study 8: Vitamin B2 Production;435
8.11.4.9.1;31.4.9.1 Riboflavin Biosynthesis;436
8.11.4.9.2;31.4.9.2 Classical Strain Development;436
8.11.5;References;436
9;Part IV Biotechnology in Industry;437
9.1;Chapter 32 Industrial Application: Biotech Industry, Markets, and Opportunities;439
9.1.1;32.1 Historical Overview and Definitions of Concepts;439
9.1.2;32.2 Areas of Industrial Application of Molecular Biotechnology;440
9.1.2.1;32.2.1 Red Biotechnology;440
9.1.2.1.1;32.2.1.1 Biopharmaceutical Drug Development;440
9.1.2.1.2;32.2.1.2 Gene and Cell Therapy;442
9.1.2.1.3;32.2.1.3 Tissue Engineering/Regenerative Medicine;445
9.1.2.1.4;32.2.1.4 Pharmacogenomics and Personalized Medicine;447
9.1.2.1.5;32.2.1.5 Molecular Diagnostic Agents;447
9.1.2.1.6;32.2.1.6 Systems Biology;448
9.1.2.1.7;32.2.1.7 Synthetic Biology;448
9.1.2.2;32.2.2 Green Biotechnology;448
9.1.2.2.1;32.2.2.1 Transgenic Plants;448
9.1.2.2.2;32.2.2.2 Genomic Approaches in Green Biotechnology;449
9.1.2.2.3;32.2.2.3 Novel Food and Functional Food;449
9.1.2.2.4;32.2.2.4 Livestock Breeding;449
9.1.2.3;32.2.3 White Biotechnology;450
9.1.3;32.3 Status Quo of the Biotech Industry Worldwide;450
9.1.3.1;32.3.1 Global Overview;450
9.1.3.2;32.3.2 United States;450
9.1.3.3;32.3.3 Europe;450
9.2;Chapter 33 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues;451
9.2.1;33.1 Patent Law;451
9.2.1.1;33.1.1 What is a Patent?;451
9.2.1.2;33.1.2 How Does One Obtain a Patent?;452
9.2.1.3;33.1.3 What is the Proper Subject Matter for a Patent?;452
9.2.1.4;33.1.4 Types of Patents in Pharmaceutical and Molecular Biotechnology;453
9.2.1.5;33.1.5 Patent Infringement;453
9.2.1.6;33.1.6 International Patent Law;454
9.2.2;33.2 Ethical and Policy Issues in Biotechnology Patents;454
9.2.2.1;33.2.1 No Patents on Nature;454
9.2.2.2;33.2.2 Threats to Human Dignity;455
9.2.2.3;33.2.3 Problems with Access to Technology;456
9.2.2.4;33.2.4 Benefit Sharing;458
9.2.3;33.3 Conclusions;459
9.2.4;Acknowledgments;459
9.3;Chapter 34 Drug Approval in the European Union and United States;461
9.3.1;34.1 Introduction;461
9.3.2;34.2 Regulation Within the European Union;461
9.3.2.1;34.2.1 The EU Regulatory Framework;461
9.3.2.2;34.2.2 The EMA and National Competent Authorities;462
9.3.2.3;34.2.3 New Drug Approval Routes;463
9.3.2.3.1;34.2.3.1 The Centralized Procedure;463
9.3.2.3.2;34.2.3.2 Decentralized Procedure and Mutual Recognition;464
9.3.3;34.3 Regulation in the United States;464
9.3.3.1;34.3.1 CDER and CBER;465
9.3.3.2;34.3.2 The Approvals Procedure;465
9.3.4;34.4 The Advent and Regulation of Biosimilars;466
9.3.5;34.5 International Regulatory Harmonization;467
9.3.6;References;468
9.4;Chapter 35 Emergence of a Biotechnology Industry;471
9.4.1;Reference;477
9.4.2;Further Reading;477
9.5;Chapter 36 The 101 of Founding a Biotech Company;479
9.5.1;36.1 First Steps Toward Your Own Company;479
9.5.2;36.2 Employees: Recruitment, Remuneration, and Participation;482
9.6;Chapter 37 Marketing;485
9.6.1;37.1 Introduction;485
9.6.2;37.2 What Types of Deals Are Possible?;486
9.6.3;37.3 What Milestone or License Fees Are Effectively Paid in a Biotech/Pharma Cooperation?;486
9.6.4;37.4 PR and IR in Biotech Companies;487
9.6.5;Further Reading;488
9.6.6;Websites;488
10;Glossary;489
11;Index;517
12;EULA;518mehr