Detail fundamental molecular biology methods and recombinant protein engineering techniques, which provides students with a solid scientific basis for the rest of the book. Present the processes of molecular biotechnology and its successes in medicine, bioremediation, raw material production, biofuels, and agriculture. Examine the intersection of molecular biotechnology and society, including regulation, patents, and controversies around genetically modified products. Filled with engaging figures that strongly support the explanations in the text, Molecular Biotechnology: Principles and Applications of Recombinant DNA presents difficult scientific concepts and technically challenging methods in clear, crisp prose. This excellent textbook is ideal for undergraduate and graduate courses in introductory biotechnology, as well as, courses dedicated to medical, agricultural, environmental, and industrial biotechnology applications.
Preface to the Fifth Edition xvii 1 The Development of Molecular Biotechnology 1 (10) Emergence of Molecular Biotechnology 1 (2) Recombinant DNA Technology 3 (1) Commercialization of Molecular Biotechnology 4 (3) Concerns and Consequences 7 (2) Summary 9 (1) References 9 (1) Review Questions 10 (1) 2 Fundamental Technologies 11 (82) Molecular Cloning 11 (21) Preparation of DNA for Cloning 11 (5) Insertion of Target DNA into a Plasmid Vector 16 (4) Transformation and Selection of Cloned DNA in a Bacterial Host 20 (4) Cloning Eukaryotic Genes 24 (4) Recombinational Cloning 28 (2) Genomic Libraries 30 (2) Genome Engineering using CRISPR Technology 32 (3) Polymerase Chain Reaction 35 (7) Amplification of DNA by PCR 36 (3) Cloning PCR Products 39 (1) Quantitative PCR 39 (3) Chemical Synthesis of DNA 42 (8) Synthesis of Oligonucleotides 42 (6) Assembling Oligonucleotides into Genes 48 (2) Gene Synthesis by PCR 50 (1) DNA Sequencing Technologies 50 (9) Dideoxynucleotide Procedure 53 (2) Pyrosequencing 55 (2) Sequencing using Reversible Chain Terminators 57 (1) Sequencing by Single Molecule Synthesis 57 (2) Sequencing Whole Genomes 59 (5) Preparation of Genomic DNA Sequencing Libraries 60 (1) High-Throughput Next-Generation Sequencing Strategies 61 (2) Genome Sequence Assembly 63 (1) Sequencing Metagenomes 64 (1) Genomics 64 (23) Transcriptomics 67 (5) Proteomics 72 (13) Metabolomics 85 (2) Summary 87 (2) References 89 (2) Review Questions 91 (2) 3 Production of Recombinant Proteins 93 (84) Protein Production in Prokaryotic Hosts 93 (27) Regulation of Transcription 94 (4) Increasing Translation Efficiency 98 (4) Increasing Protein Stability 102 (4) Increasing Protein Secretion 106 (4) Facilitating Protein Purification 110 (5) DNA Integration into the Host Chromosome 115 (5) Heterologous Protein Production in Eukaryotic Cells 120 (33) Posttranslational Modification of Eukaryotic Proteins 120 (2) General Features of Eukaryotic Expression Systems 122 (2) Yeast Expression Systems 124 (12) Baculovirus-Insect Cell Expression Systems 136 (7) Mammalian Cell Expression Systems 143 (10) Protein Engineering 153 (18) Directed Mutagenesis 154 (4) Random Mutagenesis 158 (4) Examples of Protein Engineering 162 (9) Summary 171 (2) References 173 (2) Review Questions 175 (2) 4 Molecular Diagnostics 177 (52) Immunological Approaches to Detect Protein Biomarkers 178 (15) Antibodies 178 (5) Agglutination 183 (1) Enzyme-Linked Immunosorbent Assays 183 (6) Protein Arrays to Detect Polygenic Diseases 189 (2) Immunoassays for Protein Conformation-Specific Disorders 191 (2) DNA-Based Diagnostic Approaches 193 (22) Hybridization Probes 193 (7) PCR-Based Detection Methods 200 (8) DNA Microarrays 208 (6) Whole Genome Sequencing to Assess Genetic Disease Risk 214 (1) Detecting RNA Signatures of Disease 215 (4) Detection of Disease-Associated Changes in Gene Expression Using Microarrays 215 (1) Detection of RNA Signatures of Antibiotic Resistance in Bacteria 216 (1) Detection of miRNA Signatures of Cancers 217 (2) Biofluorescent and Bioluminescent Systems 219 (5) Fluorescent Proteins 219 (2) Luciferase 221 (1) Microbial Biosensors 222 (2) Summary 224 (1) References 225 (2) Review Questions 227 (2) 5 Protein Therapeutics 229 (68) Pharmaceuticals 230 (10) Human Interferons 231 (4) Human Growth Hormone 235 (2) Tumor Necrosis Factor Alpha 237 (1) Extending Protein Half-Life 238 (2) Enzymes 240 (15) DNase I 240 (2) Alginate Lyase 242 (3) Phenylalanine Ammonia Lyase 245 (2) alpha1-Antitrypsin 247 (1) Glycosidases 248 (1) Masking Nonhuman Epitopes 249 (1) Engineering Bacteriophages 250 (3) Targeting Mitochondria 253 (2) Lactic Acid Bacteria 255 (6) Interleukin-10 255 (3) Leptin 258 (1) An HIV Inhibitor 258 (2) Insulin 260 (1) Recombinant Antibodies 261 (31) Hybrid Human-Mouse Monoclonal Antibodies 264 (4) Human Monoclonal Antibodies 268 (2) Antibody Fragments 270 (4) Combinatorial Libraries of Antibody Fragments 274 (3) A Combinatorial Library of Full-Length Antibodies 277 (1) Shuffling CDR Sequences 278 (2) Dual-Variable-Domain Antibodies 280 (1) Anticancer Antibodies 281 (3) Antibodies Against Various Diseases 284 (3) Antiobesity Antibodies 287 (3) Enhanced Antibody Half-Life 290 (2) Summary 292 (1) References 292 (3) Review Questions 295 (2) 6 Nucleic Acids as Therapeutic Agents 297 (46) Targeting Specific mRNA and DNA Sequences 299 (20) Antisense RNA 299 (3) Aptamers 302 (5) Ribozymes and DNAzymes 307 (4) Interfering RNA 311 (4) Zinc Finger Nucleases 315 (2) CRISPR-Cas System 317 (1) Nanozymes 318 (1) Nanoparticles 319 (1) Viral Delivery Systems 319 (6) Nonviral Delivery Systems 325 (10) Direct Injection 325 (2) Lipids 327 (1) Bacteria 328 (3) Dendrimers 331 (1) Antibodies 332 (1) Aptamers 332 (2) Transposons 334 (1) Gene Therapy 335 (3) Prodrug Activation Therapy 335 (2) Promoterless Gene Targeting 337 (1) Summary 338 (1) References 339 (2) Review Questions 341 (2) 7 Vaccines 343 (60) Vaccination 343 (2) Current and Future Vaccines 345 (2) Subunit Vaccines 347 (12) Herpes Simplex Virus 348 (2) Cholera 350 (1) SARS 350 (1) Staphylococcus aureus 351 (2) Human Papillomavirus 353 (1) Foot-and-Mouth Virus 354 (2) Streptococcus 356 (1) Delivery 357 (2) Peptide Vaccines 359 (4) Malaria 359 (4) Genetic Immunization: DNA Vaccines 363 (9) Delivery 364 (6) Cancer 370 (1) Dental Caries 370 (2) Attenuated Vaccines 372 (6) Herpes Simplex Virus 372 (2) Cholera 374 (1) Salmonella Species 375 (3) Leishmania Species 378 (1) Vector Vaccines 378 (18) Vaccines Directed against Viruses 378 (10) Vaccines Directed against Bacteria 388 (4) Bacteria as Antigen Delivery Systems 392 (4) Monoclonal Antibody Passive Immunity 396 (1) Influenza Virus 396 (1) Summary 397 (1) References 398 (2) Review Questions 400 (3) 8 Industrial and Environmental Uses of Recombinant Microorganisms 403 (72) Restriction Endonucleases 403 (2) Small Biological Molecules 405 (29) L-Ascorbic Acid 407 (3) Indigo 410 (2) Amino Acids 412 (5) Lycopene 417 (1) Antibiotics 418 (11) Biopolymers 429 (5) Microbial Degradation of Xenobiotics 434 (11) Genetic Engineering of Biodegradative Pathways 436 (9) Utilization of Starch and Sugars 445 (9) Commercial Production of Fructose and Alcohol 446 (2) Increasing Alcohol Production 448 (5) Improving Fructose Production 453 (1) Utilization of Cellulose and Hemicellulose 454 (13) Lignocellulosics 455 (2) Cellulase Genes 457 (5) Direct Conversion of Biomass to Ethanol 462 (2) Zymomonas mobilis 464 (3) Lipids from Cyanobacteria 467 (1) Hydrogen Production 468 (2) Summary 470 (1) References 471 (3) Review Questions 474 (1) 9 Large-Scale Production of Proteins from Recombinant Microorganisms 475 (40) Principles of Microbial Growth 476 (5) Batch Fermentation 477 (2) Fed-Batch Fermentation 479 (1) Continuous Fermentation 480 (1) Maximizing The Efficiency of The Fermentation Process 481 (10) High-Density Cell Cultures 483 (1) Increasing Plasmid Stability 484 (1) Quiescent E. Coli Cells 485 (1) Protein Secretion 486 (3) Reducing Acetate 489 (2) Bioreactors 491 (3) Typical Large-Scale Fermentation Systems 494 (7) Two-Stage Fermentation in Tandem Airlift Reactors 495 (1) Two-Stage Fermentation in a Single Stirred-Tank Reactor 496 (2) Batch versus Fed-Batch Fermentation 498 (3) Harvesting Microbial Cells 501 (1) Disrupting Microbial Cells 502 (2) Downstream Processing 504 (4) Protein Solubilization 506 (1) Utilizing an Immobilized Enzyme 507 (1) Magnetic Separation of Proteins 507 (1) Large-Scale Production of Plasmid DNA 508 (3) Summary 511 (1) References 512 (2) Review Questions 514 (1) 10 Genetic Engineering of Plants: Methodology 515 (36) Plant Transformation with the Ti Plasmid of A. Tumefaciens 516 (6) Ti Plasmid-Derived Vector Systems 522 (4) Microprojectile Bombardment 526 (1) Chloroplast Engineering 527 (5) Very High Level Protein Expression 529 (3) Use of Reporter Genes in Transformed Plant Cells 532 (1) Manipulation of Gene Expression in Plants 533 (9) Transient Gene Expression 533 (3) Plant Promoters 536 (2) Targeted Gene Editing 538 (1) Facilitating Protein Purification 539 (2) Protein Glycosylation 541 (1) Production of Marker-Free Transgenic Plants 542 (4) Removing Marker Genes from Nuclear DNA 543 (2) Removing Marker Genes from Chloroplast DNA 545 (1) Summary 546 (1) References 547 (2) Review Questions 549 (2) 11 Transgenic Plants 551 (74) Insect Resistance 551 (19) B. thuringiensis Insecticidal Toxin 551 (4) Increasing Expression of the B. thuringiensis Protoxin 555 (3) Other Strategies for Protecting Plants against Insects 558 (6) Preventing the Development of B. thuringiensis-Resistant Insects 564 (5) Targeting Aphids 569 (1) Virus Resistance 570 (8) Viral Coat Protein-Mediated Protection 570 (4) Protection by Expression of Other Genes 574 (4) Herbicide Resistance 578 (5) Fungus and Bacterium Resistance 583 (5) Salt and Drought Stress 588 (4) Fruit Ripening and Flower Wilting 592 (2) Modification of Plant Nutritional Content 594 (9) Amino Acids 594 (1) Lipids 595 (4) Vitamins 599 (2) Iron 601 (1) Gluten 602 (1) Modification of Food Plant Taste and Appearance 603 (5) Preventing Discoloration 603 (2) Starch 605 (3) Plants as Bioreactors 608 (3) Antibodies 608 (2) Poly(3-hydroxybutyric Acid) 610 (1) Eddible Vaccines 611 (4) Plant Yield 615 (4) Altering Lignin Content 615 (3) Increasing Oxygen Content 618 (1) Summary 619 (1) References 620 (4) Review Questions 624 (1) 12 Transgenic Animals 625 (54) Transgenic Mice: Methodology 626 (18) DNA Microinjection Method 627 (2) Retroviral Vector Method 629 (2) Engineered Embryonic Stem Cell Method 631 (6) Conditional Gene Inactivation with the Cre-IoxP Recombination System 637 (4) Genome Editing with the CRISPR-Cas System 641 (2) Gene Knockdown by RNA Interference 643 (1) Transgenic Mice: Applications 644 (12) Transgenic Disease Models: Alzheimer Disease 644 (3) Transgenic Mice as Test Systems 647 (4) Control of Transgene Expression 651 (3) Conditional Control of Cell Death 654 (2) Transgenic Livestock 656 (13) Cloning Livestock by Somatic Cell Nuclear Transfer 656 (2) Production of Pharmaceuticals 658 (2) Production of Donor Organs 660 (1) Disease Resistant Livestock 661 (3) Improving Milk Quality 664 (1) Improving Animal Production Traits 665 (4) Transgenic Poultry 669 (4) Transgenic Fish 673 (3) Summary 676 (1) References 676 (2) Review Questions 678 (1) 13 Molecular Biotechnology and Society 679 (46) Development of Guidelines for Recombinant DNA Research 680 (2) Deliberate Release of Genetically Modified Microorganisms 682 (2) Environmental Concerns 682 (1) Regulations 683 (1) Regulation of Genetically Modified Foods 684 (8) Food Ingredients Produced by Genetically Engineered Microorganisms 684 (3) Genetically Modified Crops 687 (4) Genetically Engineered Livestock 691 (1) Societal Concerns About Genetically Modified Foods 692 (14) Alteration of Nutritional Content of Food 692 (4) Potential for Introducing Toxins or Allergens into Food 696 (2) Potential for Transferring Transgenes from Food to Humans or Intestinal Microorganisms 698 (2) Controversy About the Labeling of Genetically Modified Foods 700 (1) Impact of Genetically Engineered Crops on Biodiversity 700 (3) Who Benefits from Production of Genetically Modified Foods? 703 (1) Environmental Benefits of Genetically Modified Crops 704 (1) How do Views about Genetically Engineered Organisms Impact Trade? 705 (1) Regulation and Safety of Medical Products of Biotechnology 706 (8) New Biological Drugs 706 (3) Genetic and Genomic Testing 709 (2) Economic Issues 711 (3) Patenting Biotechnology 714 (7) Patenting 714 (2) Patenting in Different Countries 716 (1) Patenting Nucleic Acid Sequences 717 (2) Patenting Living Organisms 719 (1) Patenting and Fundamental Research 720 (1) Summary 721 (1) References 722 (2) Review Questions 724 (1) Amino Acids of Proteins and Their Designations 725 (2) Index 727