1. Milestones of Genetic Engineering - Historical Perspective 1.1 History of Biotechnology 1.2 The Growing Significance of Biotechnology 1.3 Scope and Importance of Genetic Engineering 1.4 Agriculture and Gene Technology 1.5 Plant Transformation 1.6 Marker Assisted Selection 1.7 Challenges in Product Development using Gene Technology 1.8 Gene therapy 1.9 Biotechnology and Intellectual Property Rights 1.10 Advantages of Biotechnologies 2. Molecular Tools and Applications 2.1 Introduction 2.2 Restriction Enzymes 2.2.1 Types of Restriction Enzymes 2.2.2 Nomenclature of Restriction Enzymes 2.2.3 Restriction Enzyme Recognition Sequences 2.2.4 Properties of Restriction Enzymes 2.2.5 Patterns of DNA cutting by Restriction Enzymes 2.3 DNA Ligases 2.4 Polymerases 2.4.1 DNA Dependent DNA Polymerases 2.4.2 RNA Dependent DNA Polymerase 2.4.3 DNA Dependent RNA Polymerase 2.4.4 Bacteriophage SP6 DNA Dependent RNA Polymerase 2.4.5 Bacteriophage T7 and T3 DNA Dependent RNA Polymerase 2.4.6 Template Independent DNA Polymerase (Terminal Transferase) 2.5 Alkaline Phosphatase 3. Gene Cloning Vehicles 3.1 Introduction 3.2 Types of Vectors 3.3 Properties of a Good Vector 3.4 Plasmids 3.4.1 Properties of a Good Plasmid 3.4.2 Nomenclature of Plasmid Vectors 3.4.3 Types of Bacterial Plasmids 3.4.4 Agrobacterium based Plasmid Vectors 3.4.5 Yeast Plasmid Vectors 3.5 Phage 3.5.1 Structure of a Typical Phage 3.5.2 Lambda (l) 3.5.3 Phage M13 3.6 Host 4. Transformation 4.1 Introduction 4.2 Techniques of Introducing DNA 4.2.1 Target Cells for Transformation 4.2.2 Bacterial Transformation 4.3 Selection and Screening of Recombinant Clones 4.3.1 Genetic Methods 4.3.2 Selection of Transformed Cells and Elimination of Non-transformed Cells 4.3.3 Identification of the Clones having Recombinant Vector 4.4 Identification or Selection of Specific Clone 4.4.1 Nucleic Acid Hybridization 4.4.2 Complementation 4.4.3 Immunological Methods 4.4.4 FACS (Fluorescence Activated Cell Sorter) 5. Nucleic Acid Purification 5.1 Introduction 5.1.1 Isolation of Bacterial Chromosomal DNA 5.1.2 Isolation of Eukaryotic DNA 5.1.3 Preparation of Plant Nuclear DNA using CTAB 5.1.4 Universal and Rapid Salt Extraction of High Quality Genomic DNA 5.2 Extraction and Isolation of RNA 5.2.1 Isolation of Plant RNA 5.2.2 Isolation and Preparation of Cytoplasmic RNA from Tissue Culture Cells 5.2.3 Isolation of Total and Polysomal RNA from Plant Tissues 5.2.4 Extraction of Polysomes and Polysomal RNA 5.2.5 Isolation of Bacterial RNA 5.3 Purification of Nucleic Acids 5.3.1 Electro Elution of DNA from Agarose Gel 5.3.2 Biogel Column Purification of DNA 5.3.3 Density Gradient Centrifugation of DNA 5.3.4 Purification of DNA with PEG 5.3.5 Purification of DNA with Silica Beads 5.4 Purification of RNA 5.4.1 Extraction and Fractionation of RNA 5.4.2 Purification of mRNA 5.4.3 Purification of mRNA using Oligo(dT), Immobilized on Magnetic Beads 5.5 Yield and Yield Analyses of Nucleic Acids 5.6 Isolation of Plasmid DNA 5.6.1 Isolation of Plasmid DNA from Bacteria (E. coli) - Alkaline Lysis “Miniprep” Method 5.6.2 Isolation of Plasmid - Large Scale Preparation 5.6.3 Purification of Plasmid DNA by Precipitation with PEG 5.6.4 Characterization of Plasmid 6. DNA Sequencing Techniques 6.1 Introduction 6.2 Maxam and Gilbert’s Chemical Degradation Method 6.3 Dideoxynucleotide Chain Termination Method of DNA Sequencing 6.4 Automated DNA Sequencing 7. Restriction Enzyme Digestion and Restriction Mapping 7.1 Introduction 7.2 Restriction Enzyme Digestion 7.3 Restriction Mapping 7.4 Southern Blotting and Analysis 7.5 Autoradiography 7.6 Northern Blotting 7.7 Western Blotting 8. Genomic Libraries and Screening of Recombinants 8.1 Introduction 8.2 Screening of Recombinants 8.3 In Situ Hybridization 8.3.1 Protocol for In Situ Hybridization 8.3.2 Probe Labelling and Detection 8.3.3 Preparation of the Specimen / Biological Material 8.3.4 Spreading of Cells 8.3.5 Pretreatment of the Material 8.3.6 Denaturation 8.3.7 Hybridization 8.3.8 Post- Hybridization Washing 8.3.9 Detection of the ISH Signals 9. Gene Manipulations by Site-directed Mutagenesis - PCR Technology 9.1 Introduction 9.2 Basic Mechanism of Site-directed Mutagenesis 9.3 General Approach 9.3.1 Changing the End of a PCR Fragment 9.3.2 Changing the Middle of a Sequence by Two Consecutive Reactions; the 4 Oligo Method 9.4 PCR-based In Vitro Mutagenesis 9.4.1 Megaprimer PCR Method 9.4.2 Overlap Extension Method of Site Directed Mutagenesis 9.5 Applications of Site-directed Mutagenesis 10. cDNA Library and Reverse Transcription 10.1 Introduction 10.2 cDNA Library Construction 10.2.1 Isolation and Purification of mRNA 10.2.2 Reverse Transcription 10.2.3 Cloning of cDNA 10.3 Comparison between cDNA Library and Genomic Library 11. Genome Mapping 11.1 Introduction 11.2 Genome Maps 11.3 Molecular Markers 11.3.1 RFLP: Restriction Fragment Length Polymorphism 11.3.2 RAPD: Rapid Amplified Polymorphic DNAs 11.3.3 VNTRs: Variable Number of Tandem Repeats 11.3.4 CpG Islands 11.4 Techniques for Genome Mapping 11.4.1 Chromosome Jumping and Chromosome Walking 11.4.2 FISH - Fluorescence In Situ Hybridization 11.4.3 Microsatellite Mapping 11.5 DNA Fingerprinting 11.6 Uses of DNA Fingerprinting 12. Applications of Genetic Engineering 12.1 Introduction 12.2 Application of Genetic Engineering in Animals 12.2.1 Uses of Recombinant DNA Technology in Animal Research 12.2.2 Production of Useful Proteins 12.2.3 Genetic Engineering with Animal Viruses 12.2.4 Diagnosis of Hereditary Diseases 12.2.5 Genetic Engineering in Medicine 12.2.6 Bovine Somatotrophin (BST) 12.2.7 Rennin 12.3 Application of Genetic Engineering in Plants 12.3.1 Transgenic Plants 12.3.2 Improvement of Photosynthetic Efficiency of Plants 12.3.3 Resistance to Disease 12.3.4 Resistance to Insect 12.3.5 Resistance to Herbicide 12.3.6 Improving Plant Nutritional Value 12.3.7 Transgenic Plants for Edible Vaccines 12.3.8 Stress and Senescence Resistance Plants 12.4 Applications of Genetic Engineering in Microbes 12.5 Guidelines on the Release of Agriculture-related Genetically Modified Organisms (GMOS)
Recombinant Dna Technology
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