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About the book

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)

Academic Details

  • Phase Graduation

  • Stream Science

  • Branch Biotechnology

  • Standard/Year Thirdyear

  • Semester Iv

  • Medium English

  • Board/University Pune

  • Subject Recombinant Dna Technology


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