Cover
Contents
1. Introduction to Gene Cloning
Introduction to Gene Cloning and Genetic Engineering
What is Gene Cloning?
Gene Cloning and PCR
2. Salient Features of Cloning Vectors
Essential Components of Gene Cloning
Conjugative and Nonconjugative Plasmids
Commercially Available Plasmids
Cosmids
Preparation of Genomic DNA Libraries in Cosmid Vectors
ssDNA Phages
Filamentous Phage
Phagemid Vectors
Yeast Cloning Vectors
Cloning Vectors for Higher Organisms
Direct Transfer or Integration Vectors
3. Plasmid Biology
Structural and Functional Organisation of Plasmids
Characteristics of Plasmids
Plasmid Replication
Plasmids Carrying Conjugation or Fertility Factors
Different Types of Plasmids
4. Enzymes Used in Genetic Engineering
Introduction
DNA Polymerase
Reverse Transcriptase
Terminal Deoxynucleotidyl Transferase
RNA Polymerases
Addition of Phosphate Group/Kinasing
Dephosphorelation by Alkaline Phosphatase
Ligases
Restriction Endonuclease I and II
Nick Translation
Probe Synthesis from Phagemid
5. Isolation of Genomic and Nuclear DNA
Introduction
DNA SequencingβTo Understand the Structure of a Gene
Sequencing Gel
PCR Product Sequencing
6. Cloning and Subcloning Strategy
Construction of Recombinant DNA
Diagrammatic Representation of Different Cloning Technologies
Preparation of Competent Cell-Transformation, Transfection
Insertional Inactivation (The Other Way of Screening the Clones)
Transformation of Double-Stranded DNA
Recombinant Selection and Screening
Essential Tools Required for Cloning/Subcloning
7. Selection of rDNA Clones and Their Expression Products
Introduction
Selection
Analysis of cDNA Library
Preparation of Polyclonal Antibody
ELISA (Enzyme Linked Immunosorbent Assay)
Western Blotting
SDS β PAGE
Hybridisation
Basic Mechanism of Hybridisation
8. DNA Technology
Mutagenesis
Oligonucleotide-based Mutagenesis
PCR-based Site Directed Mutagenesis
Polymerase Chain Reaction (PCR)
Applications of rDNA Technology in Diagnostics
rDNA Technology and Forensics
DNA Fingerprinting for Sex Determination
9. The Application of Gene Cloning and DNA Analysis
Production of Protein from Cloned Genes
Cloning and Expression Vectors for System
E. Coli Promoters for Gene Expression and Protein Production
Recombinant Protein Production in : General Problems
Eukaryotes for Protein Expression and Production
Recombinant Protein Production in Pichia Pastoris, Saccharomyces Cerevisiae and Kluveromyces Lactis
Filamentous Fungi for Protein Expression and Production
Gene Cloning and DNA Analysis in Medicine
Eukaryotic Gene Synthesis and Expression in
Synthesis, Cloning and Expression of other Recombinant Proteins with Therapeutic Importance
Gene Therapy
Gene Subtraction/Antisense Technology
Glossary
Index