Recombinant DNA technology and genetic engineering

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 Sequencing–To 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 – 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