Recombinant DNA Technology

Recombinant DNA technology is a branch of molecular biology that deals with the joining of DNA molecules from two different sources and inserted into a host organism to produce a number of copies of the new genetic combinations which find applications in science, medicine, agriculture, and industry. This technology paved the way to isolate a gene, determine the nucleotide sequences, study gene transcription and protein expression, mutate genes specifically apart from many other applications. Recombinant DNA technology is not merely a collection of techniques but also an evolving science that gives us an insight into living organisms at the molecular level. The technology developed using the principles has given birth to many new streams, genomics, transcriptomics, system biology, and so on.

While teaching and taking interviews, the author came across the following statement several times: "Recombinant DNA technology is a mere technique; how would you address scientific problems with this!" This statement is the inspiration behind writing this book. The primary purpose of Recombinant DNA Technology is to make the current state of knowledge on the principles of recombinant DNA technology and its applications understandable to students, teachers, and scientists. With this book, the author hopes that students will be able to readily grasp the fundamental principles and themes presented in several chapters as well as practical applications of those themes, which have been illustrated in later chapters.

1. Recombinant DNA Technology

Introduction

Importance of Recombinant DNA

Basic Procedure of Recombinant DNA Technology

Impact of Recombinant DNA

Summary

Revision Questions


2. Methods for Creating Recombinant DNA Molecules

Introduction

Sources of DNA for Cloning

Selection of Host and Vector

Preparation of Vector and Insert DNA

Joining of DNA Molecules

Propagation of Plasmids Carrying Foreign DNA Molecules

Summary

Review Questions


3. Properties of Restriction Endonucleases

Introduction

Naming Restriction Enzymes

Types of Restriction Enzymes

Endonucleases Producing Compatible Ends

Recognition Sequence

Cleavage Pattern

Restriction Mapping

Mapping by Double Digestion

Mapping by Partial Digestion

Summary

Revision Questions


4. Screening of Recombinant DNA Molecules

Introduction

Identifying Recombinant Plasmids by Alpha(a)-complementation

Identifying Recombinant Plasmids by Hybridization

Screening by Polymerase Chain Reaction

Summary

Review Questions


5. Construction of DNA Library

Introduction

Genomic Library

Choice of Vectors

cDNA Library

Vectors Used for Cloning cDNA

Amplified DNA Libraries

Screening of Libraries

Genomic DNA Library Versus cDNA Library

Summary

Revision Questions


6. Sequencing by Sanger's Method

Introduction

Chemical DNA Sequencing Method (Maxam–Gilbert)

Principle of Sanger's Method

Procedure of Sanger's Method

Modifications

Automated DNA Sequencing

Capillary Electrophoresis

Advantages of Automated DNA Sequencing over Manual DNA Sequencing

Summary

Review Questions


7. Protein Production in Bacteria

E. coli Protein Expression System

General Characteristics of the Expression Vector Used in E. coli

Optimization of Expression Levels in E. coli Expression System

Promoter Sequences

Optimization of Other Factors Influencing Protein Expression

Strategies for Improving Protein Solubility

Improving Protein Stability

Reducing Protein Toxicity

In Vitro Expression Using E. coli Extracts

Co-expression

Protein Purification

Summary

Revision Questions


8. Site-directed Mutagenesis

Introduction

Site-directed Mutagenesis Using Single-stranded DNA

Considerations for Design of Mutagenic Oligonucleotides

Summary

Revision Questions


9. Restriction Fragment Length Polymorphism

Introduction

Basic Procedure of RFLP

Advantages and Disadvantages

Applications

Summary

Revision Questions


10. Polymerase Chain Reaction

Introduction

The Basic Reaction Method

Essential Components of PCR

Applications of PCR

Protein Expression Systems

Revision Questions


11. DNA Fingerprinting

Introduction

Illustrative Example of DNA Fingerprinting

The Procedure

Applications

Summary

Revision Questions


12. RNAi and siRNA Technology

Introduction

Discovery of RNAi

Mechanism of RNAi

siRNA Technology

Applications of RNAi–siRNA Technology

Summary

Revision Questions


13. Molecular Biology Methods

Introduction

Joining DNA Fragments with Cohesive Ends

Cloning Blunt-ended DNA Fragments

Cloning of PCR Products (TA Cloning)

Summary

Revision questions


14. Features of Commonly Used Vectors

Introduction

Plasmids as Cloning Vectors

Desirable Properties of Plasmid Cloning Vectors

Plasmid Vectors pBR322 and pUC18

Phage Vectors

M13 Vectors

Cosmid Vectors

Artificial Chromosome Vectors

Revision Questions


15. Isolation and Purification of Plasmid Vectors

Introduction

The Classical CsCl–EtBr Method

Alkaline Lysis Method

Summary

Revision Questions


16. Cloning in Cosmid Vectors

Introduction

Cosmids

Cloning Procedure

Drawbacks and Modifications of Cosmid Vectors

Summary

Revision Questions


17. Construction of Genomic DNA Libraries in Cosmid Vectors

Introduction

Cloning into Single Cos Site Vectors (pJB8)

Cloning into Double Cos Site Vectors (SuperCos-1)

Summary

Revision Questions


18. Enzymes Used in Molecular Cloning

Introduction

E. coli DNA Polymerase I (Holoenzyme)

Klenow Fragment of DNA Polymerase I

T4 DNA Polymerase

T7 DNA Polymerase

Thermostable DNA-dependent DNA Polymerases

Terminal Deoxynucleotidyl Transferase

Reverse Transcriptase

Phage RNA Polymerases: T7, T3, SP6

Poly(A) Polymerase

Alkaline Phosphatases: Bacterial Alkaline Phosphatase and

Calf Intestine Phosphatase

T4 Polynucleotide Kinase

Exonucleases

Endonucleases

S1 Nuclease

Restriction Enzymes: Digestion of DNA with Restriction Enzymes

Summary

Revision Questions


19. Agarose Gel and Polyacrylamide Gel Electrophoresis

Introduction

Principle

Agarose Gel Electrophoresis

Factors Affecting the Rate of Migration of DNA through Agarose Gel

Electrophoresis Buffer

Pulsed Field Gel Electrophoresis

Polyacrylamide Gel Electrophoresis for Small DNA Molecules

Summary

Revision Questions


20. Detection and Extraction of DNA from Gels

Introduction

Staining DNA Gels Using Ethidium Bromide

Detection of DNA in Gels Containing SYBR Gold

Photography of DNA in Gels

Extraction of DNA from Gels

Extraction of DNA (DNA Recovery)

Summary


Revision Questions

Bibliography

Glossary

Colour Plates

Index

About the Author