Genetic Engineering

Genetic Engineering

1st Edition - January 28, 1981

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  • Editor: Robert Williamson
  • eBook ISBN: 9781483214023

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Description

Genetic Engineering 1 is the first of a series containing reviews of particular topics using genetic recombinant DNA techniques. This three-chapter volume describes the construction of libraries of expressed gene sequences, the use of gene-specific probes in antenatal diagnosis, and the expression of isolated genes in cellular and cell-free systems. Chapter 1 presents particular series of steps for the preparation and screening of cDNA clone bank. Chapter 2 discusses the advances in DNA analysis techniques that have profound effects upon the understanding of some genetic diseases and on the ability to reduce the incidence of these diseases by antenatal diagnosis and therapeutic abortion. Chapter 3 considers the experimental systems for studying the expression of isolated eukaryotic genes, concentrating on microinjection into Xenopus oocytes and incubation in cell-free systems in vitro. This book is of great value to genetic engineers, geneticists, and biochemists.

Table of Contents


  • Contributors

    Preface

    The Preparation and Screening of a cDNA Clone Bank

    I Introduction

    A What is a cDNA Clone Bank?

    Β Why is a cDNA Clone Bank Useful?

    C When Might it be Preferable to Use a Genomic Bank?

    D Summary

    II The Preparation of a cDNA Clone Bank

    A General Considerations

    Β The Enzymology of cDNA Cloning

    C The Purification of Messenger RNA

    D The Synthesis of cDNA

    Ε The Synthesis of Double-Stranded cDNA

    F Cleavage of the Hairpin Loop and Removal of Single-Stranded cDNA

    G The Generation of Cohesive Ends on the Double-Stranded cDNA

    Η The Basic Principles of Cloning DNA in Bacterial Plasmids

    I Restriction Enzyme Cleavage of the Vector

    J Generation of Suitable Cohesive Ends on the Vector and the Insertion of Double-Stranded cDNA

    Κ The Relative Merits of Restriction Enzyme Linkers and Homopolymer Tails as Methods for the Insertion of Double-Stranded cDNA

    L Transformation of E. coli and Storage of the cDNA Clones

    III The Screening of a cDNA Clone Bank

    A Primary Screening

    Β Secondary Screening

    IV The Future of cDNA Cloning

    A Potential Methods of Screening of cDNA Clone Bank that do not Require Expression of the Cloned Sequence in E. coli

    Β Potential Methods of Screening a cDNA Clone Bank that do Require Expression of the Cloned Sequence in E. coli

    V References

    DNA Analysis and the Antenatal Diagnosis of Hemoglobinopathies

    I Introduction

    A The Clinical Problem of the Hemoglobinopathies

    II Globin Proteins

    A Problems of Detection in the Fetus

    Β The Inherited Hemoglobinopathies

    C The Antenatal Diagnosis of Hemoglobinopathies — Why DNA?

    III DNA Structural Analysis

    A Techniques

    Β Methodology

    C Timing and Reliability

    D DNA Structures and their Detection

    IV Antenatal Diagnosis by the Direct Detection of Dysfunctional Genes

    A The Detection of Gene Deletions

    Β The Detection of Point Mutations — The Structural Variants

    V Antenatal Diagnosis by the Indirect Detection of Dysfunctional Genes — The Use of Polymorphisms

    A Allele-Specific Linked Polymorphisms

    Β Linked Polymorphisms

    C How Useful are Linked Polymorphisms in Antenatal Diagnosis?

    VI The Application and Future Applications of DNA Analysis in Antenatal Diagnosis

    A Genetic Counselling

    Β The Implementation of the Techniques of DNA Analysis in a Clinical Setting

    C Improvements in the Technique

    D Extension to Other Genes

    VII Conclusion

    VIII Acknowledgements

    IX References

    Expression of Cloned Genes in Cell-Free Systems and in Microinjected Xenopus Oocytes

    I Introduction

    II Transcription of Isolated Genes by RNA Polymerase III

    A Templates and Transcripts

    Β Expression in Microinjected Oocytes

    C Expression in Vitro

    D Transcription Signals Encoded in the DNA Sequence

    Ε Factors Which Confer Transcriptional Specificity

    F In Vitro Analysis of the Developmental Regulation of 5s RNA Synthesis

    G Post-Transcriptional Processing of Polymerase III Transcripts

    III Transcription of Isolated Genes by RNA Polymerase II

    A Templates and Transcripts

    Β Transcription of Isolated Chromatin in Vitro

    C The Endogenous Activities of Isolated Nuclei

    D In Vitro Transcription in Cellular Extracts

    Ε Transcription by Polymerase II in Microinjected Xenopus Oocytes

    F Transcription Signals Encoded in the Template

    G RNA Processing

    Η Prospects for Reconstituting Regulation

    IV Transcription of Isolated Genes by RNA Polymerase I

    A Templates and Transcripts

    Β Transcription of Ribosomal RNA Genes in Isolated Nuclei and Nucleoli

    C Transcription of Isolated Genes by RNA Polymerase I

    D Regulation of Polymerase I Transcription

    V Conclusions and Prospects

    A The Activities of Expression Systems

    Β Analysis of Gene Regulation

    VI Acknowledgements

    VII References

Product details

  • No. of pages: 180
  • Language: English
  • Copyright: © Academic Press 1981
  • Published: January 28, 1981
  • Imprint: Academic Press
  • eBook ISBN: 9781483214023

About the Editor

Robert Williamson

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