Chromosome Microdissection and Cloning

Chromosome Microdissection and Cloning

A Practical Guide

1st Edition - August 26, 1993

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  • Author: Nabil Hagag
  • eBook ISBN: 9780323160773

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Description

Chromosome Microdissection and Cloning: A Practical Guide is a straightforward guide to chromosome microdissection and cloning. It presents an overview of the procedures and briefly reviews a few areas of research in which these techniques are applied. Topics range from preparation of chromosomes for microdissection to molecular cloning of microdissected chromosomal DNA. Methods of chromosome microdissection, including video microscope method and oil chamber method, are described. Comprised of five chapters, this book begins with an overview of the structure and organization of chromosomes, followed by a description of methods for preparing and preserving chromosomal DNA in a manner that is useful for cloning and direct analysis. Microdissection of metaphase chromosomes and isolation of fragments can be accomplished in one of the three ways described in the next chapter: by microdissection using an upright microscope and glass capillaries in an oil chamber; by laser microbeam; and with the use of an inverted microscope equipped with a video camera and high magnification-high resolution lenses. A step-by-step guide to these techniques and solutions for common problems are given following each method. Protocols for cloning and identifying genetic sequences from defined chromosome regions, particularly using the polymerase chain reaction, are also discussed. The final chapter focuses on applications of chromosome microdissection, such as cloning of disease-specific genes and generating "sequence tagged sites" to be used in large DNA sequencing projects. This monograph will be particularly helpful to investigators setting up microdissection systems de novo.

Table of Contents


  • Preface

    Chapter 1 Introduction to Chromosome Microdissection

    Chromosome Organization

    Cloning DNA from Chromosome Fragments

    References

    Chapter 2 Preparation of Chromosomes for Microdissection

    Introduction

    Critical Aspects of Chromosome Preparation

    Enrichment of Metaphase Spreads

    Hypotonic Treatment

    Chromosome Fixing and Spreading

    Aging, Storing, and Staining of Metaphase Spreads

    Reagents

    Equipment

    Protocols

    Protocol 1. Preparation of Chromosomes from Peripheral Blood T Lymphocytes (Whole Blood Microculture Method)

    Protocol 2. Preparation of Chromosomes from Monolayer Tissue Culture Cell Lines

    Protocol 3. Preparation of Chromosomes from Monolayer Cells Grown on Coverslips

    Protocol 4. Preparation of Chromosomes from Dipteran Salivary Glands

    Protocol 5. Solid Staining and GTG Banding of Metaphase Chromosomes

    References

    Chapter 3 Methods of Chromosome Microdissection

    Introduction

    Methods

    Video Microscope Method

    Oil Chamber Method

    Laser Microdissection Method

    Summary of Chromosome Microdissection and Collection for DNA Cloning

    Reagents and Equipment

    Protocol

    References

    Chapter 4 Molecular Cloning of Microdissected Chromosomal DNA

    Introduction

    Cloning DNA from Microdissected Chromosomal DNA Fragments

    Method 1. Direct Cloning of DNA from Microdissected Chromosomal Fragments

    Method 2. Ligation of Microdissected Chromosomal DNA with Plasmid Vector or Linker-Adaptor and PCR Amplification

    Method 3. PCR Amplification of Microdissected Chromosomal DNA Fragments Followed by Probing a Complete Recombinant Library

    Analysis of Recombinant Clones Derived from Microdissected Chromosomal DNA

    Determination of DNA Insert Size Range

    Determination of the Percentage of Recombinant Clones Containing Repeat and Unique Sequences

    Calculation of the Percentage of Total Microdissected DNA Cloned

    Determination of Potential Structural Gene Sequences

    Localization of Recombinant Clones Using in Situ Hybridization

    References

    Chapter 5 Applications of Chromosome Microdissection

    Direct Analysis of the PCR Product of Microdissected Chromosome Fragments

    Gene Mapping

    Mapping Sites of Chromosome Rearrangement and Deletions

    Determination of Coupling Phase

    Recombinant DNA Libraries Generated from Microdissected Chromosome Fragments

    Genetic Analysis of Specialized Chromosome Structures

    Applications in Genomic Sequencing Projects

    Characterization of Disease-Related Genetic Loci

    Study of Chromosome Abnormalities in Cancer Cells

    Gene Transfer Using Chromosome Fragments

    References

    Appendix: List of Suppliers and Addresses

    Glossary

    Index

Product details

  • No. of pages: 170
  • Language: English
  • Copyright: © Academic Press 1993
  • Published: August 26, 1993
  • Imprint: Academic Press
  • eBook ISBN: 9780323160773

About the Author

Nabil Hagag

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