Chromosome Microdissection and Cloning

A Practical Guide

1st Edition - August 26, 1993
Author: Nabil Hagag
Language: English
eBook ISBN:
9 7 8 - 0 - 3 2 3 - 1 6 0 7 7 - 3

Chromosome Microdissection and Cloning: A Practical Guide is a straightforward guide to chromosome microdissection and cloning. It presents an overview of the procedures and… Read more

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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.

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