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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.
Chapter 1 Introduction to Chromosome Microdissection
Cloning DNA from Chromosome Fragments
Chapter 2 Preparation of Chromosomes for Microdissection
Critical Aspects of Chromosome Preparation
Enrichment of Metaphase Spreads
Chromosome Fixing and Spreading
Aging, Storing, and Staining of Metaphase Spreads
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
Chapter 3 Methods of Chromosome Microdissection
Video Microscope Method
Oil Chamber Method
Laser Microdissection Method
Summary of Chromosome Microdissection and Collection for DNA Cloning
Reagents and Equipment
Chapter 4 Molecular Cloning of Microdissected Chromosomal DNA
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
Chapter 5 Applications of Chromosome Microdissection
Direct Analysis of the PCR Product of Microdissected Chromosome Fragments
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
Appendix: List of Suppliers and Addresses
- No. of pages:
- © Academic Press 1993
- 26th August 1993
- Academic Press
- eBook ISBN:
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