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Chromosome microdissection and cloning : a practical guide

Author: Nabil G Hagag; Michael V Viola
Publisher: San Diego : Academic Press, 1993.
Edition/Format:   Print book : EnglishView all editions and formats
Summary:

This manual has been designed to aid the novice, as well as the experienced researcher, in setting up laboratories from "scratch" for the study of genetic abnormalities and chromosome aberrations.  Read more...

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Genre/Form: Laboratory Manuals
Additional Physical Format: Online version:
Chromosome microdissection and cloning.
San Diego : Academic Press, 1993
(OCoLC)643888121
Material Type: Internet resource
Document Type: Book, Internet Resource
All Authors / Contributors: Nabil G Hagag; Michael V Viola
ISBN: 0123133203 9780123133205
OCLC Number: 27816787
Description: x, 160 pages : illustrations ; 24 cm
Contents: Introduction to chromosome microdissection --
Chromosome organization --
Cloning DNA from chromosome fragments --
Preparation of chromosomes for microdissection --
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 --
Methods of chromosome microdissection --
Methods --
Video microscope method --
Oil chamber method --
Laser microdissection method --
Summary of chromosome microdissection and collection for DNA cloning --
Reagents and equipment --
Protocol. (cont) Molecular cloning of microdissected chromosal DNA --
Cloning of DNA from microdissected chromosomal DNA fragments --
Method 1. Direct cloning of DNA from microdissected chromosomal fragments --
Direct cloning into [gamma] phage --
Method 2. Ligation of microdissected chromosomal DNA with plasmid vector or linker-adaptor and PCR amplification --
Protocol 2.1. Ligation of microdissected DNA with plasmid vector, PCR amplification, and cloning --
Protocol 2.2. Ligation of microdissected DNA with linker-adaptor, PCR amplification, and cloning --
Method 3. PCR amplification of microdissected chromosomal DNA fragments followed by probing a complete recombinant library --
Protocol 3.1. Preparation of chromosomal DNA for amplification --
Protocol 3.2. PCR amplification of microdissected chromosomal DNA using "universal" primers --
Protocol 3.3. PCR amplification using human Alu sequence-based primers --
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. (cont) Protocol 4.1. Assay for repeat sequences --
Protocol 4.2. Assay for 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 --
Protocol 5.1. DNA probe labeling for fluorescence in situ hybridization --
Protocol 5.2. Fluorescent in situ hybridization to metaphase chromosome spreads --
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.
Responsibility: [edited by] Nabil G. Hagig and Michael V. Viola.

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