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Chromosome microdissection and cloning : a practical guide /
| Clasificación: | Libro Electrónico |
|---|---|
| Otros Autores: | , |
| Formato: | Electrónico eBook |
| Idioma: | Inglés |
| Publicado: |
San Diego :
Academic Press,
1993.
|
| Temas: | |
| Acceso en línea: | Texto completo |
Tabla de Contenidos:
- 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.