Authors
Patrizia Gasparini and Omar Malazzi
Summary
Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used for the detection of specific chromosomal rearrangements and applicable to many different specimen types. FISH is widely used for several diagnostic applications: identification of numerical and structural abnormalities characterization of marker chromosomes, monitoring of effects of therapy, detection of minimal residual disease, origin of cells after bone marrow transplantation, identification of regions of deletion or amplification, chromosomes abnormalities in non-dividing or terminally differentiated cells, determination of lineage involvement of clonal cells. Moreover it has many applications in research: identification of non-random chromosome rearrangements, identification of translocation molecular breakpoint, identification of commonly deleted regions, gene mapping, characterization of somatic cells hybrids, identification of amplified genes, study of mechanism of rearrangements, RNA-FISH to study gene expression of 3D chromosome organization in interphase. DNA is a double stranded molecule, and when it is chemically denatured and separated into two strands, it quickly reanneals into a double stranded conformation. Thus, when a single stranded probe is incubated with a single-stranded (denatured) metaphase chromosome (or interphase), the probe will bind to complementary DNA sequences to reform the double stranded molecule. Overall, the most critical step when using FISH is the choice of adequate probes. A DNA probe is defined according to its target or complementary DNA in metaphase and interphase cells: (1) repetitive sequence probe, (2) whole chromosome (painting), (3) locus-specific probes. Technically the ideal probes especially for interphase FISH should give strong, specific signals with no backgrounds and should have a high hybridization efficiency (> 90%).Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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