What is FISH?
FISH is widely used for several diagnostic applications: identification of numerical and structural abnormalities, characterization of marker chromosomes, monitoring the effects of therapy, detection of minimal residual disease, tracking the origin of cells after bone marrow transplantation, identification of regions of deletion or amplification, detection of chromosome 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 the mechanism of rearrangements. FISH is also used to compare the genomes of two biological species to deduce evolutionary relationships.
How does FISH work?
FISH is useful, for example, to help a researcher identify where a particular gene falls within an individual’s chromosomes. Here’s how it works:
o Make a probe complementary to the known sequence. When making the probe, label it with a fluorescent marker, e.g. fluorescein, by incorporating nucleotides that have the marker attached to them.
o Put the chromosomes on a microscope slide and denature them.
o Denature the probe and add it to the microscope slide, allowing the probe hybridize to its complementary site.
o Wash off the excess probe and observe the chromosomes under a fluorescent microscope. The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to
How many types of probes for FISH?
Generally, researchers use three different types of FISH probes, each of which has a different application:
Locus specific probes bind to a particular region of a chromosome. This type of probe is useful when researchers have isolated a small portion of a gene and want to determine on which chromosome the gene is located.
Alphoid or centromeric repeat probes are generated from repetitive sequences found in the middle of each chromosome. Researchers use these probes to determine whether an individual has the correct number of chromosomes. These probes can also be used in combination with “locus specific probes” to determine whether an individual is missing genetic material from a particular chromosome.
Whole chromosome probes are actually collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome. Using multiple probes labeled with a mixture of different fluorescent dyes, scientists are able to label each chromosome in its own unique color. The resulting full-color map of the chromosome is known as a spectral karyotype. Whole chromosome probes are particularly useful for examining chromosomal abnormalities, for example, when a piece of one chromosome is attached to the end of another chromosome.