Fluorescent In Situ Hybridization (FISH) Introduction

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What is FISH?[ TOP ]

Fluorescence in situ hybridization (FISH) is a kind of cytogenetic technique which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. This technique provides a novel way for researchers to visualize and map the genetic material in an individual cell, including specific genes or portions of genes. It is an important tool for understanding a variety of chromosomal abnormalities and other genetic mutations. Different from most other techniques used for chromosomes study, FISH has no need to be performed on cells that are actively dividing, which makes it a very versatile procedure.

FISH Fig. 1 Scheme of the principle of the FISH experiment to localize a gene in the nucleus.

How does FISH work?[ TOP ]

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.

National Human Genome Research Institute Fig. 2 The five basic steps of FISH. (Oliveira and French 2005)

What is FISH used for?[ TOP ]

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, etc. 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 many types of probes for FISH?[ TOP ]

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.

Our Services and Products:[ TOP ]

Creative Biolabs offers a full array of custom fluorescence in situ hybridization (FISH) service from probe design, chromosome/cell preparation to expert result interpretation.

A.	Chromosome/Cell Preparation: We can help you performing cell chromosome FISH (FISH on interphase, metaphase and cultured cells), tissue chromosome FISH (FISH on formalin-fixed, paraffin-embedded tissue or cell slides) and RNA-FISH (FISH to study intracellular RNA localization, RNA processing, quantitation). We performed numerous tests aimed to improve the efficiency of cytogenetic slide preparation and to increase FISH signals. Several modifications of the general protocol resulted in better chromosome spreading, better chromosome morphology and shorter hybridization times, while yielding brighter FISH signals.
B.	Probes Design, Labeling, Purification and Test: Our FISH probes are synthetic pieces of DNA that couple with a fluorescent indicator, so that the chromosomes or genes that they bind to can be directly visualized under our imaging analysis system. Our well-established standard operating procedure allows efficient optimization of labeling a variety of fluorescent probes. Also, we can provide experimental services to meet your special needs. Customized probes are available as well. The choice of directly or indirectly labeled SpectrumOrange, SpectrumGreen, or SpectrumAqua probes is dependent upon the use and combination of other fluorophores in the FISH assay. o Whole chromosome painting probes (WPP) o Chromosome arm painting probes (APP) o Chromosome terminal band painting probes (TPP) o Chromosome enumeration probes (CEP) o Chromosome subtelomere probes (CSP) o chromosome loci specific probes (CLP)
C.	In Situ Hybridization: We can optimize the FISH hybridization conditions and provide a full array of FISH services.
D.	Result Interpretation: Our FISH imaging analysis system consists of a fluorescent microscope, a charge-coupled-device (“CCD”) camera and imaging analysis software. All three components are provided by our component suppliers with internal modifications. Through the use of FISH probes, the imaging analysis system enables medical practitioners to detect and localize the presence or absence of specific DNA sequences in chromosomes.
E.	Prenatal Diagnosis: Our optimized prenatal diagnosis detection kits offer innovative technologies and standard methods that are applied to the diagnosis of fetal disorders. o Trisomy (21/18/13/12/9/8) o Turner Syndrome o Trisomy X o XYY Syndrome o DiGeorge Syndrome o Triploidy
F.	Cancer Diagnosis and Prognosis: Many cancer diagnosis testing kits are available in our company. You can also custom other cancer diagnosis probes according to your needs. o Bladder Cancer o Breast Cancer o Cervical Cancer o Chronic Lymphocytic Leukemia o Chronic Granulocytic Leukemia o Multiple Myeloma
G.	Reagents for FISH: Our optimized reagents and solutions will help you perform your FISH experiments more smoothly and efficiently.
H.	Features: The advantages of our services and products are as follows: o Simple, easy, fast and repeatable o Sensitive o Competitive price o Easy to process multiple slides simultaneously o High quality

Citations/References[ TOP ]

Brandriff, B., Gordon, L. and Trask, B. (1991) 'DNA sequence mapping by fluorescence in situ hybridization', Environmental and molecular mutagenesis, 18(4), 259-262.

Fan, Y.-S., Davis, L. M. and Shows, T. B. (1990) 'Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes', Proceedings of the National Academy of Sciences, 87(16), 6223-6227.

Henegariu, O., Artan, S., Greally, J. M., Chen, X.-N., Korenberg, J. R., Vance, G. H., Stubbs, L., Bray-Ward, P. and Ward, D. C. (2001) 'Cryptic translocation identification in human and mouse using several telomeric multiplex fish (TM-FISH) strategies', Laboratory investigation, 81(4), 483-491.

Henegariu, O., Bray-Ward, P., Artan, S., Vance, G. H., Qumsyieh, M. and Ward, D. C. (2001) 'Small marker chromosome identification in metaphase and interphase using centromeric multiplex FISH (CM-FISH)', Laboratory investigation, 81(4), 475-481.

Henegariu, O., Dunai, J., Chen, X.-N., Korenberg, J. R., Ward, D. C. and Greally, J. M. (2001) 'A triple color FISH technique for mouse chromosome identification', Mammalian Genome, 12(6), 462-465.

Henegariu, O., Heerema, N. A., Bray-Ward, P. and Ward, D. C. (1999) 'Colour-changing karyotyping: an alternative to M-FISH/SKY', Nature genetics, 23(3), 263-264.

Henegariu, O., Heerema, N. A., Lowe Wright, L., Bray-Ward, P., Ward, D. C. and Vance, G. H. (2001) 'Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing', Cytometry, 43(2), 101-109.

Oliveira, A. M. and French, C. A. (2005) 'Applications of fluorescence in situ hybridization in cytopathology', Acta cytologica, 49(6), 587-594.

Tepperberg, J., Pettenati, M., Rao, P., Lese, C., Rita, D., Wyandt, H., Gersen, S., White, B. and Schoonmaker, M. (2001) 'Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature', Prenatal diagnosis, 21(4), 293-301.

FISH Probe samples:

Human chromosome 1 painting probe (green)	Human chromosome 1 arm painting probe
Human chromosome 1 terminal band painting probe	Human chromosome 3 centromere probe
Human chromosome 1 subtelomere probes	Hybridization signal of human XIST gene probe (red)

TABLE 1: Custom FISH Services
SERVICE	DESCRIPTION	Price
Chromosome/Cell Preparation	We can help you performing cell chromosome FISH (FISH on interphase, metaphase and cultured cells) and tissue chromosome FISH (FISH on formalin-fixed, paraffin-embedded tissue or cell slides).	ORDERING
Probes Design, Labeling, Purification and Test	Our well-established standard operating procedure allows efficient optimization of labeling variety of fluorescent probes. Customized probes are available as well. The choice of directly labeled SpectrumOrange, SpectrumGreen, or SpectrumAqua probes is dependent upon the use and combination of other fluorophores in the FISH assay.	ORDERING
In Situ Hybridization	We have optimized the FISH hybridization conditions and can provide all kinds of FISH service for researchers.	ORDERING
Result Interpretation	Our FISH imaging analysis system consists of a fluorescent microscope, a charge-coupled-device (“CCD”) camera and imaging analysis software. All three components are provided by our component suppliers with internal modifications. Through the use of FISH probes, the imaging analysis system enables medical practitioners to detect and localize the presence or absence of specific DNA sequences in chromosomes.	ORDERING

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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