FLUOROSCENT INSITU FLUOROSCENT INSITU HYBRIDIZATIONHYBRIDIZATION

Fluorescent in situ hybridization (Fluorescent in situ hybridization (FISHFISH) is a ) is a powerful technique for detecting RNA or DNA powerful technique for detecting RNA or DNA sequences in cells, tissues, and tumors. FISH sequences in cells, tissues, and tumors. FISH provides a unique link among the studies of provides a unique link among the studies of cell biologycell biology, cytogenetics, and molecular , cytogenetics, and molecular geneticsgenetics.. ItIt is a technique in which single-is a technique in which single-stranded nucleic acids (usually DNA, but RNA stranded nucleic acids (usually DNA, but RNA may also be used) are permitted to interact so may also be used) are permitted to interact so that complexes, or hybrids, are formed by that complexes, or hybrids, are formed by molecules with sufficiently similar, molecules with sufficiently similar, complementarycomplementary sequences.sequences.

By By nucleic acidnucleic acid hybridization, the degree of hybridization, the degree of sequence identity can be determined, and sequence identity can be determined, and specific sequences can be detected and located specific sequences can be detected and located on a given on a given chromosomechromosome The method comprises of three basic steps: The method comprises of three basic steps: fixation of a specimen on a fixation of a specimen on a microscopemicroscope slide, slide, hybridization of labeled probe to homologous hybridization of labeled probe to homologous fragments of genomic DNA, and enzymatic fragments of genomic DNA, and enzymatic detection of the tagged target hybridsdetection of the tagged target hybrids

The probe sequences were initially detected with The probe sequences were initially detected with isotopic reagents, nonisotopic hybridization has isotopic reagents, nonisotopic hybridization has become increasingly popular, with fluorescent become increasingly popular, with fluorescent hybridization now a common choice. Protocols hybridization now a common choice. Protocols involving nonisotopic probes are considerably involving nonisotopic probes are considerably faster, with greater signal resolution, and provide faster, with greater signal resolution, and provide options to visualize different targets options to visualize different targets simultaneously by combining various detection simultaneously by combining various detection methods. methods.

The detection of sequences on the target The detection of sequences on the target chromosomes is performed indirectly, commonly chromosomes is performed indirectly, commonly with biotinylated or digoxigenin-labeled probes with biotinylated or digoxigenin-labeled probes detected via a fluorochrome-conjugated detected via a fluorochrome-conjugated detection reagent, such as an antibody detection reagent, such as an antibody conjugated with fluorescein, as a result, the conjugated with fluorescein, as a result, the direct visualization of the relative position of the direct visualization of the relative position of the probes is possible. probes is possible.

nucleic acid probes labeled directly with nucleic acid probes labeled directly with fluorochromes are used for the detection of large fluorochromes are used for the detection of large target sequences. This method takes less target sequences. This method takes less timetime and results in lower background; however, lower and results in lower background; however, lower signal intensity is generated. Higher sensitivity signal intensity is generated. Higher sensitivity can be obtained by building layers of detection can be obtained by building layers of detection reagents, resulting in amplification of the signal. reagents, resulting in amplification of the signal. Using such means, it is possible to detect single-Using such means, it is possible to detect single-copy sequences on chromosome with probes copy sequences on chromosome with probes shorter than 0.8 kb. shorter than 0.8 kb.

Probes can vary in length from a few base pairs Probes can vary in length from a few base pairs for synthetic oligonucleotides to larger than one for synthetic oligonucleotides to larger than one Mbp. Probes of different types can be used to Mbp. Probes of different types can be used to detect distinct DNA types. PCR-amplified detect distinct DNA types. PCR-amplified repeated DNA sequences, oligonucleotides repeated DNA sequences, oligonucleotides specific for repeat elements, or cloned repeat specific for repeat elements, or cloned repeat elements can be used to detect clusters of elements can be used to detect clusters of repetitive DNA in heterochromatin blocks or repetitive DNA in heterochromatin blocks or centromeric regions of individual chromosomes centromeric regions of individual chromosomes

FISH is useful in determining aberrations in the FISH is useful in determining aberrations in the number of chromosomes present in a cell. In number of chromosomes present in a cell. In contrast, for detecting single contrast, for detecting single locuslocus targets, targets, cDNAs or pieces of cloned genomic DNA, from cDNAs or pieces of cloned genomic DNA, from 100bp to 1Mbp in size, can be used. To detect 100bp to 1Mbp in size, can be used. To detect specific chromosomes or chromosomal regions, specific chromosomes or chromosomal regions, chromosome-specific DNA libraries can be used chromosome-specific DNA libraries can be used as probes to delineate individual chromosomes as probes to delineate individual chromosomes from the full chromosomal complement Specific from the full chromosomal complement Specific probes have been commercially available for probes have been commercially available for each of the human chromosomes since 1991. each of the human chromosomes since 1991.

A given A given tissuetissue or cell source, such as sections of or cell source, such as sections of frozen tumors, imprinted cells, cultured cells, or frozen tumors, imprinted cells, cultured cells, or embedded sections, may be hybridized. The embedded sections, may be hybridized. The DNA probes are hybridized to chromosomes DNA probes are hybridized to chromosomes from dividing (metaphase) or non-dividing from dividing (metaphase) or non-dividing (interphase) cells. The observation of the (interphase) cells. The observation of the hybridized sequences is done using hybridized sequences is done using epifluorescence epifluorescence microscopymicroscopy. White . White lightlight from a from a source lamp is filtered so that only the relevant source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent wavelengths for excitation of the fluorescent molecules reach the molecules reach the samplesample or non-dividing or non-dividing (interphase) cells. (interphase) cells.

The light emitted by fluorochromes is generally The light emitted by fluorochromes is generally of larger wavelengths, which allows the of larger wavelengths, which allows the distinction between excitation and distinction between excitation and emissionemission light by means of a second optical light by means of a second optical filter.Therefore, it is possible to see bright filter.Therefore, it is possible to see bright colored signals on a dark background. It is also colored signals on a dark background. It is also possible to distinguish between several possible to distinguish between several excitation and emission bands, thus between excitation and emission bands, thus between several fluorochromes, which allows the several fluorochromes, which allows the observation of many different probes on the observation of many different probes on the same target. same target.

APPLICATIONAPPLICATION

FISH has a large number of applications in FISH has a large number of applications in molecular biologymolecular biology and medical science, and medical science, including including genegene mapping, mapping, diagnosisdiagnosis of of chromosomal abnormalitieschromosomal abnormalities, and studies of , and studies of cellular structure and function. Chromosomes in cellular structure and function. Chromosomes in three-dimensionally preserved nuclei can be three-dimensionally preserved nuclei can be "painted" using FISH. In clinical research, FISH "painted" using FISH. In clinical research, FISH can be used for prenatal diagnosis of inherited can be used for prenatal diagnosis of inherited chromosomal aberrations, postnatal diagnosis of chromosomal aberrations, postnatal diagnosis of carriers of genetic carriers of genetic diseasedisease, diagnosis of , diagnosis of infectious disease, viral and bacterial disease, infectious disease, viral and bacterial disease, tumor,tumor, cytogenetic diagnosis, and detection of cytogenetic diagnosis, and detection of aberrant gene expression .aberrant gene expression .

In laboratory research, FISH can be used for In laboratory research, FISH can be used for mapping chromosomal genes, to study the mapping chromosomal genes, to study the evolutionevolution of genomes (Zoo FISH), analyzing of genomes (Zoo FISH), analyzing nuclear organization, visualization of nuclear organization, visualization of chromosomal territories and chromosomal territories and chromatinchromatin in in interphase cells, to analyze dynamic nuclear interphase cells, to analyze dynamic nuclear processes, somatic processes, somatic hybridhybrid cells, replication, cells, replication, chromosome sorting, and to study tumor chromosome sorting, and to study tumor biologybiology . .It can also be used in developmental It can also be used in developmental biology to study the temporal expression of biology to study the temporal expression of genes during differentiation and development. genes during differentiation and development. Recently, high resolution FISH has become a Recently, high resolution FISH has become a popular method for ordering genes or DNA popular method for ordering genes or DNA markers within chromosomal regions of interest markers within chromosomal regions of interest