FISH

Fluorescence in situ hybridization (FISH) has become an important adjunct to routine karyotyping and has greatly expanded the power of cytogenetic analysis.

A major limitation of karyotyping is that it is applicable only to cells that are dividing or can be induced to divide in vitro. This problem can be overcome with DNA probes that recognize chromosome-specific sequences.

Such probes are labeled with fluorescent dyes and applied to interphase nuclei. The probe binds to its complementary sequence on the chromosome and thus labels the specific chromosome, which can then be visualized under a fluorescent microscope. Thus, FISH can be used to enumerate chromosomes in interphase nuclei.

The application of FISH is not limited to interphase nuclei, however. By using DNA probes that are specific for defined regions of the chromosomes, FISH can be used to demonstrate subtle microdeletions, complex translocations, and telomere alterations that are not readily detectable by routine karyotyping.

In addition to its diagnostic utility, FISH can also be used as a tool to physically map newly isolated genes of clinical interest. Novel DNA sequences are labeled with a fluorescent dye and then applied to a metaphase spread.

The DNA binds to its complementary sequence and thus pinpoints the localization of the gene to a specific site. Chromosome painting is an extension of FISH, whereby whole chromosomes can be labeled with a series of fluorescent DNA probes that bind to multiple sites along a particular chromosome.

The number of chromosomes that can be detected simultaneously by chromosome painting is limited by the availability of fluorescent dyes that excite different wavelengths of visible light.

 Thus, chromosome painting has limited ability to visualize all 46 human chromosomes simultaneously. This hurdle has been overcome by the introduction of spectral karyotyping (SKY). By using a combination of five fluorochromes and appropriate computer-generated signals, the entire human genome can be visualized (SKY).

 Another recent FISH application directly compares the DNA content of differentially labeled normal and tumor cell populations by their co-hybridization to normal metaphase chromosome spreads (comparative genomic hybridization or CGH) or to a series of genomic DNA clones aligned on glass slides (array comparative genomic hybridization). In this manner, tumor-specific alterations in gene copy number can be ascertained.

FISH use in oncology

 6p25 : RREB1
 6q2 3: MYB
 8q24 : MYC
 9p21 : CDKN2A ( p16 )
 11q13 : CCDN1 ( cyclin-D1 )
 ch.6 centromere : CEP6
 ch.9 centromere : CEP9

Types

 nuclear extraction-based fluorescence in situ hybridization (NE-FISH)

References

 Lichter P, Ward DC. Is non-isotopic in situ hybridization finally coming of age? Nature 1990;345:93-94. PMID: 1691830