Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) uses fluores-cently tagged DNA or RNA probes to identify genomic


Degrade RNases and target Probes release RNA


Target preamplifiers probe „


Surface of microwell bDNA amplifier ^

Hybridize bDNA amplifiers

Surface of microwell bDNA amplifier ^

Hybridize bDNA amplifiers

Add substrate and measure light

Figure 2-7. Diagram of branched DNA (bDNA) method. (Reprinted with permission of Bayer Healthcare.)

sequences of interest.78,79 The major advantages of FISH are the ability to utilize FFPE tissue sections, allowing correlation of probe hybridization with tissue morphology, and the increased resolution provided by FISH for identification of specific abnormalities when partnered with conventional cytogenetics. The number and location of the fluorescent signal(s) can identify chromosomal abnormalities including gene amplification, gene deletion, or structural rearrangements such as translocations.

FISH is similar to Southern blot in that sequential steps of denaturation, hybridization, and washing are involved. Slides are prepared in the cytogenetic or histology laboratory. Probe(s) are then applied to the slide along with a nuclear counterstain and reagents to enhance denaturation and reduce background. The slides are sealed and incubated (usually overnight) in a humid environment at high temperature. These conditions denature the probe and patient DNA, allowing hybridization to occur between the probe and its complementary DNA sequence without binding to nonspecific sites. Excess nonspecifically bound probe is washed away, and the pattern of fluorescence is read by fluorescence microscopy. The fluorescent signal(s) can be enhanced by the use of a digital imaging system and computer software.

The specificity of FISH is largely based on the selection of the probe. Ideally, the probe is complementary to the gene of interest; however, if the disease gene is unknown, satellite probes that identify a chromosomal region linked to the disease may be used. Labeled bacterial or yeast artificial chromosomes (BAC or YAC, respectively) are typically used as FISH probes, but short oligonucleotides also can be used with signal amplification techniques. Probes that identify individual whole chromosomes or chromosomal arms are often called "painting probes" due to the colorful patterns they generate. Other probes that hybridize to a specific gene can be used for the detection of deletions or duplications and are called single-copy gene probes. Probes that hybridize to the alpha-satellite regions near centromeres are used in clinical cytogenetics to identify and count individual chromosomes. Probes that hybridize to the subtelomeric portions of chromosomes are used to identify cryptic telomeric abnormalities such as translocations.

Dual-color FISH (dFISH) employs two probes with different fluorescence wavelengths to identify structural chromosomal rearrangements. Each probe generates a characteristic color by itself (split signal) and a third color when the two probes are juxtaposed (fusion signal). A fusion signal indicating chromosomal rearrangement is used to identify disease-causing mutations that predictably involve only two partner genes, for example,BCR and ABL. However, when a particular gene with multiple potential translocation partners is tested, it is more efficient to have both probes bind to the 5' and 3' ends of the particular gene such that the normal allele shows the fusion signal and the rearranged allele shows two split signals. This is the technique used for the MLL gene, which is rearranged to over 30 different partner genes in various types of leukemia.

Spectral karyotyping (SKY) and multiplex FISH (M-FISH) are relatively new advancements of conventional FISH that utilize multiple fluorochromes, specialized optics, and image analysis that can simultaneously identify all chromosomes.80 Comparative genomic hybridization (CGH), also called "copy number karyotyping," is a variation of FISH that detects relative gains or losses of the genome.81 This method compares the ratios of patient specimen DNA, labeled with one fluorochrome, to that of normal DNA, labeled with a different fluorochrome when hybridized to control chromosomes. CGH is used predominantly in research to identify possible pathways involved in tumor progression, recurrence, or metastasis. Colorimetric probes are used in chro-mogenic in situ hybridization (CISH), which has the advantage that the signal does not fade with time or require fluorescence microscopy for analysis.

Examples of Applications of FISH

1. Detection of BCR/ABL in chronic myelogenous leukemia

2. Detection of HER2 gene amplification for breast cancer diagnosis and prognosis

3. Detection of MYCN amplification in neuroblastoma82

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