FISH: Fluorescence In Situ Hybridization

FISH: Fluorescence In Situ Hybridization

FISH, fluorescence in situ hybridization, is a cytogenetic technique that enables “mapping” of the genetic material of cells

Arun Kumar, PhD

What is FISH?

FISH, fluorescence in situ hybridization, is a cytogenetic technique enabling “mapping” of the genetic material of cells. It is commonly used to label DNA providing information on the location, length, and number of copies of specific genes or chromosome portions. Additionally, it can be applied to all types of RNA, and is foremost utilized to detect copy numbers and location of mRNA to visualize cellular transcription activity. FISH is based on the specific interaction between a fluorescence-labeled probe and a specific target sequence in cellular DNA or RNA. FISH was first described in 1969 by two independent research groups. It is one of the oldest cytogenetics methods and was used as early as 1993 to determine aneuploidy for preimplantation diagnostics.

How Does it Work?

Image Courtesy of Enzo Life Sciences

Here’s the basic principle:

  • A probe is generated with a complementary sequence to that of the sequence of interest. The probe is fluorescently labeled by the incorporation of nucleotides conjugated with fluorescent markers.
  • The chromosomes are denatured by exposing them to heat and chemicals that break the hydrogen bonds holding the double helix structure together. Later, in proper conditions, chromosomes will go back to their original state.
  • The probe is also denatured and then added to the specimen for the hybridization, that is the specific binding to its complementary sequence on the chromosome.
  • The excess probe is washed off and the chromosomes are observed under a fluorescent microscope. The sites of interest bound by the probe will fluoresce!
  • FISH

What is the Difference Between FISH and ISH?

FISH and ISH both use the same concept of in situ hybridization, but FISH does so with the addition of fluorescent probes. This enables direct detection. Fluorescence allows the visualization of probes in combination with the surrounding cells and tissues. Looking for a particular DNA sequence within whole chromosomes has been described as looking for a needle in a haystack. Attaching markers that cause the segments of interest to pop out with color gives a much clearer picture of where they are and can help us picture why their location on the chromosome might be significant.

FISH Detection. Fluorescent labels are attached to a probe which will hybridize to a target DNA strand. Then the fluorescent probe-target hybrids can be detected under a microscope immediately post-hybridization washes, using a fluorescent microscope.

ISH Detection. The probe is labeled with a non-fluorescent hapten like Biotin, Digoxigenin or DNP. Streptavidin or an antibody linked to an enzyme (horseradish peroxidase or alkaline phosphate) binds the non-fluorescence hapten with high specificity. Then the enzyme converts a substrate (chromogen) into an insoluble color product, which can be detected using a bright-field microscope.



What Sample Types are Used in FISH?

FISH can be used with tissue samples, chromosome spreads and cell cultures, such as:

  • Blood and Bone Marrow
  • Urine and Fecal Samples
  • Bacterial Cultures
  • Mucosal Samples
  • Even Sediment!

What Information does FISH Actually Provide?

FISH provides a visual, color-coded map of DNA segments of interest within chromosomes. It tells us where they are along the chromosome and approximately how many segments are present and have been bound. Because FISH allows for a fluorescent copy of the DNA of interest, it can be used for a multitude of different applications, including:

  • Chromosome "painting"
    • "Paints" can be formed with hybridization probes matching sequences along the length of a particular chromosome in a process called multicolor FISH. This causes each chromosome to appear a different color, allowing rapid detection of large chromosomal changes.
  • Gene mapping on chromosomes
    • FISH helps to identify gene locations.
  • Analyzing cells not currently undergoing mitosis
    • Other techniques can analyze only metaphase chromosomes, but FISH can analyze both interphase and metaphase chromosomes. This means that cells don’t need to be cultured far in advance before chromosomal analysis, and cells that don’t divide frequently, such as solid tumor cells, may also be analyzed. 
  • Detecting chromosomal abnormalities
    • In combination with karyotyping, FISH can detect deletions, translocations, and duplications of genes.

Most importantly, FISH helps us understand the organization, regulation, and function of genes, which in turn gives us valuable information on how to treat genetic diseases. Using this technique, we can examine chromosomal integrity or localize and measure DNA/RNAs within tissues, giving us the tools to catch diseases early, understand how they work, and ultimately find cures.

Gene mapping on chromosome. FISH probes hybridize to C-MYC (red signal) and to centromere 8 (green signal) in metaphase spread human chromosomes.
Chromosome painting. FISH probes specific to each the chromo- somes hybridized to DNA in metaphase spread. The combination of colors and specific probes that hybridize to a particular chromosome allows for easy detection of deletions and translocations among chromosomes. A fragment (arrow head) can be identified as an extra piece of chromosome, since it is the same aqua color as the two normal copies of chromosome 15 (arrows).
Image from: Uhrig, S. et al. (1999) Am J Hum Genet. Aug; 65(2):448-62
Detecting chromosomal abnormalities. Acute Lymphoblastic Leukemia (ALL) tumor cell line cells hybridized with FISH probes specific to the CDKN2A gene (red) and Centromere 9 (green) showing hemizygous loss of CDKN2A as indicated by only one red CDKN2A and one green CEN 9 signal in the nucleus.
Normal cell not currently undergoing mitosis. FISH probe specific to the CDKN2A gene (red) and Centromere 9 (green) hybridizing to normal interphase cells as indicated by two red and two green signals in the nucleus.