Model Answer
0 min readIntroduction
Fluorescence In Situ Hybridization (FISH) is a powerful cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Developed in the 1980s, FISH revolutionized genetic diagnostics by allowing visualization of DNA sequences directly within cells, offering a significant advantage over traditional karyotyping. It utilizes fluorescent probes that bind to complementary DNA sequences, enabling the identification of chromosomal abnormalities, gene copy number variations, and even specific gene mutations. This technique has become indispensable in clinical genetics, cancer research, and prenatal diagnostics.
Principle of FISH
The fundamental principle of FISH relies on the hybridization of a labeled DNA probe to its complementary sequence on a target chromosome or DNA sequence. The probe, a short single-stranded DNA fragment, is labeled with a fluorescent dye (fluorophore). When the probe encounters its complementary sequence, it binds (hybridizes) through base pairing (A with T, and G with C). This hybridization is then visualized using a fluorescence microscope, where the fluorophore emits light upon excitation, revealing the location of the target sequence.
Procedure of FISH
The FISH procedure involves several key steps:
- Probe Preparation: DNA probes are designed to target specific genomic regions. These probes are labeled with fluorophores like FITC, Texas Red, or Cy5, each emitting a different color.
- Sample Preparation: Cells or tissues are fixed (e.g., with formaldehyde) to preserve their morphology. Chromosomes are often denatured (separated into single strands) by heating.
- Hybridization: The denatured probe is added to the denatured sample. Hybridization occurs under controlled conditions (temperature, salt concentration) to allow specific binding.
- Washing: Unbound probe is removed by washing.
- Detection & Visualization: The sample is counterstained with a DNA dye (e.g., DAPI) to visualize all chromosomes. A fluorescence microscope is used to detect the fluorescent signal from the hybridized probe.
Applications of FISH
FISH has a wide range of applications across various fields:
- Chromosomal Aberration Detection: Identifying aneuploidies (abnormal chromosome number, e.g., Down syndrome – Trisomy 21), deletions, duplications, and translocations.
- Cancer Diagnostics: Detecting gene amplifications (e.g., HER2/neu in breast cancer) and chromosomal rearrangements associated with specific cancers.
- Prenatal Diagnosis: Rapidly detecting chromosomal abnormalities in fetal cells obtained through amniocentesis or chorionic villus sampling.
- Gene Mapping: Locating specific genes on chromosomes.
- Species Identification: Identifying different species based on their unique DNA sequences.
- Microbial Detection: Identifying specific bacteria or viruses in clinical samples.
Comparative Genomic Hybridization (CGH) using FISH (FISH-CGH): A variation of FISH used to detect copy number variations across the entire genome. It compares the genomic DNA of a test sample to a normal reference sample.
| Application | Specific Example |
|---|---|
| Cancer Diagnostics | Detecting the Philadelphia chromosome (translocation between chromosomes 9 and 22) in Chronic Myeloid Leukemia (CML). |
| Prenatal Diagnosis | Identifying Trisomy 18 (Edwards syndrome) in a fetus. |
| Gene Mapping | Confirming the location of the BRCA1 gene on chromosome 17. |
Conclusion
FISH remains a cornerstone of modern cytogenetics and molecular diagnostics. Its ability to visualize specific DNA sequences directly within cells provides invaluable information for diagnosing genetic disorders, understanding cancer development, and advancing our knowledge of genome organization. Continued advancements in probe technology and imaging techniques are expanding the applications of FISH, promising even more precise and efficient genetic analysis in the future. The technique’s versatility ensures its continued relevance in both research and clinical settings.
Answer Length
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