11. Cytogenetic methods. FISH (fluorescence in situ hybridization).
Covers FISH, a molecular cytogenetic method that uses fluorescent DNA probes to locate specific sequences on chromosomes, compared with conventional G-banding. It walks through the steps from sample preparation to fluorescence visualisation, then the four probe types, and their clinical uses in genetics, cancer, prenatal diagnosis and research. A comparison table sets FISH against karyotyping, aCGH and NGS, and the note ends with its advantages, limitations and clinical relevance.
Topic 11 of 65 in the MU Sofia syllabus for Genetics (Medicine, Year 3). The free AceMed note covers it in 10 sections, about 416 words (3 min of reading), with 14 flashcards.
In this note
Cytogenetic Methods: FISH (Fluorescence In Situ Hybridization)
I. Introduction
II. Principle of FISH
III. Steps in FISH Procedure
IV. Types of FISH Probes
V. Applications of FISH
VI. Advantages of FISH
VII. Limitations of FISH
VIII. Comparison with Other Methods
IX. Clinical Relevance
I. Introduction
Cytogenetics = study of chromosomes and their abnormalities.
Traditional cytogenetics (light microscopy + G-banding) allows detection of large chromosomal rearrangements (>5–10 Mb).
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that increases resolution by using fluorescently labeled DNA probes to visualize specific DNA sequences on chromosomes or nuclei.
Applications: detection of microdeletions, duplications, translocations, and gene amplifications.
II. Principle of FISH
Uses DNA probes labeled with fluorescent dyes that hybridize (bind) to complementary DNA sequences within the genome.
Under a fluorescence microscope, the hybridized probe produces a signal at the location of the target DNA sequence.
The number, size, and arrangement of signals indicate normal or abnormal chromosome structure.
III. Steps in FISH Procedure
1. Sample preparation
Cells obtained from blood, bone marrow, amniotic fluid, solid tissue, or tumor biopsies.
Chromosomes fixed on slides (metaphase spreads) or nuclei in interphase.
2. Denaturation
Both the target DNA (chromosomes) and the fluorescent probe DNA are denatured (heated) → single-stranded.
3. Hybridization
Fluorescent probe binds specifically to its complementary DNA sequence.
4. Washing
Removes nonspecifically bound probes.
5. Visualization
Fluorescence microscope detects signals.
Different dyes (green, red, orange) can be used → allows multicolor FISH for complex rearrangements.
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