FISH - Fluorescent In Situ Hybridization

Summary

This video introduces Fluorescent In Situ Hybridization (FISH), a cytogenetic technique used to visualize specific chromosomal locations. FISH employs fluorescent probes that bind to specific DNA regions to detect chromosomal abnormalities, which may lead to diseases. The video explains how probes are designed, their preparation process, and how hybridization with target DNA is achieved. It also highlights the medical significance of FISH, such as its role in diagnosing conditions like Down syndrome and identifying genetic mutations. Viewers are encouraged to ask questions for further clarification.

Takeaways

• 🔬 Fluorescent in situ hybridization (FISH) is a cytogenetic technique used to visualize specific chromosome locations using fluorescent probes.
• 🧬 FISH helps detect chromosomal abnormalities that may lead to diseases, providing valuable information for medical diagnosis.
• 🧫 The first step in the FISH process is the fixation of cells using a formaldehyde-based fixative to preserve cellular structures.
• 🔖 Probes used in FISH must be complementary to the target chromosomal region and can be designed using either RNA or DNA sequences.
• ✂️ Special enzymes like DNAse create nicks in the DNA, and fluorescent nucleotides are inserted to label the probes.
• 🔥 Denaturation of both the probe and the target DNA is achieved by heating cells to around 95°C, allowing the probe to bind to the chromosome sequence.
• 🧪 Unbound probes are washed away, and the results are analyzed using a fluorescence microscope to detect chromosomal mutations or abnormalities.
• 🔍 A fluorescent signal indicates successful hybridization, while no signal suggests a mutation or deletion in the gene of interest.
• 👶 FISH is used in karyotyping to detect chromosomal disorders, such as trisomy 21 (Down syndrome), by staining whole chromosomes in different colors.
• 🧬 FISH is also utilized to visualize genetic mutations with shorter probes designed to target specific genes for research and medical purposes.

Q & A

What is the main purpose of Fluorescent In Situ Hybridization (FISH)?

The main purpose of FISH is to detect chromosomal abnormalities by visualizing specific chromosomal locations using a fluorescent probe, helping to identify mutations that could cause diseases.

How does the fluorescent probe work in FISH?

The fluorescent probe works by binding to a specific chromosomal region. It is complementary to the target sequence, and after hybridization, the probe emits fluorescence, allowing visualization of the region under a microscope.

What is the role of the fixative in the FISH process?

The fixative, typically formalin-based, is used to fix the cells by causing protein-protein and protein-nucleic acid cross-links. This stabilizes the cells and prepares them for the hybridization process.

How are probes designed for FISH?

Probes are designed to be complementary to the chromosomal region of interest. This can involve either short double-stranded RNA or DNA probes, which are labeled with fluorescent nucleotides using an enzyme such as DNAse to create random cuts, or 'nicks,' in the DNA.

What is the purpose of denaturation in the FISH process?

Denaturation involves heating the target DNA and the probe to around 95°C to separate their strands. This allows the labeled probe to bind specifically to the complementary sequence of the target DNA during hybridization.

What happens if the probe does not bind to the target DNA?

If the probe does not bind, it indicates that the target sequence may have a mutation or deletion. In such cases, no fluorescence signal is detected, as hybridization does not occur.

How is fluorescence used to detect genetic abnormalities?

Fluorescence is used to detect whether the probe successfully hybridized to the target sequence. If the chromosomal region is intact, the probe binds and emits fluorescence. If there is a mutation or deletion, no signal will be observed.

What are some medical applications of FISH?

FISH is commonly used in medicine to detect chromosomal abnormalities, such as trisomy 21 (Down syndrome), and genetic mutations linked to diseases. It is also used to create karyotypes for detailed chromosomal analysis.

Why are short probes often used in FISH for detecting genetic mutations?

Short probes are often used when only a specific gene needs to be observed, as they can precisely target smaller chromosomal regions where mutations may occur.

How are the results of FISH analyzed?

The results of FISH are analyzed using a fluorescence microscope. If the probe binds to the chromosomal region of interest, the specific location will appear as a fluorescent signal under the microscope.