Explain and describe the tools and techniques of FISH.

Principles of FISH

FISH is based on the hybridization of a labeled DNA probe to a complementary DNA sequence on a chromosome. The probe, typically a short, single-stranded DNA sequence, is labeled with a fluorescent dye (fluorophore). When the probe binds to its target sequence, the fluorophore emits light when excited by a specific wavelength of light, allowing visualization of the target sequence under a fluorescence microscope.

Tools and Techniques of FISH

1. Probe Preparation

The DNA probe is the core component of FISH. Probes can be generated using several methods:

• Cosmid clones: These are bacterial artificial chromosomes (BACs) containing large DNA fragments.
• Plasmids: Smaller DNA fragments cloned into plasmids.
• PCR products: Amplified DNA fragments generated using Polymerase Chain Reaction.
• Synthetic oligonucleotides: Short, chemically synthesized DNA sequences (typically 20-50 base pairs).

The probe is then labeled with a fluorophore. Common fluorophores include:

• FITC (Fluorescein Isothiocyanate): Emits green light.
• Texas Red: Emits red light.
• Cy3 and Cy5: Emit green and red light, respectively.

2. Target DNA Preparation

The target DNA must be accessible to the probe. This involves several steps:

• Fixation: Cells are fixed (e.g., with formaldehyde) to preserve their morphology and prevent DNA degradation.
• Permeabilization: The cell membrane and nuclear envelope are permeabilized to allow the probe to enter.
• Denaturation: Both the target DNA and the probe are denatured (separated into single strands) by heating.

3. Hybridization

The denatured probe is mixed with the denatured target DNA and incubated under specific conditions (temperature, salt concentration, formamide concentration) that promote hybridization. The hybridization temperature and salt concentration are crucial for specificity and efficiency. Hybridization typically takes place overnight.

4. Post-Hybridization Washes

After hybridization, stringent washes are performed to remove unbound probe. These washes involve increasing salt concentrations to remove non-specifically bound probes, leaving only the specifically hybridized probes.

5. Signal Detection

The hybridized probe is visualized using a fluorescence microscope. The fluorophore emits light when excited by a specific wavelength of light. The signal intensity and location are analyzed to determine the presence or absence of the target sequence.

Variations of FISH

a) Metaphase FISH

This is the traditional FISH technique performed on metaphase chromosomes. It allows for the visualization of the entire chromosome complement and the detection of large chromosomal abnormalities like deletions, duplications, and translocations.

b) Interphase FISH

This technique is performed on interphase cells (cells not undergoing division). It is useful for detecting gene amplifications and deletions in tumor cells. It is faster than metaphase FISH but provides less information about the overall chromosome complement.

c) Comparative Genomic Hybridization (CGH) FISH

CGH-FISH combines the principles of FISH and comparative genomic hybridization. It uses two differently labeled probes – one from the patient’s sample and one from a normal control sample. This allows for the detection of gains and losses of chromosomal regions in the patient’s sample.

d) Multicolor FISH (M-FISH)

M-FISH uses multiple probes, each labeled with a different fluorophore, to paint each chromosome a unique color. This allows for the simultaneous visualization of all chromosomes and the detection of complex chromosomal rearrangements.

e) SKY (Spectral Karyotyping)

SKY is a more advanced version of M-FISH that uses a wider range of fluorophores and spectral imaging to differentiate all human chromosomes. It is particularly useful for identifying complex chromosomal rearrangements.