Fluorescent In Situ Hybridization (FISH) | Zoology Optional Notes for UPSC PDF Download

About: Fluorescent In Situ Hybridization (FISH)

Fluorescent In Situ Hybridization (FISH) is a powerful technique used to detect specific DNA sequences on chromosomes from a pool of fragments of similar sizes. It involves using fluorescent probes that attach to chromosomal regions with matching base sequences. This method enables the detection of base sequences on a combed DNA molecule by hybridizing known base sequences (the probes) with the target DNA (matrixDNA or target DNA).

1. Fixation: A specimen is secured on a microscope slide.

2. Hybridization: Labeled probes bind to homologous fragments of genomic DNA.

3. Fluorescent Detection: Enzymatic detection is used to visualize the tagged target hybrids.

FISH typically involves the following specimen types:

• Frozen Sections: Fresh tissue is rapidly frozen and embedded in a support medium for cryosectioning. Sections are fixed prior to hybridization.
• Paraffin-Embedded Sections: Sections are fixed in formalin and embedded in wax paraffin before being sectioned.
• Cells in Suspension: Cells can be cytospun onto glass slides and fixed with methanol.

Types of Probes

Probes are fragments of cloned DNA used for hybridization. Four common types of probes are used in in situ hybridization:

1. Oligonucleotide Probes: Synthetically produced with known nucleotide sequences. They are small (around 40-50 base pairs), stable, and resistant to RNases. Suitable for recognizing closely related gene families.
2. Single-Stranded DNA Probes: Larger (around 200-500 base pairs) and can be obtained through reverse transcription of RNA or PCR-generated fragments. They require more time and resources.
3. Double-Stranded DNA Probes: Produced by incorporating sequences of interest into bacterial cells and extracting the DNA. PCR can also be used for rapid probe generation. Denaturation is required before hybridization.
4. RNA Probes (cRNA Probes or Riboprobes): RNA probes are thermostable and resistant to RNases. They allow for post-hybridization digestion to reduce background staining. However, they are sensitive to RNases.

Labeling of Probes

Probes need to be labeled for detection after hybridization. Probes can be labeled at the 5' or 3' ends or tailed with labeled nucleotides. Labels can be radiolabels (e.g., 35Sulphur) or non-radioactive labels.

• Radiolabeled Probes: Visualized using photographic film after exposure.
• Non-radioactive Labels: Can be incorporated during probe synthesis, allowing detection using fluorescent microscopy.

Hybridization Process

Hybridization involves mixing single-stranded probes with denatured target DNA, separating the two strands and allowing the single-strand probes to bind to their complementary target sequences. Factors affecting hybridization include temperature, pH, monovalent cation concentration, and the presence of organic solvents.
Typical hybridization solution components include dextran sulfate, formamide, dithiothreitol (DTT), NaCl+ sodium citrate (SSC), and EDTA.

Observation

Hybridized sequences are observed using epifluorescence microscopy. Fluorescent labels emit light at wavelengths distinguishable from the excitation light, allowing bright signals to be seen against a dark background. Multiple probes can be visualized simultaneously.

• Radiolabeled probes are detected using photographic film or emulsion.
• Fluorescent labels, such as FITC or biotin, require an intermediate step for detection and are observed with a fluorescent microscope.

Overall, FISH is a versatile technique for visualizing specific DNA sequences in various specimen types, providing valuable insights into chromosomal structures and genetic information.