Molecular Cytogenetics: FISH Techniques

Molecular Cytogenetics (FISH)

• Molecular cytogenetics is a powerful technique used to detect and map specific DNA sequences within chromosomes.
• It has broad applications spanning clinical diagnostics, cancer research, and evolutionary biology.
• FISH (Fluorescence in situ hybridization) is a key method within molecular cytogenetics.

FISH Procedure

• Samples are prepared by fixing and treating the cells to allow DNA accessibility. This often involves chemical treatments to permeabilize the cells.
• Fluorescently-labeled DNA probes (FISH probes) are key to the process.
• Hybridization: The probes are added to the prepared cells allowing them to bind or adhere to the target DNA sequences on the chromosome.
• Washing: Unbound probes are removed via carefully controlled washing steps.
• Visualization: Specimens are viewed under a fluorescence microscope. Specific fluorescent signals (colors) indicate target DNA locations.

FISH Probes

• FISH probes are essential molecular tools in the procedure.
• They are short strands of DNA or RNA that are complementary to the targeted DNA sequence.
• These target specific chromosomal regions.
• Probe design is critical; the probes need to be precisely engineered to bind to the intended locations. This often involves using probes that are complementary to particular genes or chromosomal regions.
• Fluorescent dyes (fluorochromes) are attached to the probes to enable visualization. Different probes are often labeled with different fluorochromes to allow for simultaneous detection of multiple chromosomal regions during a single experiment.
• Different types of probes cater to different research questions.
• Specific types include: whole chromosome probes, locus-specific probes (e.g., locus-specific DNA probes), and spectral karyotyping probes.
• Whole chromosome probes usually cover entire sections of chromosomes for overall chromosomal assessment.
• Locus-specific probes are more targeted, illuminating specific gene positions or regions.
• Spectral karyotyping (SKY) probes use multiple colors to distinguish all chromosomes; this technique is especially helpful in identifying abnormalities.
• Important Considerations:
  • Stringency conditions during hybridization play a role. Stringency refers to the temperature and salt concentration during probe hybridization. Optimal conditions are critical for efficient binding and to minimize background staining.
  • The strength of binding between the probe and the target DNA is influenced by similar characteristics such as sequence complementarity and the hybridization temperature.
  • Probe concentration and hybridization time influence the signal intensity.
• Applications:
  • Detecting chromosomal abnormalities in various diseases (e.g., cancer, genetic disorders).
  • Determining gene location in a chromosome.
  • Identifying structural rearrangements within chromosomes.
  • Following cell division and gene expression.
  • Evaluating genetic diversity and conservation across species.
  • Examining chromosomal changes during development.