Molecular Cytogenetics: FISH Techniques

Study Notes

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.

Description

This quiz explores the vital techniques of molecular cytogenetics, specifically focusing on FISH (Fluorescence in situ hybridization). Delve into the procedure, from sample preparation to visualization, and understand the significance of FISH probes in mapping DNA sequences within chromosomes. Test your knowledge on this powerful analytical method used in clinical diagnostics and research.