FISH presentation from hngu university (department of life science)

Fish presentation

1. Department of life sciences FISH TECHNIQUE Presented by – vandana vaghela
Roll number – 22
Semester – 2
M.Sc. Microbiology

2. Content - Introduction Types of FISH What is probes? Principle of FISH Procedure Applications of FISH Advantages of FISH Limitations of FISH

3. Introduction - FISH (Fluorescence in situ hybridization) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. Fluorescent labels quickly replaced radioactive labels in hybridization probes because of their greater safety, stability, and ease of detection.

4. Types of FISH - A variety of FISH procedures are available to cytogeneticists, who use them to diagnose many types of chromosomal abnormalities in patients. 1) Direct FISH : The probe is labeled directly with a fluorescent dye. 2) Indirect FISH : The probe is labeled with a molecule that can be detected by a fluorescently labeled antibody. 3) Multi-color FISH: Multiple probes labeled with different fluorescent dyes are used to detect multiple targets simultaneously.

5. What is probes - In the FISH (Fluorescence In Situ Hybridization) technique, probes are short, single-stranded DNA sequences that are labeled with a fluorescent dye. These probes are designed to be complementary to specific DNA sequences on a chromosome. When the probe is added to a sample of cells or tissues, it will hybridize (bind) to its complementary sequence. This allows researchers to visualize the location of the specific DNA sequence on the chromosome using a fluorescence microscope.

6. Principle of FISH 1. Probe Design: A DNA probe is created, which is a short, single-stranded DNA sequence complementary to the target DNA sequence you want to find.
This probe is labeled with a fluorescent dye (fluorophore). 2. Hybridization: The target DNA (in chromosomes or cell nuclei) is denatured (made single-stranded) to allow the probe to bind.
The probe is added and allowed to hybridize (base-pair) with its complementary target sequence on the chromosome. 3. Visualization: Fluorescence microscopy is used to visualize the location of the probe. The fluorescent dye attached to the probe emits light when excited by a specific wavelength of light, indicating the location of the target DNA sequence.

7. Procedure- The basic elements of FISH are a DNA probe and a target sequence. Before hybridization, the DNA probe is labeled , Two labeling strategies are commonly used: indirect labeling (left panel) and direct labeling (right panel). For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore. The labeled probe and the target DNA are denatured. Combining the denatured probe and target allows the annealing of complementary DNA sequences. If the probe has been labeled indirectly, an extra step is required for visualization of the nonfluorescent hapten that uses an enzymatic or immunological detection system.

8. Applications of FISH - ★ Genetic Diagnostics: • Detecting chromosomal abnormalities like aneuploidy (e.g., Down syndrome)
• Identifying gene deletions or duplications (e.g., DiGeorge syndrome) ★ Gene Mapping: • Determining the precise location of genes on chromosomes
• Ordering genes within a chromosome. ★ Evolutionary Studies: • Comparing DNA sequences between species to understand evolutionary relationships.

9. Advantages of FISH - ★ High Sensitivity and Specificity : FISH can detect even small DNA sequence changes with high accuracy. ★ Direct Visualization: It allows direct visualization of the target DNA sequence within the cell or chromosome. ★ Relatively Fast: The FISH procedure can be completed relatively quick. ★ Multiple Targets: Different probes with different fluorescent labels can be used simultaneously to detect multiple targets in the same sample.

10. Limitations of FISH - ★ Signal Intensity Variation: The intensity of the fluorescent signal can vary, making it challenging to quantify the amount of target DNA accurately. ★ Cost : FISH can be more expensive than some other genetic testing methods. ★ Limited Resolution: The resolution of FISH is limited by the size of the fluorescent spot, making it difficult to distinguish closely spaced DNA sequences.

11. References Wilson, K., & Walker, J. M. (Eds.). (2010). Principles and Techniques of Biochemistry and Molecular Biology (7 th ed.). Cambridge University Press. Speicher, M. R., & Carter, N. P. The new cytogenetics: Blurring the boundaries with molecular biology. Nature Reviews Genetics 6, 782–792 (2005) doi:10.1038/nrg1692. Biotechnology and genetics in fisheries and aquaculture ; by A. R. Beaumont and K. Hoare ; pg 86 .