Karyotype and FISH
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Transcript of Karyotype and FISH
Karyotype and FISH
Prof. Laila Anjuman Banu
Chairman
Department of Anatomy, BSMMU
Dr. Subarna DasResident, MS AnatomyPhase-A, Year-1, Block-2
Guided by-
Presented by-
Objectives
• Define karyotype and FISH
• Describe the procedure of karyotyping and FISH
• Explain chromosomal abnormalities through karyotyping and FISH
• Describe the principles of FISH
What is a Karyotype?
Type of samples
• Peripheral blood
• Cord blood
• Bone marrow blood
• Amniotic fluid
• Products of conception
Overview of the procedure
1. Collection of blood
2. Cell culture
3. Stopping the cell division at Metaphase
4. Hypotonic treatment of blood cells
Overview of the procedure
5. Fixation
6. Slide preparation
7. Slide dehydration
8. Treatment with enzyme
9. Staining
Contd….
Karyotyping Reagents
1. Phytohemagglutinin M-form
2. Colcemid™ Solution in PBS
3. Potassium Chloride Solution
4. Giemsa Stain Solution
5.Trypsine
6.Methylalcohol and acetic acid
Procedure of karyotyping
Slide dehydration
• Dry slides on slide rack in 60oC oven
• Bake for 3 days
• Allow to cool before proceeding to the next
step
Treatment with enzyme
• Prepare 0.025% trypsin solution fresh, by mixing 5 ml of 0.25% trypsin with 45 ml Hank’s solution
• Immerse slide in 0.025 % trypsin for 10-120 seconds
• Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsinaction
Contd….
Staining
• Prepare a dilution of Giemsa stain by mixing 1 part of Giemsa stain with 3 parts of Phosphate buffer
• Flood slide with Giemsa stain for 2 minutes
• Rinse slides thoroughly with distilled water
• Allow slides to drain, then place on 60oC slide warming tray until completely dry
Contd…
Screening
How chromosomes are identified?
Normal male
Down syndrome
Turner syndrome
Klinefelter syndrome
FISH
Principles of FISH
• Basic elements of FISH are: DNA probe and target sequences
• Before hybridization, the DNA probe is labeled nick translation, random primed labeling or PCR
•The labeled probe and the target DNA are denatured
• The denatured probe and target allows the annealing of complementary DNA sequences.
Contd…
The procedure of FISH
• Denature the chromosomes
• Denature the probe
• Hybridization
• Fluorescence staining
• Examine slides or store in the dark
Technique
Screening
Multi-Color FISH
Translocation
THANK YOU