karyotype and fish
TRANSCRIPT
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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-
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Objectives
• Define karyotype and FISH
• Describe the procedure of karyotyping and FISH
• Explain chromosomal abnormalities through karyotyping and FISH
• Describe the principles of FISH
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What is a Karyotype?
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Type of samples
• Peripheral blood
• Cord blood
• Bone marrow blood
• Amniotic fluid
• Products of conception
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Overview of the procedure
1. Collection of blood
2. Cell culture
3. Stopping the cell division at Metaphase
4. Hypotonic treatment of blood cells
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Overview of the procedure
5. Fixation
6. Slide preparation
7. Slide dehydration
8. Treatment with enzyme
9. Staining
Contd….
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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
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Procedure of karyotyping
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Slide dehydration
• Dry slides on slide rack in 60oC oven
• Bake for 3 days
• Allow to cool before proceeding to the next
step
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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
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• Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsinaction
Contd….
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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
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• Rinse slides thoroughly with distilled water
• Allow slides to drain, then place on 60oC slide warming tray until completely dry
Contd…
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Screening
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How chromosomes are identified?
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Normal male
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Down syndrome
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Turner syndrome
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Klinefelter syndrome
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FISH
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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
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•The labeled probe and the target DNA are denatured
• The denatured probe and target allows the annealing of complementary DNA sequences.
Contd…
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The procedure of FISH
• Denature the chromosomes
• Denature the probe
• Hybridization
• Fluorescence staining
• Examine slides or store in the dark
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Technique
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Screening
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Multi-Color FISH
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Translocation
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THANK YOU