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Karyotype and FISH
The document outlines the procedures and principles of karyotyping and fluorescence in situ hybridization (FISH), detailing sample types, step-by-step methods, and reagents used. It explains the identification of chromosomal abnormalities, such as Down syndrome and Turner syndrome, through these techniques. Key procedures include cell culture, slide preparation, enzyme treatment, and staining, alongside basic principles of FISH involving DNA probes and hybridization.
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Karyotype and FISH
- 1. Karyotype and FISH Prof.Laila Anjuman Banu Chairman Department of Anatomy, BSMMU Dr. Subarna Das Resident, MS Anatomy Phase-A, Year-1, Block-2 Guided by- Presented by-
- 2. Objectives • Define karyotypeand FISH • Describe the procedure of karyotyping and FISH • Explain chromosomal abnormalities through karyotyping and FISH • Describe the principles of FISH
- 3. What is aKaryotype?
- 4. Type of samples •Peripheral blood • Cord blood • Bone marrow blood • Amniotic fluid • Products of conception
- 5. Overview of theprocedure 1. Collection of blood 2. Cell culture 3. Stopping the cell division at Metaphase 4. Hypotonic treatment of blood cells
- 6. Overview of theprocedure 5. Fixation 6. Slide preparation 7. Slide dehydration 8. Treatment with enzyme 9. Staining Contd….
- 7. Karyotyping Reagents 1. PhytohemagglutininM-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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- 9. Slide dehydration • Dryslides on slide rack in 60oC oven • Bake for 3 days • Allow to cool before proceeding to the next step
- 10. 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
- 11. • Remove slidefrom trypsin and immediately immerse in phosphate buffer to stop trypsin action Contd….
- 12. Staining • Prepare adilution of Giemsa stain by mixing 1 part of Giemsa stain with 3 parts of Phosphate buffer • Flood slide with Giemsa stain for 2 minutes
- 13. • Rinse slidesthoroughly with distilled water • Allow slides to drain, then place on 60oC slide warming tray until completely dry Contd…
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- 22. 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
- 23. •The labeled probeand the target DNA are denatured • The denatured probe and target allows the annealing of complementary DNA sequences. Contd…
- 25. The procedure ofFISH • Denature the chromosomes • Denature the probe • Hybridization • Fluorescence staining • Examine slides or store in the dark
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