Karyotype and FISH

Define karyotype and FISH
Describe the procedure of karyotyping and FISH
Explain chromosomal abnormalities through karyotyping and FISH
Describe the principles of FISH

Presented by-
Dr. Subarna Das
Resident, MS Anatomy
Phase-A, Year-1, Block-2

Guided by-
Prof. Laila Anjuman Banu
Chairman
Department of Anatomy, BSMMU

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 karyotype and FISH Describe the procedure of karyotyping and FISH Explain chromosomal abnormalities through karyotyping and FISH Describe the principles of FISH

3. What is a Karyotype ?

4. Type of samples Peripheral blood Cord blood Bone marrow blood Amniotic fluid Products of conception

5. Overview of the procedure 1. Collection of blood 2. Cell culture 3. Stopping the cell division at Metaphase 4. Hypotonic treatment of blood cells

6. Overview of the procedure 5. Fixation 6. Slide preparation 7. Slide dehydration 8. Treatment with enzyme 9. Staining Contd ….

7. 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

8. Procedure of karyotyping

9. Slide dehydration Dry slides 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 slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action Contd ….

12. 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

13. Rinse slides thoroughly with distilled water Allow slides to drain, then place on 60oC slide warming tray until completely dry Contd …

14. Screening

15. How chromosomes are identified?

16. Normal male

17. Down syndrome

18. Turner syndrome

19. Klinefelter syndrome

20. FISH

21. 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

22. The labeled probe and the target DNA are denatured The denatured probe and target allows the annealing of complementary DNA sequences. Contd …

23. The procedure of FISH Denature the chromosomes Denature the probe Hybridization Fluorescence staining Examine slides or store in the dark

24. Technique

25. Screening

26. Multi-Color FISH

27. Translocation

28. THANK YOU