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