karyotyping

KARYOTYPING

Definition of Karyotype

  • Karyotype: A photomicrograph of chromosomes arranged according to a standard classification.

Chromosome Arrangement

  • Chromosomes are digitally arranged to match with their homologue or partner chromosome.

  • Homologous Chromosomes:

    • Characteristics: Same size and shape, carry the same genes, one inherited from each parent.

    • Numbering: Chromosomes are numbered according to size.

Sex Determination using Karyotypes

  • A karyotype with 23 exact pairs indicates that the individual is female.

  • Notably, the #23 chromosomes are both X chromosomes.

CYTOGENETICS

  • Definition: Cytogenetics is the study of the structure and properties of chromosomes. It includes:

    • Chromosomal behavior during mitosis and meiosis.

    • Chromosomal influence on the phenotype.

    • Factors causing chromosomal changes (Hare and Singh, 1979).

Nomenclature of Chromosomes

  • Metaphase Chromosome Structure:

    • Centromere: The central part of a chromosome.

    • Sister Chromatids: Duplicated chromosomes attached at the centromere.

    • Short Arm (p) and Long Arm (q) of Chromosomes.

Centromeric Positions and Arm Length Classifications

  • Types of chromosomes based on centromeric position:

    • Metacentric: Centromere roughly in the middle.

    • Submetacentric: Centromere slightly off-center.

    • Acrocentric: Centromere significantly off-center.

    • Telocentric: Centromere at one end.

Techniques in Karyotyping

Amniocentesis

  • Definition: A procedure to obtain amniotic fluid which contains fetal cells.

    • Procedure:

    • Insert needle into the uterus.

    • Extract amniotic fluid.

Chorionic Villus Sampling (CVS)

  • Definition: A technique for obtaining fetal tissue by removing cells from the chorion.

Preparation of Chromosomes

  • Steps to prepare chromosomes for karyotyping:

    1. Add tissue sample.

    2. Culture in a growth medium.

    3. Add chemical to stimulate mitosis.

    4. Incubate for 2-3 days.

    5. Stop mitosis at metaphase using chemicals.

    6. Centrifuge for layering and concentrate cells.

    7. Put cells onto a microscope slide.

    8. Add stain to enhance visibility.

    9. Cut out, photograph, and arrange chromosomes into karyotype.

    10. Utilize fixative for preservation.

DNA Sample Collection for Karyotypes

  • Sources: Packed red and white blood cells (including lymphocytes).

  • Procedures:

    1. Centrifuge the blood sample.

    2. Use hypotonic solution to lyse cells and obtain supernatant.

    3. Apply fixative and stain for visibility.

Methodology for Karyotyping

  • Aseptic Precautions: Necessary to prevent contamination.

  • Cell Culture Preparation:

    • Prepare RPMI 1640 medium.

    • Collection of 10ml of blood with heparin.

    • Setting of culture involves:

    • 8 ml of medium.

    • 0.1 ml of PHA-M.

    • 0.5 ml of blood/plasma.

    • 2 ml of autologous plasma/FCS.

  • Incubation: At 37°C for 72 hours.

Harvesting of Culture

  • Spindle Inhibitors: Use of colchicine/colcemid (0.1 mg/ml).

  • Hypotonic Treatment: Use 0.075M KCl.

  • Fixation Process: (3:1 methanol : acetic acid).

  • Slide Preparation and Staining:

    • Stain slides with 4% Giemsa for 20-25 minutes.

    • Screening slides to study chromosome morphology.

    • Construction of karyotypes after staining.

Human Male Karyotype Example

  • Shows typical layout for karyotype with X and Y chromosomes.

Chromosome Banding Techniques

Types of Banding Methods:

  1. G-Banding

  2. Q-Banding

  3. C-Banding

  4. R-Banding

  5. T-Banding

  6. NOR-Banding

  7. High-Resolution Banding

  8. Restriction Endonuclease Banding

Q-Banding Procedure

  1. Dehydrate slides in decreasing concentrations of alcohol (90%, 70%, 50%).

  2. Rinse in distilled water.

  3. Wash in phosphate buffer at pH 6.8.

  4. Stain with quinacrine mustard or quinacrine dihydrochloride for 20 minutes.

  5. Rinse in phosphate buffer and mount in the same buffer.

  6. Examine under a fluorescent microscope.

C-Banding Procedure

  1. Treat slides in 0.2 N HCl for one hour at room temperature.

  2. Rinse in de-ionized water.

  3. Immerse in 1% barium hydroxide at 50°C for 5-15 minutes.

  4. Rinse in de-ionized water.

  5. Incubate at 60°C in 2XSSC buffer for one hour.

  6. Rinse, stain in 4% Giemsa stain for 90 minutes.

  7. Rinse, dry, and examine under oil immersion.

R-Banding Procedure

  1. Age slides for 7-10 days.

  2. Place slides in phosphate buffer pH 6.5 at 85°C, incubate for 20-25 minutes.

  3. Stain in 0.01% acridine orange for 4-6 minutes.

  4. Rinse, mount and examine under a fluorescent microscope.

T-Banding Procedure

  1. Age slides for 7 days.

  2. Place in PBS pH 5.0 at 87°C for 20-60 minutes.

  3. Stain in 3% Giemsa in phosphate buffer pH 6.8 at 87°C, leave for 5-30 minutes.

  4. Rinse in phosphate buffer, examine under a fluorescent microscope.

  5. Alternatively, use UV lamp after covering slides with a cover slip.

G-Banding Technique

  • Aging of good slides for 10 days followed by:

  1. Use of normal saline.

  2. Treated with 0.25% trypsin solution for 10-15 seconds.

  3. Immerse in 70% ethanol for a few minutes.

  4. Stain with 10% Giemsa for 6-10 minutes.

  5. Capture microphotographs of good spreads.

  6. Construction of G-banded karyotype.

Conclusion

  • Thank you for your kind attention!