Karyotyping – 1

What is a Karyotype? A display or photomicrograph of an individual’s somatic-cell metaphase chromosomes that are arranged in a standard sequence (usually based on number, size, and type)

How Do Scientists Identify Chromosomes? Three key features to identify chromosomes similarities and differences: Size. This is the easiest way to tell two different chromosomes apart. Banding pattern. The size and location of Giemsa bands on chromosomes make each chromosome pair unique. Centromere position. Centromeres are regions in chromosomes that appear as a constriction.

Metacentric chromosomes, the centromere lies near the center of the chromosome. Submetacentric , have a centromere that is off-center, so that one chromosome arm is longer than the other. Acrocentric chromosomes, the centromere resides very near one end.

Chromosome Groups Group Chromosomes Description A 1–3 Largest; 1 and 3 are metacentric but 2 is submetacentric B 4,5 Large; submetacentric with two arms very different in size C 6–12,X Medium size; submetacentric D 13–15 Medium size; acrocentric E 16–18 Small; 16 is metacentric but 17 and 18 are submetacentric F 19,20 Small; metacentric G 21,22,Y Small; acrocentric Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller than chromosome 22.

Chromosome Groups

Chromosomes Banding Chromosomes are stained with various dyes enabling the chromosome segments to be identified G – Banding It is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes by Giemsa stain or dye. The Dye gives chromosomes a striped appearance because it stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs.

G – Banding The Dye stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs. AT rich regions appear as Dark bands ( G+ bands) AT Poor regions appear as Light bands (G- bands)

Peripheral Blood Cultured Skin Fibroblast Bone Marrow Amniotic Fluid Lab Procedure SPECIMENS USED Peripheral Blood Cultured Skin Fibroblast Bone Marrow Amniotic Fluid

Lab Procedure SPECIMENS USED Product of conception (POV)

Lab Procedure for blood speciemens Vinous Blood collection with sterile condetions in Heparine tube but not EDTA Cell culture (the target cells are the lymphocytes) Culture the blood samples (lymphocytes)in a growth medium for 2 – 3 days at 37 degree and stimulate mitosis The growth culture contains A growth base like RPMI L-glutamine  support cell growth Bovine serum albumine Antibiotics Phytohaemagglutinin as lymphocytes growth stimulus or mitogen

Lab Procedure for blood speciemens Stopping the cell division at Metaphase by adding Colchicine after 72 hours, Colchicine stops the cell division by inhibiting the microtubules polymerization at the Metaphase so that the chromosomes sticks in the metaphase and do not separate

Lab Procedure for blood speciemens Hypotonic treatment of red & white blood cells Potassium chloride This solution causes the white blood cells to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic solution

Lab Procedure for blood speciemens Fixation : the cells are Fixed a solution which contain (3:1 methanol to glacial acetic acid) Slides preparation Dropping the swelled & fixed cells on the glass slides by pasture pipit  this leads to cells explosion and the chromosomes spreads on the slides Air dry of the slides Heat dry at oven

Lab Procedure for blood speciemens Slides staining Using Giemsa stain that stains the AT rich regions and establish the banding pattern of the chromosomes

Reading a Karyotype slides Search for a good 10 – 20 views Select the chromosomes one by one and do the following Count the number of chromosome in each view field Draw them on a paper (now days by using the digital camera the technician can take a photo for the field) Arrange them in order by writing the order or number of each chromosome near it on the paper (now days this is computerized)

Reading a Karyotype slides

Chromosomes shapes

Chromosomes shapes