Human Chromosomes Identification by G-Banding Karyotyping Mazen Zaharna Molecular Biology 1/2009

Experiment Objectives Preparing, Staining and Observing G-banding human chromosomes Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases. MOLECULAR BIOLOGY

Human Chromosomes A “normal” human carries 23 PAIRS of chromosomes (1 set came from the mother, 1 set came from the father) 22 of these sets are called autosomes (or “self chromosomes”) 1 set are the sex chromosomes A female carries two X chromosomes (XX) A male carries an X chromosome and a Y chromosome (XY) MOLECULAR BIOLOGY

Chromosome abnormalities Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural as in translocations, inversions, large scale deletions or duplications. nversions are of two types: paracentric and pericentric. chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologouschromosomes MOLECULAR BIOLOGY

Chromosomal abnormalities that can be detected by karyotyping t(9;22)(q34;q11) Philadelphia Chromosome - CML MOLECULAR BIOLOGY

cri-du-chat syndrome Chromosome 5p deletion in cri-du-chat syndrome Mazen Zaharna Molecular Biology 1/2009

Chromosomal Abnormalities Alterations in chromosome number. Euploid - normal set (2n) Polyploidy – extra set of the entire genome. (3n, 4n etc) Aneuploidy – the number of chromosomes is not a multiple of the normal haploid number. Monosomy one member of a chromosome pair is missing, (2n-1) Trisomy one chromosome set consists of 3 copies of a chromosome, (2n+1) MOLECULAR BIOLOGY

Chromosomal abnormalities that can be detected by karyotyping MOLECULAR BIOLOGY

Why do scientists look at chromosomes? Scientists can diagnose or predict genetic disorders by looking at chromosomes. This kind of analysis is used in prenatal testing and in diagnosing certain disorders, such as Down syndrome, or in diagnosing a specific types of leukemia. MOLECULAR BIOLOGY

Situations where analysis is strongly recommended Problems with early growth & development Fertility problems Neoplasia Pregnancy in older women Chorionic villus sampling (CVS) is a form of prenatal diagnosis to determine chromosomal or genetic disorders in the fetus. It entails getting a sample of the chorionic villus ( placental tissue) and testing it. The advantage of CVS is that it can be carried out 10-13 weeks after the last period, earlier than amniocentesis (which is carried out at 15-18 weeks). MOLECULAR BIOLOGY

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) MOLECULAR BIOLOGY

Mazen Zaharna Molecular Biology 1/2009 21 22 x y Mazen Zaharna Molecular Biology 1/2009

How Do Scientists Identify Chromosomes? Three key features to identify their 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. Using these key features, scientists match up the 23 pairs MOLECULAR BIOLOGY

Mazen Zaharna Molecular Biology 1/2009 In metacentric chromosomes, the centromere lies near the center of the chromosome. Submetacentric & very Submetacentric chromosomes, have a centromere that is off-center, so that one chromosome arm is longer than the other. In acrocentric chromosomes, the centromere resides very near one end. Mazen Zaharna Molecular Biology 1/2009

Performing a Karyotype The slides are scanned for metaphase spreads and usually 10 to 30 cells are analyzed under the microscope by a cytogeneticist. When a good spread (minimum number of overlapping chromosomes) is found, a photograph is taken or the analysis is done by a computer. The chromosomes are arranged in a standard presentation format of longest to shortest. MOLECULAR BIOLOGY

Chromosome banding Chromosomes are stained with various dyes enabling the chromosome segments to be identified Most methods can distinguish 550 bands/ haploid set High resolution methods can distinguish up to 850 bands/ haploid set that can allow identification of small interstitial deletions G-Banding, C-Banding, Q-Banding, R-Banding, T-Banding Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes:[16] G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands - the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich. This method will normally produce 300-400 bands in a normal, human genome. R-banding is the reverse of G-banding (the R stands for "reverse"). The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres. Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding. T-banding: visualize telomeres. Silver staining: Silver nitrate stains the nucleolar organization region-associated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR. MOLECULAR BIOLOGY

G-Banding Dye gives chromosomes a striped appearance because it stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs. MOLECULAR BIOLOGY

G-Banding Regions that stain as dark G bands replicate late in S phase of the cell cycle and contain more condensed chromatin, While light G bands generally replicate early in S phase, and have less condensed chromatin. MOLECULAR BIOLOGY

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 with satellites E 16–18 Small; 16 is metacentric but 17 and 18 are submetacentric F 19,20 Small; metacentric G 21,22,Y Small; acrocentric, with satellites on 21 and 22 but not on the Y Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller than chromosome 22. MOLECULAR BIOLOGY

Overview of Procedure Collection of blood Cell culture Stopping the cell division at Metaphase Hypotonic treatment of red & white blood cells Fixation Slide preparation MOLECULAR BIOLOGY

Overview of Procedure Slide dehydration Treatment with enzyme Staining MOLECULAR BIOLOGY

Monitor the quality of chromosome spreading Monitor the quality of chromosome spreading under phase contrast. Chromosomes should be well spread without visible cytoplasm, should appear dark grey under phase contrast MOLECULAR BIOLOGY

7- Slide dehydration Place fixed, dry slides on slide rack in 60oC oven Bake for 3 days Allow to cool before proceeding to the next step MOLECULAR BIOLOGY

8- 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 trypsin action MOLECULAR BIOLOGY

Determination of Trypsin and Staining time Trypsin Time (seconds) Staining Time (minutes) Cell Source Lymphoblastoid 30 4.0 Blood Lymphocytes 15 3.0 Age of Oven Dried Slides 0-3 days 3-20 days 3.5 20+ days 45 Cell Concentration < 20 mitosis 20-50 mitosis 50+ mitosis 4.5 MOLECULAR BIOLOGY

9- 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 MOLECULAR BIOLOGY

Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 DOWN SYNDROME Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 DOWN SYNDROME Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 TURNER SYNDROM Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 DELETION IN X Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 DEL IN X Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 RING X Mazen Zaharna Molecular Biology 1/2009

Robertsonian translocation IN 14  occurs in the five acrocentric chromosome pairs, namely 13, 14, 15, 21 and22.  Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 KLINFILTER SYNDROME Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 Triple X syndrome  Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009 DOUBLE YY SYNDROME Mazen Zaharna Molecular Biology 1/2009

Edwards syndrome TRISOMY 18 Mazen Zaharna Molecular Biology 1/2009

Patau Syndrome TRISOMY 13 Mazen Zaharna Molecular Biology 1/2009