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Exp. 4 karyotyping
This document describes the process of karyotyping and identifying human chromosomes through G-banding. Key points include: - Karyotyping involves preparing, staining, and observing human chromosomes to diagnose genetic disorders and identify chromosomal abnormalities. - Humans normally have 23 pairs of chromosomes, including 22 autosomes and 1 sex chromosome. G-banding allows identification of chromosomes based on size, banding pattern, and centromere position. - The karyotyping process involves culturing cells, arresting cell division, staining chromosomes, and analyzing spreads under a microscope to identify numerical or structural abnormalities. G-banding produces dark and light bands that make each chromosome pair unique. - Common chrom
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Exp. 4 karyotyping
- 1. Human Chromosomes Identification byG-Banding Karyotyping Mazen Zaharna Molecular Biology 1/2009
- 2. Experiment Objectives • Preparing,Staining and Observing Gbanding human chromosomes • Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases. Mazen Zaharna Molecular Biology 1/2009
- 3. 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) Mazen Zaharna Molecular Biology 1/2009
- 4. Why do scientistslook 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. Mazen Zaharna Molecular Biology 1/2009
- 5. Chromosome abnormalities • Chromosomeabnormalities can be – numerical, as in the presence of • extra • or missing chromosomes, – or structural as in translocations, inversions, large scale deletions or duplications. Mazen Zaharna Molecular Biology 1/2009
- 6. Situations where analysisis strongly recommended Problems with early growth & development Fertility problems Neoplasia Pregnancy in older women Mazen Zaharna Molecular Biology 1/2009
- 7. What is aKaryotype? 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) Mazen Zaharna Molecular Biology 1/2009
- 8. 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. Mazen Zaharna Molecular Biology 1/2009
- 9. How Do ScientistsIdentify 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 Mazen Zaharna Molecular Biology 1/2009
- 10. 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
- 11. Chromosome banding • Chromosomesare 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 Mazen Zaharna Molecular Biology 1/2009
- 12. G-Banding Dye gives chromosomesa striped appearance because it stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs. Mazen Zaharna Molecular Biology 1/2009
- 13. G-Banding • Regions thatstain 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. Mazen Zaharna Molecular Biology 1/2009
- 14. 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. Mazen Zaharna Molecular Biology 1/2009
- 15. Chromosomal Abnormalities • Alterations inchromosome 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) Mazen Zaharna Molecular Biology 1/2009
- 16. Chromosomal abnormalities thatcan be detected by karyotyping Mazen Zaharna Molecular Biology 1/2009
- 17. Chromosomal abnormalities thatcan be detected by karyotyping Mazen Zaharna Molecular Philadelphia Chromosome - CML Biology 1/2009
- 18. Overview of Procedure 1. 2. 3. 4. Collectionof blood Cell culture Stopping the cell division at Metaphase Hypotonic treatment of red & white blood cells 5. Fixation 6. Slide preparation Mazen Zaharna Molecular Biology 1/2009
- 19. Overview of Procedure 7.Slide dehydration 8. Treatment with enzyme 9. Staining Mazen Zaharna Molecular Biology 1/2009
- 20. Monitor the qualityof 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 Mazen Zaharna Molecular Biology 1/2009
- 21. 7- Slide dehydration •Place fixed, dry slides on slide rack in 60 oC oven • Bake for 3 days • Allow to cool before proceeding to the next step Mazen Zaharna Molecular Biology 1/2009
- 22. 8- Treatment withenzyme • 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 10120 seconds • Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action Mazen Zaharna Molecular Biology 1/2009
- 23. Determination of Trypsinand Staining time Trypsin Time (seconds) Staining Time (minutes) Lymphoblastoid 30 4.0 Blood Lymphocytes 15 3.0 0-3 days 15 3.0 3-20 days 30 3.5 20+ days 45 4.0 Previously Banded 45 4.0 < 20 mitosis 15 3.0 20-50 mitosis 30 3.5 Cell Source Age of Oven Dried Slides Cell Concentration 50+ mitosis Mazen Zaharna Molecular 45 Biology 1/2009 4.5
- 24. 9- Staining • Preparea 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 60 oC slide warming tray until completely dry Mazen Zaharna Molecular Biology 1/2009
- 25. Mazen Zaharna Molecular 21 Biology1/2009 22 x y
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Editor's Notes
- #7 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).
- #12 G-Banding, C-Banding, Q-Banding, R-Banding, T-Banding