1 / 28

Human Chromosomes Identification by G-Banding

Human Chromosomes Identification by G-Banding. Karyotyping. Experiment Objectives. Preparing, Staining and Observing G-banding human chromosomes Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases. Human Chromosomes.

dyllis
Télécharger la présentation

Human Chromosomes Identification by G-Banding

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. Experiment Objectives • Preparing, Staining and Observing G-banding 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 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. Mazen Zaharna Molecular Biology 1/2009

  5. 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. Mazen Zaharna Molecular Biology 1/2009

  6. Situations where analysis is strongly recommended • Problems with early growth & development • Fertility problems • Neoplasia • Pregnancy in older women Mazen Zaharna Molecular Biology 1/2009

  7. 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) 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 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 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 • 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 Mazen Zaharna Molecular Biology 1/2009

  12. 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. Mazen Zaharna Molecular Biology 1/2009

  13. 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. Mazen Zaharna Molecular Biology 1/2009

  14. Chromosome Groups Mazen Zaharna Molecular Biology 1/2009

  15. 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) Mazen Zaharna Molecular Biology 1/2009

  16. Chromosomal abnormalities that can be detected by karyotyping Mazen Zaharna Molecular Biology 1/2009

  17. Chromosomal abnormalities that can be detected by karyotyping Philadelphia Chromosome - CML Mazen Zaharna Molecular Biology 1/2009

  18. Overview of Procedure • Collection of blood • Cell culture • Stopping the cell division at Metaphase • Hypotonic treatment of red & white blood cells • Fixation • Slide preparation Mazen Zaharna Molecular Biology 1/2009

  19. Overview of Procedure • Slide dehydration • Treatment with enzyme • Staining Mazen Zaharna Molecular Biology 1/2009

  20. 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 Mazen Zaharna Molecular Biology 1/2009

  21. 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 Mazen Zaharna Molecular Biology 1/2009

  22. 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 Mazen Zaharna Molecular Biology 1/2009

  23. Determination of Trypsin and Staining time Mazen Zaharna Molecular Biology 1/2009

  24. 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 Mazen Zaharna Molecular Biology 1/2009

  25. 21 22 x y Mazen Zaharna Molecular Biology 1/2009

  26. Mazen Zaharna Molecular Biology 1/2009

  27. Mazen Zaharna Molecular Biology 1/2009

  28. Mazen Zaharna Molecular Biology 1/2009

More Related