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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.

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human chromosomes identification by g banding

Human Chromosomes Identification by G-Banding

Karyotyping

Mazen Zaharna Molecular Biology 1/2009

experiment objectives
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

human chromosomes
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

why do scientists look at chromosomes
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

chromosome abnormalities
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

situations where analysis is strongly recommended
Situations where analysis is strongly recommended
  • Problems with early growth & development
  • Fertility problems
  • Neoplasia
  • Pregnancy in older women

Mazen Zaharna Molecular Biology 1/2009

what is a karyotype
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

performing a karyotype
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

how do scientists identify chromosomes
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

slide10
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

chromosome banding
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

g banding
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

g banding1
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

chromosome groups
Chromosome Groups

Mazen Zaharna Molecular Biology 1/2009

chromosomal abnormalities
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

chromosomal abnormalities that can be detected by karyotyping
Chromosomal abnormalities that can be detected by karyotyping

Mazen Zaharna Molecular Biology 1/2009

chromosomal abnormalities that can be detected by karyotyping1
Chromosomal abnormalities that can be detected by karyotyping

Philadelphia Chromosome - CML

Mazen Zaharna Molecular Biology 1/2009

overview of procedure
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

overview of procedure1
Overview of Procedure
  • Slide dehydration
  • Treatment with enzyme
  • Staining

Mazen Zaharna Molecular Biology 1/2009

monitor the quality of chromosome spreading
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

7 slide dehydration
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

8 treatment with enzyme
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

determination of trypsin and staining time
Determination of Trypsin and Staining time

Mazen Zaharna Molecular Biology 1/2009

9 staining
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

slide25
21 22 x y

Mazen Zaharna Molecular Biology 1/2009

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