Human chromosomes identification by g banding
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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

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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


Human chromosomes identification by g banding 1173352

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


Human chromosomes identification by g banding 1173352

21 22 x y

Mazen Zaharna Molecular Biology 1/2009


Human chromosomes identification by g banding 1173352

Mazen Zaharna Molecular Biology 1/2009


Human chromosomes identification by g banding 1173352

Mazen Zaharna Molecular Biology 1/2009


Human chromosomes identification by g banding 1173352

Mazen Zaharna Molecular Biology 1/2009


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