UNIT 7.1 : Karyotyping Procedure

Question 1

The number and appearance of chromosomes in the nucleus of a eukaryotic cell

Answer 1

Karyotype

Question 2

Information on the karyotype

Answer 2

• Size of chromosome
• Position of centromere
• Presence of secondary constrictions
• Size of satellites

Question 3

Comes from the Greek word “Karyon” which means nucleus

Answer 3

Karyotype

Question 4

The study of whole sets of chromosomes

Answer 4

Karyology

Question 5

The standard format of representing chromosomes as diagrams when the haploid set of chromosomes of an organism are ordered in a series of decreasing size

Answer 5

Idiogram or Karyogram

Question 6

Which type of Karyotype is this?

Show larger differences between smaller and larger chromosome in a set

Answer 6

Asymmetric Karyotype

Question 7

Which type of Karyotype is this?

Show lesser difference between smaller and larger chromosome in a set

Answer 7

Symmetric Karyotype

Question 8

Which type of Karyotype is this?

Have more acrocentric chromosomes (fewer metacentric chromosomes) and relatively advanced feature

Answer 8

Asymmetric Karyotype

Question 9

Which type of Karyotype is this?

Have more metaphase chromsomes and no advanced feature

Answer 9

Symmetric Karyotype

Question 10

a Russian scientist who suggested that in flowering plants, there is a predominant trend towards karyotype asymmetry

Answer 10

GA Levitzky (1931)

Question 11

Associated with more advanced features in an organism

Answer 11

Asymmetric karyotype

because it has evolved through structural chromosome changes

Question 12

Proportion of metacentric, acrocentric chromosomes in a set

Answer 12

Degree of Asymmetry

Question 13

Ratio between size of largest and smallest chromosomes in a set

Answer 13

Degree of Asymmetry

Question 14

How would you interpret the Degree of Asymmetry?

Answer 14

The Higher the proportion of acrocentric chromosomes, the greater the value of size ratio, more asymmetrical is a karyotype

Question 15

The process of pairing and ordering all the chromosomes of an organism, thus providing a genome-wide snapshot of an individual’s chromosomes

Answer 15

Karyotyping

Question 16

Preparing Karyotypes uses what standardized procedures that reveal characteristic structural features for each chromosome?

Answer 16

Staining procedures

Question 17

Karyotyping analysis can reveal what?

e.g. chromosomal translocations, deletions, duplications, or inversions

Answer 17

Subtle structural changes

Question 18

• It is stained darkly
• Mostly contain DNA repeating sequences

Answer 18

Heterochromatic band

Question 19

• Stain lightly
• Mainly contain protein and coding genes (play a large role in translation)

Answer 19

Euchromatic band

Question 20

Are less compact and more evenly spaced

makes DNA segments available for transcription and translation

Answer 20

Euchromatins

Question 21

Tightly packed together

Transcription factors cannot readily access DNA sequences, so they play a lesser role in transcription and translation

Answer 21

Heterochromatins

Question 22

The level of compaction is driven by what?

Answer 22

The methylation of DNA

Question 23

• Causes nucleosomes to pack tightly together
• Transcription factors cannot bind the DNA, and genes are not expressed

Answer 23

Methylation (DNA inaccessible, gene inactive)

Question 24

• Results in loose packing of nucleosomes
• Transcription factors can bind the DNA and genes are expressed

Answer 24

Acetylation (DNA accessible, gene active)

Question 25

What are the **Materials** used in the Karyotyping procedure?

Answer 25

1. Sterile 5mL syringe 2. 21-gauge syringe needle 3. Conical tubes (15mL) 4. Green-top vacutube (heparin) 5. Glass slides 6. Pasteur pipette 7. Pipettor and pipette tips 8. Serological pipettes

Question 26

What are the **Reagents** used in the Karyotyping procedure?

Answer 26

1. Glacial Acetic acid 2. Methanol 3. KCl (hypotonic solution) 4. RPMI growth medium 5. Fetal bovine serum 6. Phytohemagglutinin 7. Colcemid (Colchicine) 8. GIEMSA dye 9. Trypsin

Question 27

What are the **Equipment** used in the Karyotyping procedure?

Answer 27

1. Centrifuge 2. Incubator at 37°C CO2 3. Refrigerator 4. Inverted microscope

Question 28

What are the 12 steps in Karyotyping procedure?

Answer 28

1. Draw 10 to 20 mL of blood 2. Add few drops of blood 3. Add phutohemagglutinin to stimulate mitosis 4. Incubate at 37°C for 2 to 3 days 5. Add Colcemid to culture for 1 to 2 hours to stop mitosis in metaphase 6. Transfer cells to tube 7. Centrifuge to concentrate cells. Add low-salt solution to eliminate red blood cells and swell lymphocytes 8. Transfer to tube containing fixative 9. Stain slide with Giemsa 10. Drop cells onto microscope slide 11. Examine with microscope 12. Digitize chromosome images processed to make karyotype

Question 29

What are the **5 major steps** in Karyotyping?

Answer 29

1. Short term lymphocyte culture 2. Harvesting of Lymphocytes 3. Fixing the cells 4. Making the Chromosome slides 5. Slide analysis

Question 30

# (1)Regarding Short term Lymphocyte culture The reagent that **induces mitotic activity**

Answer 30

phytohemagglutinin (PHA)

Question 31

# (1) Regarding Short term Lymphocyte culture is typically RPMI which allows cells to grow

Answer 31

Cell culture growth medium

Question 32

# (1) Regarding Short term Lymphocyte culture A supplement that supports the growth of blood | provides nutrients to allow cells to survive

Answer 32

Fetal bovine serum

Question 33

# (1) Regarding Short term Lymphocyte culture Prevent microbial contamination ## Footnote Contamination invalidates the test

Answer 33

Antibiotics

Question 34

# (1) Regarding Short term Lymphocyte culture The cultured blood cells will be grown at __ °C incubator for __ days

Answer 34

37°C incubator for 3 days ## Footnote Temp is set at 37°C to mimic the normal body temperature

Question 35

# (2) Regarding Harvesting of Lymphocytes Addition of ____ ,the reagent that arrests the cell cycle at metaphase stage, into the culture and incubate for ___ mins

Answer 35

Colcemid (aka Colchicine) 15 minutes

Question 35

# (1) Regarding Short term Lymphocyte culture Cells must be in **what phase** because splitting of a cell line 2 days before harvesting and changing rthe medium 1 day before harvesting, stimulates cell proliferation significantly

Answer 35

Logarithmic phase ## Footnote The logarithmic phase means that there are more cells that are alive and are actively dividing compared to the cells that are dying

Question 36

# (2) Regarding Harvesting of Lymphocytes Colchicine causes ____, once the cells undergo this, no cell division occurs

Answer 36

spindle disruption

Question 37

# (2) Regarding Harvesting of Lymphocytes Optimal exposure time to colcemid requires a balance between what?

Answer 37

Between profilerative activity index of cells and concentration of colcemid ## Footnote If the cells have a HIGH proliferative index ( they can divide rapidly), they need a shorter exposure to a high concentration of colcemid.

Question 38

# (2) Regarding Harvesting of Lymphocytes Centrifuge the tube at ____ RPM for ____ minutes

Answer 38

1000 RPM 10 minutes

Question 39

# (2) Regarding Harvesting of Lymphocytes The cell pellet was resuspended in warm ____ solution (can be KCl or sodium citrate)

Answer 39

warm hypotonic solution ## Footnote The cells need to be swelled because it is easier to visualize the chromosomes if the cells are larger.

Question 40

# (2) Regarding Harvesting of Lymphocytes Incubate at room temperature for how many minutes?

Answer 40

15 minutes

Question 41

# (2) Regarding Harvesting of Lymphocytes can siginificantly increase the total yield of metaphase chromosomes

Answer 41

Cell synchronization

Question 42

# (3) FIxing the Cells The cell suspension in hypotonic state will be centrifuged for ____ RPM for ____ mins

Answer 42

1200 RPM 5 minutes

Question 43

# (3) FIxing the Cells The cell pellet will be treated **with what** and will be centrifuged at 1200 RPM for 5 mins

Answer 43

treated with fixative solution or Carnoy's fixative

Question 44

# (3) FIxing the Cells The process will be repeated ____ times, then the final addition of fixative solution will require incubation at ____ °C for ____ minutes

Answer 44

repeated 3 times incubation at 4°C 10 minutes

Question 45

# (4) Making the Chromosome Slides How many cold slides will be layered next to each other in a paper towel?

Answer 45

5 or 6 cold slides

Question 46

# (4) Making the Chromosome Slides How many drops of the samples will be dropped onto each slide and dry them spontaneously

Answer 46

2 or 3 drops

Question 47

# (4) Making the Chromosome Slides The slides will be stained by?

Answer 47

GTG-banding (G-bands by Trypsin using Giemsa)

Question 48

# (4) Making the Chromosome Slides most common method of staining chromosomes for differentation which uses trypsin | Trypsin is an enzyme ## Footnote Trypsin digests the chromosomes at regions rich in basic amino acids (Arg and Lys)

Answer 48

GTG-banding

Question 49

# (5) Slide Analysis Slides that will be choses for analysis and visualization must be?

Answer 49

* Properly trypsinized chromosomes * Clearly defined metaphase spreading

Question 50

# (5) Slide Analysis Slide analysis requires a microscope with automated computer software program primarily what software?

Answer 50

Cytovision™ by Applied Imaging Inc. ## Footnote which follows the International System of Human Cytogenetic Nomenclature (ISCN) that arrange chromosomes according to size and banding patterns