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mt 6315 (cytogenetics) > UNIT 7.1 : Karyotyping Procedure > Flashcards

UNIT 7.1 : Karyotyping Procedure Flashcards

1
Q

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

A

Karyotype

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2
Q

Information on the karyotype

A
  • Size of chromosome
  • Position of centromere
  • Presence of secondary constrictions
  • Size of satellites
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3
Q

Comes from the Greek word “Karyon” which means nucleus

A

Karyotype

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4
Q

The study of whole sets of chromosomes

A

Karyology

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5
Q

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

A

Idiogram or Karyogram

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6
Q

Which type of Karyotype is this?

Show larger differences between smaller and larger chromosome in a set

A

Asymmetric Karyotype

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7
Q

Which type of Karyotype is this?

Show lesser difference between smaller and larger chromosome in a set

A

Symmetric Karyotype

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8
Q

Which type of Karyotype is this?

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

A

Asymmetric Karyotype

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9
Q

Which type of Karyotype is this?

Have more metaphase chromsomes and no advanced feature

A

Symmetric Karyotype

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10
Q

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

A

GA Levitzky (1931)

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11
Q

Associated with more advanced features in an organism

A

Asymmetric karyotype

because it has evolved through structural chromosome changes

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12
Q

Proportion of metacentric, acrocentric chromosomes in a set

A

Degree of Asymmetry

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13
Q

Ratio between size of largest and smallest chromosomes in a set

A

Degree of Asymmetry

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14
Q

How would you interpret the Degree of Asymmetry?

A

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

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15
Q

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

A

Karyotyping

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16
Q

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

A

Staining procedures

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17
Q

Karyotyping analysis can reveal what?

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

A

Subtle structural changes

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18
Q
  • It is stained darkly
  • Mostly contain DNA repeating sequences
A

Heterochromatic band

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19
Q
  • Stain lightly
  • Mainly contain protein and coding genes (play a large role in translation)
A

Euchromatic band

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20
Q

Are less compact and more evenly spaced

makes DNA segments available for transcription and translation

A

Euchromatins

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21
Q

Tightly packed together

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

A

Heterochromatins

22
Q

The level of compaction is driven by what?

A

The methylation of DNA

23
Q
  • Causes nucleosomes to pack tightly together
  • Transcription factors cannot bind the DNA, and genes are not expressed
A

Methylation (DNA inaccessible, gene inactive)

24
Q
  • Results in loose packing of nucleosomes
  • Transcription factors can bind the DNA and genes are expressed
A

Acetylation (DNA accessible, gene active)

25
Q

What are the Materials used in the Karyotyping procedure?

A
  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
26
Q

What are the Reagents used in the Karyotyping procedure?

A
  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
27
Q

What are the Equipment used in the Karyotyping procedure?

A
  1. Centrifuge
  2. Incubator at 37°C CO2
  3. Refrigerator
  4. Inverted microscope
28
Q

What are the 12 steps in Karyotyping procedure?

A
  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
29
Q

What are the 5 major steps in Karyotyping?

A
  1. Short term lymphocyte culture
  2. Harvesting of Lymphocytes
  3. Fixing the cells
  4. Making the Chromosome slides
  5. Slide analysis
30
Q

(1)Regarding Short term Lymphocyte culture

The reagent that induces mitotic activity

A

phytohemagglutinin (PHA)

31
Q

(1) Regarding Short term Lymphocyte culture

is typically RPMI which allows cells to grow

A

Cell culture growth medium

32
Q

(1) Regarding Short term Lymphocyte culture

A supplement that supports the growth of blood

provides nutrients to allow cells to survive

A

Fetal bovine serum

33
Q

(1) Regarding Short term Lymphocyte culture

Prevent microbial contamination

Contamination invalidates the test

A

Antibiotics

34
Q

(1) Regarding Short term Lymphocyte culture

The cultured blood cells will be grown at __ °C incubator for __ days

A

37°C incubator for 3 days

Temp is set at 37°C to mimic the normal body temperature

35
Q

(2) Regarding Harvesting of Lymphocytes

Addition of ____ ,the reagent that arrests the cell cycle at metaphase stage, into the culture and incubate for ___ mins

A

Colcemid (aka Colchicine)
15 minutes

35
Q

(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

A

Logarithmic phase

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

36
Q

(2) Regarding Harvesting of Lymphocytes

Colchicine causes ____, once the cells undergo this, no cell division occurs

A

spindle disruption

37
Q

(2) Regarding Harvesting of Lymphocytes

Optimal exposure time to colcemid requires a balance between what?

A

Between profilerative activity index of cells and concentration of colcemid

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

38
Q

(2) Regarding Harvesting of Lymphocytes

Centrifuge the tube at ____ RPM for ____ minutes

A

1000 RPM
10 minutes

39
Q

(2) Regarding Harvesting of Lymphocytes

The cell pellet was resuspended in warm ____ solution (can be KCl or sodium citrate)

A

warm hypotonic solution

The cells need to be swelled because it is easier to visualize the chromosomes if the cells are larger.

40
Q

(2) Regarding Harvesting of Lymphocytes

Incubate at room temperature for how many minutes?

A

15 minutes

41
Q

(2) Regarding Harvesting of Lymphocytes

can siginificantly increase the total yield of metaphase chromosomes

A

Cell synchronization

42
Q

(3) FIxing the Cells

The cell suspension in hypotonic state will be centrifuged for ____ RPM for ____ mins

A

1200 RPM
5 minutes

43
Q

(3) FIxing the Cells

The cell pellet will be treated with what and will be centrifuged at 1200 RPM for 5 mins

A

treated with fixative solution or Carnoy’s fixative

44
Q

(3) FIxing the Cells

The process will be repeated ____ times, then the final addition of fixative solution will require incubation at ____ °C for ____ minutes

A

repeated 3 times
incubation at 4°C
10 minutes

45
Q

(4) Making the Chromosome Slides

How many cold slides will be layered next to each other in a paper towel?

A

5 or 6 cold slides

46
Q

(4) Making the Chromosome Slides

How many drops of the samples will be dropped onto each slide and dry them spontaneously

A

2 or 3 drops

47
Q

(4) Making the Chromosome Slides

The slides will be stained by?

A

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

48
Q

(4) Making the Chromosome Slides

most common method of staining chromosomes for differentation which uses trypsin

Trypsin is an enzyme

Trypsin digests the chromosomes at regions rich in basic amino acids (Arg and Lys)

A

GTG-banding

49
Q

(5) Slide Analysis

Slides that will be choses for analysis and visualization must be?

A
  • Properly trypsinized chromosomes
  • Clearly defined metaphase spreading
50
Q

(5) Slide Analysis

Slide analysis requires a microscope with automated computer software program primarily what software?

A

Cytovision™ by Applied Imaging Inc.

which follows the International System of Human Cytogenetic Nomenclature (ISCN) that arrange chromosomes according to size and banding patterns

mt 6315 (cytogenetics) (11 decks)

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