UNIT 7.1 : Karyotyping Procedure Flashcards
The number and appearance of chromosomes in the nucleus of a eukaryotic cell
Karyotype
Information on the karyotype
- Size of chromosome
- Position of centromere
- Presence of secondary constrictions
- Size of satellites
Comes from the Greek word “Karyon” which means nucleus
Karyotype
The study of whole sets of chromosomes
Karyology
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
Idiogram or Karyogram
Which type of Karyotype is this?
Show larger differences between smaller and larger chromosome in a set
Asymmetric Karyotype
Which type of Karyotype is this?
Show lesser difference between smaller and larger chromosome in a set
Symmetric Karyotype
Which type of Karyotype is this?
Have more acrocentric chromosomes (fewer metacentric chromosomes) and relatively advanced feature
Asymmetric Karyotype
Which type of Karyotype is this?
Have more metaphase chromsomes and no advanced feature
Symmetric Karyotype
a Russian scientist who suggested that in flowering plants, there is a predominant trend towards karyotype asymmetry
GA Levitzky (1931)
Associated with more advanced features in an organism
Asymmetric karyotype
because it has evolved through structural chromosome changes
Proportion of metacentric, acrocentric chromosomes in a set
Degree of Asymmetry
Ratio between size of largest and smallest chromosomes in a set
Degree of Asymmetry
How would you interpret the Degree of Asymmetry?
The Higher the proportion of acrocentric chromosomes, the greater the value of size ratio, more asymmetrical is a karyotype
The process of pairing and ordering all the chromosomes of an organism, thus providing a genome-wide snapshot of an individual’s chromosomes
Karyotyping
Preparing Karyotypes uses what standardized procedures that reveal characteristic structural features for each chromosome?
Staining procedures
Karyotyping analysis can reveal what?
e.g. chromosomal translocations, deletions, duplications, or inversions
Subtle structural changes
- It is stained darkly
- Mostly contain DNA repeating sequences
Heterochromatic band
- Stain lightly
- Mainly contain protein and coding genes (play a large role in translation)
Euchromatic band
Are less compact and more evenly spaced
makes DNA segments available for transcription and translation
Euchromatins
Tightly packed together
Transcription factors cannot readily access DNA sequences, so they play a lesser role in transcription and translation
Heterochromatins
The level of compaction is driven by what?
The methylation of DNA
- Causes nucleosomes to pack tightly together
- Transcription factors cannot bind the DNA, and genes are not expressed
Methylation (DNA inaccessible, gene inactive)
- Results in loose packing of nucleosomes
- Transcription factors can bind the DNA and genes are expressed
Acetylation (DNA accessible, gene active)
What are the Materials used in the Karyotyping procedure?
- Sterile 5mL syringe
- 21-gauge syringe needle
- Conical tubes (15mL)
- Green-top vacutube (heparin)
- Glass slides
- Pasteur pipette
- Pipettor and pipette tips
- Serological pipettes
What are the Reagents used in the Karyotyping procedure?
- Glacial Acetic acid
- Methanol
- KCl (hypotonic solution)
- RPMI growth medium
- Fetal bovine serum
- Phytohemagglutinin
- Colcemid (Colchicine)
- GIEMSA dye
- Trypsin
What are the Equipment used in the Karyotyping procedure?
- Centrifuge
- Incubator at 37°C CO2
- Refrigerator
- Inverted microscope
What are the 12 steps in Karyotyping procedure?
- Draw 10 to 20 mL of blood
- Add few drops of blood
- Add phutohemagglutinin to stimulate mitosis
- Incubate at 37°C for 2 to 3 days
- Add Colcemid to culture for 1 to 2 hours to stop mitosis in metaphase
- Transfer cells to tube
- Centrifuge to concentrate cells. Add low-salt solution to eliminate red blood cells and swell lymphocytes
- Transfer to tube containing fixative
- Stain slide with Giemsa
- Drop cells onto microscope slide
- Examine with microscope
- Digitize chromosome images processed to make karyotype
What are the 5 major steps in Karyotyping?
- Short term lymphocyte culture
- Harvesting of Lymphocytes
- Fixing the cells
- Making the Chromosome slides
- Slide analysis
(1)Regarding Short term Lymphocyte culture
The reagent that induces mitotic activity
phytohemagglutinin (PHA)
(1) Regarding Short term Lymphocyte culture
is typically RPMI which allows cells to grow
Cell culture growth medium
(1) Regarding Short term Lymphocyte culture
A supplement that supports the growth of blood
provides nutrients to allow cells to survive
Fetal bovine serum
(1) Regarding Short term Lymphocyte culture
Prevent microbial contamination
Contamination invalidates the test
Antibiotics
(1) Regarding Short term Lymphocyte culture
The cultured blood cells will be grown at __ °C incubator for __ days
37°C incubator for 3 days
Temp is set at 37°C to mimic the normal body temperature
(2) Regarding Harvesting of Lymphocytes
Addition of ____ ,the reagent that arrests the cell cycle at metaphase stage, into the culture and incubate for ___ mins
Colcemid (aka Colchicine)
15 minutes
(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
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
(2) Regarding Harvesting of Lymphocytes
Colchicine causes ____, once the cells undergo this, no cell division occurs
spindle disruption
(2) Regarding Harvesting of Lymphocytes
Optimal exposure time to colcemid requires a balance between what?
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.
(2) Regarding Harvesting of Lymphocytes
Centrifuge the tube at ____ RPM for ____ minutes
1000 RPM
10 minutes
(2) Regarding Harvesting of Lymphocytes
The cell pellet was resuspended in warm ____ solution (can be KCl or sodium citrate)
warm hypotonic solution
The cells need to be swelled because it is easier to visualize the chromosomes if the cells are larger.
(2) Regarding Harvesting of Lymphocytes
Incubate at room temperature for how many minutes?
15 minutes
(2) Regarding Harvesting of Lymphocytes
can siginificantly increase the total yield of metaphase chromosomes
Cell synchronization
(3) FIxing the Cells
The cell suspension in hypotonic state will be centrifuged for ____ RPM for ____ mins
1200 RPM
5 minutes
(3) FIxing the Cells
The cell pellet will be treated with what and will be centrifuged at 1200 RPM for 5 mins
treated with fixative solution or Carnoy’s fixative
(3) FIxing the Cells
The process will be repeated ____ times, then the final addition of fixative solution will require incubation at ____ °C for ____ minutes
repeated 3 times
incubation at 4°C
10 minutes
(4) Making the Chromosome Slides
How many cold slides will be layered next to each other in a paper towel?
5 or 6 cold slides
(4) Making the Chromosome Slides
How many drops of the samples will be dropped onto each slide and dry them spontaneously
2 or 3 drops
(4) Making the Chromosome Slides
The slides will be stained by?
GTG-banding (G-bands by Trypsin using Giemsa)
(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)
GTG-banding
(5) Slide Analysis
Slides that will be choses for analysis and visualization must be?
- Properly trypsinized chromosomes
- Clearly defined metaphase spreading
(5) Slide Analysis
Slide analysis requires a microscope with automated computer software program primarily what software?
Cytovision™ by Applied Imaging Inc.
which follows the International System of Human Cytogenetic Nomenclature (ISCN) that arrange chromosomes according to size and banding patterns
