MT6315 L7 KARYOTYPING Flashcards
Defined as the number and appearance of chromosome in the nucleus of a eukaryotic cell
Karyotype
Karyotypes are usually used in what kind of cytogenetics?
Classical
A karyotype is a product of?
Karyotyping
Usual information seen in a karyotype includes?
Size of chromosome
Position of chromosome
Presence of secondary constrictions
Size of satellites
What are satellites?
Regions of the chromosome near the centromere
Contains repeated DNA that do not code for proteins
Centromere
T or F: Karyotyping starts from staining and viewing
F, starts from cell harvesting or culturing
If the chromosomes are almost the same size, how do you determine the chromosomal order?
Determining the position of the centromeres
Karyotype comes from the Greek word _____ which means ______
Karyon
nucleus
Study of whole sets of chromosomes
Karyology
What are commonly viewed in karyotypes?
Aneuploidies
What stage of the cell is usually needed in karyotyping?
Metaphase (condensed form of the cell)
Standard format of representing chromosomes as diagram when the haploid set of chromosomes of an organism are ordered in a series of decreasing size.
Idiogram or Karyogram
Idiogram or Karyogram is a standard format of representing chromosomes as diagram when the ______ set of chromosomes of an organism are ordered in a series of ______ size.
haploid
decreasing
Asymmetric or Symmetric Karyotype: Show larger differences between smaller and larger chromosome in a set
Asymmetric
Asymmetric or Symmetric Karyotype: Have more acrocentric chromosomes and relatively advanced feature.
Asymmetric
Asymmetric or Symmetric Karyotype: Show lesser difference between smaller and larger chromosome in a set.
Symmetric
Asymmetric or Symmetric Karyotype: Have more metaphase chromosomes and no advanced feature.
Symmetric
Who suggested that in flowering plants there is a predominant trend towards karyotype asymmetry?
G.A. Levitzky
What did G.A. Levitzky propose or suggest regarding flowering plants?
There is a predominant trend towards karyotype asymmetry.
The “trend” proposed by G.A. Levitzky was carefully studied in?
the genus Crepis of the family compositae.
Species showing a greater asymmetry is (more/less) advanced.
More
Degree of asymmetry is the proportion of?
Metacentric and acrocentric chromosomes in a set
Ratio between size of largest and smallest chromosomes in a set.
Degree of asymmetry
What factors contribute to the increased asymmetry of a chromosome?
Higher the proportion of acrocentric chromosomes, Greater the value of size ratio
Process of pairing and ordering all the chromosomes of an organism
Karyotyping
Provides a genome-wide snapshot of an individual’s
chromosomes.
Karyotyping
How can screening for aneuploidies pre-natally happen?
via Amniocentesis
Karyotypes are prepared using?
Standardized staining procedures that reveal characteristic structural features for each chromosome.
What is the most commonly used standardized staining procedure for Karyotypes?
Giemsa staining
What can karyotyping analysis reveal?
subtle structural changes such as chromosomal translocations, deletions, duplications, or inversions.
changes in chromosome number associated with aneuploid conditions.
Light staining band is (heterochromatic/euchromatic)
Euchromatic
________ consists of less amount of DNA lightly compressed with the histone proteins.
Euchromatin
_________ consists of more amount of DNA tightly compressed with the histone proteins.
Heterochromatin
Which has more proteins, euchromatin or heterochromatin?
Heterochromatin
Causes nucleosomes to pack tightly together.
Transcription factors cannot bind the DNA and genes are not expressed.
Methylation
Results in loose packing of nucleosomes.
Transcription factors can bind the DNA and genes are expressed.
Acetylation
What are the materials used in karyotyping?
Sterile 5 mL syringe
21-gauge syringe needle
Conical tubes (15 mL)
Green-top Vacutube
Glass slides
Pasteur Pipette
Pipettor and Pipette tips
Serological pipettes
What are the reagents used in karyotyping?
Glacial acetic acid
Methanol
KCl (hypotonic solution)
RPMI Growth medium
Fetal Bovine serum
Phytohemagglutinin
Colcemid
Giemsa dye
Trypsin
What are the equipment used in karyotyping?
Centrifuge
Incubator (37C at CO2)
Refrigerator
Inverted microscope
What reagent bursts unneeded cells?
Glacial acetic acid
What lyses or bursts cells?
KCl
What ensures lymphocyte survival, only has decarbonate and no CO2?
RPMI
What reagent is a mitogen and lectin?
Phytohemagglutinin
What are the 2 kinds of giemsa dye?
Ethanol blue
Eosin
What kind of cells are used in karyotypes and contain mature RBCs?
Nucleated cells
What are the steps in karyotyping? (Hint: sa picture)
- Draw blood (10mL - 20mL)
- Add few drops of blood
- Add phytohemagglutinin to stimulate mitosis
- Incubate for 37C for 2-3 days
- Add colcemid to culture for 1-2 hours to stop mitosis in metaphase
- Transfer cells to tube
- Transfer to tube containing fixative
- Stain slide with Giemsa
- Drop on the slide
- Centrifuge to concentrate cells, add low-salt solution to eliminate RBCs and swell lymphocytes
- Examine with microscope
- Digitized 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
The collected blood will be grown in vitro by adding what reagents?
cell culture growth medium, fetal bovine serum, antibiotics, and phytohemagglutinin (PHA)
What reagent induces mitotic activity?
PHA
The cultured blood cells will be grown at ____ °C incubator for __ days
37
3
The cells must be in what phase to stimulate short term lymphocyte culture?
Logarithmic phase
Why must the cells (in step 1 of karyotyping) be in the logarithmic phase?
because splitting of a cell line 2 days before harvesting, and changing the medium 1 day before harvesting stimulates cell proliferation significantly.
What derivate of colchicine arrests the cell cycle at metaphase stage?
Pre-warmed Colcemid
How long should the cell in colcemid be incubated?
15mins
Optimal exposure time to colcemid requires a balance between?
proliferative activity index of cells and concentration of colcemid
The tube should be centrifuged for how many RPM and for low long?
1000 RPM
10mins
After centrifugation (step 2/5), the cell pellets must be?
resuspended in warm hypotonic solution (can be KCl or sodium citrate) and the solution was mixed.
In step 2 of karyotyping, the tube must be incubated at what temperature for how long?
Room temp
15mins
What are the additional modifications that allow for enrichment of long (prometaphase) chromosomes?
using Actinomycin D or ethidium bromide (added before harvesting), or bromodeoxyuridine (BrdU), added before colcemid treatment.
What can significantly increase the total yield of metaphase chromosomes?
Cell synchronization
Cells are arrested at [what phase] by adding an [what for how long]. After this, the block is released by [what process] and adding [what reagent] for [how many hours] before [what treatment].
S phase
excess amount of BrdU overnight (16 h)
washing the cells
thymidine
5.5 h
colcemid treatment
The cell suspension in hypotonic state will be centrifuged for [what RPM for how long].
1200 RPM for 5 mins.
The cell pellet will be treated with fixative solution [composed of what] or [what fixative] and will be centrifuged at [how much RPM and how long]
absolute methanol:glacial acetic acid; 3:1
Carnoy’s fixative
1200 RPM for 5 mins
The process of fixing the cells will be repeated [how many times] and the final addition of fixative solution will require incubation at [what temperature for how long]
3x
4 °C for 10 mins.
In making the chromosome slides, how many cold slides will be layered next to each other and how many drops of the samples will be dropped on each slide?
5-6 cold slides
2-3 drops
The chromosome slide will be stained by _______________, the most common method of staining chromosomes for differentiation which uses _____ that digests the chromosomes at regions rich in ____________.
GTG-banding (G-bands by Trypsin using Giemsa)
trypsin
basic amino acids (Arg and Lys).
Slides that will be chosen for analysis and visualization must be?
Properly trypsinized chromosomes
Clearly defined metaphase spreading
Slide analysis requires what equipment?
microscope with automated computer software program primarily, CytovisionTM by Applied Imaging Inc. which follows the International System of Human Cytogenetic Nomenclature (ISCN) that arrange chromosomes according to size and banding patterns.
What is a part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or lighter with various banding methods?
A band
Who published the first paper describing the use of quinacrine mustard to stain chromosome thereby ushered in a new era of chromosome banding?
Caspersson et al in 1958
What did Caspersson et al’s paper describe?
use of quinacrine mustard to stain chromosome thereby ushered in a new era of chromosome banding
First attempt to provide nomenclature for chromosome banding in any species and thus its recommendations have been adopted to nonhuman species as well.
The Paris report (1971)
What was described in The Paris report (1971)?
first attempt to provide nomenclature for chromosome banding in any species and thus its recommendations have been adopted to nonhuman species as well.
What is the purpose of studying the chromosomal banding pattern?
to see smaller pieces of the chromosome, to identify smaller structural chromosome abnormalities not visible on a routine analysis.
Banding techniques can be classified based on?
GC and AT rich regions.
Constitutive Heterochromatin Region.
What type of chromosomes are always used for banding techniques?
Always metaphase chromosomes whose size has condensed and whose diameter is increased
Who discovered Q banding and when?
Casperson et.al (1958)
Who discovered G banding?
Summer et.al (1971)
Who discovered N banding?
Matsui & Sasaki (1973)
Who discovered C banding?
Linde & Laursen (1978)
What is C banding (what does it stand for)?
Centromeric banding
What kind of banding: AT-rich regions stain darker than GC-rich regions
G banding
What kind of banding: Quinacrine fluorescent dye stains AT-rich regions
Q banding
What kind of banding: Banding pattern is opposite G-banding
R banding
What kind of banding: Stains heterochromatic regions close to the centromeres
C banding
What kind of banding: Usually stains the entire long arm of the Y chromosome
C banding
In G-banding, what regions stain darker than which regions?
AT-rich stain darker than GC-rich
Quinacrine fluorescent dye stains what regions?
AT-rich
What does C-banding technique usually stain?
Heterochromatic regions close to the centromeres
Entire long arm of the Y chromosome
What are the steps in Q banding technique?
Chromosome -> Stain with Quinacrine mustard -> Subject to UV light -> Banding pattern
In Q-banding, if the region is rich in AT-bases, what are the steps/results?
Dark stain -> AT region quenches dye and fluorescence, situated in heterochromatin region
In Q-banding, if the region is rich in GC-bases, what are the steps/results?
Light staining -> GC region quenches dye but do not fluorescence, situated in euchromatin region
What are the advantages of Q banding?
Simple and versatile
Used where G band is not accepted.
Used in study of chromosome heteromorphism.
What are the disadvantages of Q banding?
Tendency to fade during examination.
Photo-degradation
Chromopore- absorb light of a particular wavelength due to a chemical bond formed between dye and light.
UV light breaks the chemical bond.
Term used for the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number.
Karyotype
Asymmetric chromosomes have advanced through?
Structural chromosome changes
Analysis of a chromosome
Karyotyping
Can detect gross genetic changes—anomalies involving several megabases or more of DNA
Karyotyping analysis
C banding is centromere staining that results from?
Akali treatment
Results from heat treatment in a phosphate buffer followed by staining with Giesma dyes.
R banding
In G-banding, dark bands are ____ rich
A-T
In G-banding, light bands are ___ rich
G-C
Principle of G-banding is?
Proteolysis with trypsin followed by giemsa staining
Principle of R-banding is?
Heat denaturation followed by giemsa staining
In R-banding, dark bands are __ rich
G-C
In R-banding, light bands are __ rich
A-T
Principle behind Q banding?
Staining with quinacrine mustard dye
In Q banding, dark bands are ____ rich
A-T
In Q banding, light bands are ____ rich
G-C
Principle behind C banding?
Denaturation with barium hydroxide followed by giemsa staining
In C banding, dark bands represent?
Heterochromatin
Steps in G banding?
Chromosome -> Weak trypsin/urea/protease -> Treated with Giemsa -> banding pattern
Why is trypsin used in G banding?
To denature the protein
Why is G banding treated with Giemsa?
The interaction of the DNA with thiazine & eosin components of stain brightens sulfur rich regions
Then anding pattern has what dyes?
Methylene Azure+
Methylene Violet+
Methylene Blue+
What are the advantages of G banding?
Used in identification of bands rich in Sulfur content.
Used in the identification of chromosomal abnormalities
Gene Mapping.
What are the disadvantages of G banding?
Not used in plants.
N banding steps?
Chromosome -> Air dried -> Treated with 5% Trichloroacetic acid @ 95ᵒC for 30 min. -> Treated with 0.1N HCl @ 60ᵒC for 30 min. -> Banding pattern in Structural non-histone proteins linked to NOR region
Advantages of N banding?
Used in the identification of Nucleolar organizer region.
Superior banding pattern for plants.
Steps in C banding?
Chromosome
Treating solution with Alkali solution
Washing with Sodium citrate @ 60ᵒC for 30 min.
Staining with Giemsa Solution
Banding pattern at heterochromatin region
In C banding, why is the chromosome treated with an alkali solution?
For DNA denaturing
In C banding, why is the chromosome washed with Na citrate at 60C for 30mins?
Repetitive DNA renature but unique DNA do not renature
C banding advantages?
Identification of chromosomes particularly in insects and plants.
Identification of bivalents at diakinesis using both centromere position.
Paternity testing.
Gene mapping.
Each chromosome arm is divided into regions, or ____________, that can be seen using a _________ and ___________.
cytogenetic bands
microscope and special stains.
How are cytogenetic bands labeled?
Counting from the centromere out toward the telomeres.
At higher resolutions, what can be seen within the bands?
Sub-bands
How are sub-bands numbered/labeled?
Numbered from the centromere out toward the telomere.
What is ISCN?
International System for Human Cytogenetic Nomenclature
How does the ISCN arrange chromosomes?
Each area of chromosome given number
Lowest number of closest (proximal) to centromere
Highest number at tips (distal) to centromere
Prader-Willi syndrome results from the absence or nonexpression of?
Most cases of Prader-Willi syndrome (about 70%) occur when?
a group of genes on chromosome 15
a segment of the paternal chromosome 15 is deleted in each cell.
ALL of the following information can be found on the karyotype EXCEPT:
A. Size of chromosome
B. Sex of organism
C. Presence of point mutations
D. Presence of satellites
C
Which is NOT a characteristic of Symmetric Karyotype?
A. Have more metacentric chromosomes
B. Have more acrocentric chromosomes
C. Lesser difference between larger and smaller chromosomes
D. No relatively advanced feature
B I think
Which reagent in the karyotyping procedure cause the cells to swell to release the chromosomes?
A. Phytohemagglutinin (PHA)
B. KCl solution
C. Colcemid
D. Actinomycin D
I think B
Which chromosome banding technique can be used for paternity testing?
A.Q-banding
B. NOR-banding
C. G-banding
D. C-banding
D
Which chromosome banding technique uses proteolytic enzymes to expose sulfur rich regions?
A.Q-banding
B. NOR-banding
C. G-banding
D. C-banding
C
- Arrange the following events in correct order:
I. Addition of Carnoy’s fixative to pelleted cells.
II. Resuspension of cells with Sodium Citrate
III. Treatment of cultured follicular cells with PHA
IV. Sorting of air-dried microscopic slides and locking them into the microscope for analysis
V. Addition of colcemid to stimulated follicular cells
VI. Staining of slides with GTG-banding
Correct answer: III -> V -> II -> I -> VI -> IV
