MT6315 L7 KARYOTYPING Flashcards
1
Q
Defined as the number and appearance of chromosome in the nucleus of a eukaryotic cell
A
Karyotype
2
Q
Karyotypes are usually used in what kind of cytogenetics?
A
Classical
3
Q
A karyotype is a product of?
A
Karyotyping
4
Q
Usual information seen in a karyotype includes?
A
Size of chromosome
Position of chromosome
Presence of secondary constrictions
Size of satellites
5
Q
What are satellites?
A
Regions of the chromosome near the centromere
6
Q
Contains repeated DNA that do not code for proteins
A
Centromere
7
Q
T or F: Karyotyping starts from staining and viewing
A
F, starts from cell harvesting or culturing
8
Q
If the chromosomes are almost the same size, how do you determine the chromosomal order?
A
Determining the position of the centromeres
9
Q
Karyotype comes from the Greek word _____ which means ______
A
Karyon
nucleus
10
Q
Study of whole sets of chromosomes
A
Karyology
11
Q
What are commonly viewed in karyotypes?
A
Aneuploidies
12
Q
What stage of the cell is usually needed in karyotyping?
A
Metaphase (condensed form of the cell)
13
Q
Standard format of representing chromosomes as diagram when the haploid set of chromosomes of an organism are ordered in a series of decreasing size.
A
Idiogram or Karyogram
14
Q
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.
A
haploid
decreasing
15
Q
Asymmetric or Symmetric Karyotype: Show larger differences between smaller and larger chromosome in a set
A
Asymmetric
16
Q
Asymmetric or Symmetric Karyotype: Have more acrocentric chromosomes and relatively advanced feature.
A
Asymmetric
17
Q
Asymmetric or Symmetric Karyotype: Show lesser difference between smaller and larger chromosome in a set.
A
Symmetric
18
Q
Asymmetric or Symmetric Karyotype: Have more metaphase chromosomes and no advanced feature.
A
Symmetric
19
Q
Who suggested that in flowering plants there is a predominant trend towards karyotype asymmetry?
A
G.A. Levitzky
20
Q
What did G.A. Levitzky propose or suggest regarding flowering plants?
A
There is a predominant trend towards karyotype asymmetry.
21
Q
The “trend” proposed by G.A. Levitzky was carefully studied in?
A
the genus Crepis of the family compositae.
22
Q
Species showing a greater asymmetry is (more/less) advanced.
A
More
23
Q
Degree of asymmetry is the proportion of?
A
Metacentric and acrocentric chromosomes in a set
24
Q
Ratio between size of largest and smallest chromosomes in a set.
A
Degree of asymmetry
25
What factors contribute to the increased asymmetry of a chromosome?
Higher the proportion of acrocentric chromosomes, Greater the value of size ratio
26
Process of pairing and ordering all the chromosomes of an organism
Karyotyping
27
Provides a genome-wide snapshot of an individual’s
chromosomes.
Karyotyping
28
How can screening for aneuploidies pre-natally happen?
via Amniocentesis
29
Karyotypes are prepared using?
Standardized staining procedures that reveal characteristic structural features for each chromosome.
30
What is the most commonly used standardized staining procedure for Karyotypes?
Giemsa staining
31
What can karyotyping analysis reveal?
subtle structural changes such as chromosomal translocations, deletions, duplications, or inversions.
changes in chromosome number associated with aneuploid conditions.
32
Light staining band is (heterochromatic/euchromatic)
Euchromatic
33
________ consists of less amount of DNA lightly compressed with the histone proteins.
Euchromatin
34
_________ consists of more amount of DNA tightly compressed with the histone proteins.
Heterochromatin
35
Which has more proteins, euchromatin or heterochromatin?
Heterochromatin
36
Causes nucleosomes to pack tightly together.
Transcription factors cannot bind the DNA and genes are not expressed.
Methylation
37
Results in loose packing of nucleosomes.
Transcription factors can bind the DNA and genes are expressed.
Acetylation
38
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
39
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
40
What are the equipment used in karyotyping?
Centrifuge
Incubator (37C at CO2)
Refrigerator
Inverted microscope
41
What reagent bursts unneeded cells?
Glacial acetic acid
42
What lyses or bursts cells?
KCl
43
What ensures lymphocyte survival, only has decarbonate and no CO2?
RPMI
44
What reagent is a mitogen and lectin?
Phytohemagglutinin
45
What are the 2 kinds of giemsa dye?
Ethanol blue
Eosin
46
What kind of cells are used in karyotypes and contain mature RBCs?
Nucleated cells
47
What are the steps in karyotyping? (Hint: sa picture)
1. Draw blood (10mL - 20mL)
2. Add few drops of blood
3. Add phytohemagglutinin to stimulate mitosis
4. Incubate for 37C for 2-3 days
5. Add colcemid to culture for 1-2 hours to stop mitosis in metaphase
6. Transfer cells to tube
7. Transfer to tube containing fixative
8. Stain slide with Giemsa
9. Drop on the slide
10. Centrifuge to concentrate cells, add low-salt solution to eliminate RBCs and swell lymphocytes
11. Examine with microscope
12. Digitized chromosome images processed to make karyotype
48
What are the 5 major steps in karyotyping?
1. Short term lymphocyte culture
2. Harvesting of lymphocytes
3. Fixing the cells
4. Making the chromosome slides
5. Slide Analysis
49
The collected blood will be grown in vitro by adding what reagents?
cell culture growth medium, fetal bovine serum, antibiotics, and phytohemagglutinin (PHA)
50
What reagent induces mitotic activity?
PHA
51
The cultured blood cells will be grown at ____ °C incubator for __ days
37
3
52
The cells must be in what phase to stimulate short term lymphocyte culture?
Logarithmic phase
53
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.
54
What derivate of colchicine arrests the cell cycle at metaphase stage?
Pre-warmed Colcemid
55
How long should the cell in colcemid be incubated?
15mins
56
Optimal exposure time to colcemid requires a balance between?
proliferative activity index of cells and concentration of colcemid
57
The tube should be centrifuged for how many RPM and for low long?
1000 RPM
10mins
58
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.
59
In step 2 of karyotyping, the tube must be incubated at what temperature for how long?
Room temp
15mins
60
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.
61
What can significantly increase the total yield of metaphase chromosomes?
Cell synchronization
62
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
63
The cell suspension in hypotonic state will be centrifuged for [what RPM for how long].
1200 RPM for 5 mins.
64
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
65
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.
66
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
67
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).
68
Slides that will be chosen for analysis and visualization must be?
Properly trypsinized chromosomes
Clearly defined metaphase spreading
69
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.
70
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
71
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
72
What did Caspersson et al's paper describe?
use of quinacrine mustard to stain chromosome thereby ushered in a new era of chromosome banding
73
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)
74
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.
75
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.
76
Banding techniques can be classified based on?
GC and AT rich regions.
Constitutive Heterochromatin Region.
77
What type of chromosomes are always used for banding techniques?
Always metaphase chromosomes whose size has condensed and whose diameter is increased
78
Who discovered Q banding and when?
Casperson et.al (1958)
79
Who discovered G banding?
Summer et.al (1971)
80
Who discovered N banding?
Matsui & Sasaki (1973)
81
Who discovered C banding?
Linde & Laursen (1978)
82
What is C banding (what does it stand for)?
Centromeric banding
83
What kind of banding: AT-rich regions stain darker than GC-rich regions
G banding
84
What kind of banding: Quinacrine fluorescent dye stains AT-rich regions
Q banding
85
What kind of banding: Banding pattern is opposite G-banding
R banding
86
What kind of banding: Stains heterochromatic regions close to the centromeres
C banding
87
What kind of banding: Usually stains the entire long arm of the Y chromosome
C banding
88
In G-banding, what regions stain darker than which regions?
AT-rich stain darker than GC-rich
89
Quinacrine fluorescent dye stains what regions?
AT-rich
90
What does C-banding technique usually stain?
Heterochromatic regions close to the centromeres
Entire long arm of the Y chromosome
91
What are the steps in Q banding technique?
Chromosome -> Stain with Quinacrine mustard -> Subject to UV light -> Banding pattern
92
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
93
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
94
What are the advantages of Q banding?
Simple and versatile
Used where G band is not accepted.
Used in study of chromosome heteromorphism.
95
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.
96
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
97
Asymmetric chromosomes have advanced through?
Structural chromosome changes
98
Analysis of a chromosome
Karyotyping
99
Can detect gross genetic changes—anomalies involving several megabases or more of DNA
Karyotyping analysis
100
C banding is centromere staining that results from?
Akali treatment
101
Results from heat treatment in a phosphate buffer followed by staining with Giesma dyes.
R banding
102
In G-banding, dark bands are ____ rich
A-T
103
In G-banding, light bands are ___ rich
G-C
104
Principle of G-banding is?
Proteolysis with trypsin followed by giemsa staining
105
Principle of R-banding is?
Heat denaturation followed by giemsa staining
106
In R-banding, dark bands are __ rich
G-C
107
In R-banding, light bands are __ rich
A-T
108
Principle behind Q banding?
Staining with quinacrine mustard dye
109
In Q banding, dark bands are ____ rich
A-T
110
In Q banding, light bands are ____ rich
G-C
111
Principle behind C banding?
Denaturation with barium hydroxide followed by giemsa staining
112
In C banding, dark bands represent?
Heterochromatin
113
Steps in G banding?
Chromosome -> Weak trypsin/urea/protease -> Treated with Giemsa -> banding pattern
114
Why is trypsin used in G banding?
To denature the protein
115
Why is G banding treated with Giemsa?
The interaction of the DNA with thiazine & eosin components of stain brightens sulfur rich regions
116
Then anding pattern has what dyes?
Methylene Azure+
Methylene Violet+
Methylene Blue+
117
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.
118
What are the disadvantages of G banding?
Not used in plants.
119
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
120
Advantages of N banding?
Used in the identification of Nucleolar organizer region.
Superior banding pattern for plants.
121
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
122
In C banding, why is the chromosome treated with an alkali solution?
For DNA denaturing
123
In C banding, why is the chromosome washed with Na citrate at 60C for 30mins?
Repetitive DNA renature but unique DNA do not renature
124
C banding advantages?
Identification of chromosomes particularly in insects and plants.
Identification of bivalents at diakinesis using both centromere position.
Paternity testing.
Gene mapping.
125
Each chromosome arm is divided into regions, or ____________, that can be seen using a _________ and ___________.
cytogenetic bands
microscope and special stains.
126
How are cytogenetic bands labeled?
Counting from the centromere out toward the telomeres.
127
At higher resolutions, what can be seen within the bands?
Sub-bands
128
How are sub-bands numbered/labeled?
Numbered from the centromere out toward the telomere.
129
What is ISCN?
International System for Human Cytogenetic Nomenclature
130
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
131
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.
132
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
133
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
134
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
135
Which chromosome banding technique can be used for paternity testing?
A.Q-banding
B. NOR-banding
C. G-banding
D. C-banding
D
136
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
137
6. 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
