Karyotyping (from Estandarte) Flashcards
Chromosomes continue to condense until it reaches the ___ state
Metaphase
___ allows the proper visualization of chromosomes
Staining
___ can lead to banding
Staining
A consequence of differential staining along the length of the chromosome
Banding
T/F: Banding provides more information about the chromosome
T
Visible dyes such as Giemsa are used in ___ microscopy, while quinacrine are used in ___ microscopy
Light
Fluorescence
Heavy metal complexes are used as stains in ___ and ___ microscopy
Fluorescence
Electron
Advantage of light microscopy
Little damage on the chromosome
Advantage of fluorescence microscopy
High sensitivity and specificity
T/F: Both light and fluorescence microscopy have limited resolution
T
Electron microscopy includes ___ and ___ microscopy
TEM (transmission electron)
SEM (scanning electron)
The sample thickness of TEM is limited to approximately ___ nm, while SEM is only ___-___
100
surface-sensitive
___ ___ ___ ___ provides high resolution and 3D-images of the chromosome structure
Coherent x-ray diffraction imaging (CDI)
Enhance the contrast between different cellular components
Staining
T/F: Banding can be used to study abnormalities in the chromosome such as deletions, insertions, or translocations
T
___ heterochromatin consists of satellite DNAs
Constitutive
___ heterochromatin is a condensed and transcriptionally silent chromatin region
Facultative
Banding technique for constitutive heterochromatin
C-banding
Banding technique for facultative heterochromatin
G- or Q-banding
Banding technique for euchromatin
R-banding
___ interactions usually involve metal stains wherein the metal coordinates with the nitrogen atoms of the base pairs of the DNA
Covalent
The coordination of metal with nitrogen atoms often occurs at position ___ of ___ or ___
7
adenine or guanine
The coordination of metal with nitrogen atoms often occurs at position 7 of adenine or guanine occurs at some ___ stains and results to ___ ___ binding
Platinum
Major groove
___ ___ occurs with stains that have a functional group containing an electronegative atom such as nitrogen or oxygen
Hydrogen bonding (H-bonding)
During hydrogen bonding, the sites are position of __ of adenine, position __ of cytosine, and position __ of guanine
4
6
2
H-bonding leads to ___ ___ or ___ ___ binding
Major groove
Minor groove
____ ____ involve stains that have cationic groups
Electrostatic interactions
Electrostatic interactions result to ___ binding
external
Pi-pi interactions occur with stains that have ___ ___ groups
planar aromatic
___ is a visible light dye that binds to DNA through intercalation and thus, is used for chromosome staining
Giemsa
Giemsa is a mixture of cationic thiazine dyes, most importantly ___ __ and anionic eosin dyes such as ___ __
Azure B
Eosin Y
Staining of the chromosomes involves the formation of thiazine-eosin precipitate in a __ molar ratio
2:1
The formation of the precipitate in Giemsa staining is favored on a ___ (hydrophobic or hydrophilic) environment
Hydrophobic
The most widely used banding method for cytogenetic analysis that was first developed by Seabright
G-banding
This banding technique is advantageous in the aspect of stability and resolution of the band produced
G-banding
T/F: Fluorochromes are more stable and capable of producing clearer bands than visible light dyes
F (visible light dyes are more stable and capable)
Most common pretreatment method used in the digestion of the chromosomes in G-banding is with a ___ such as ___
protease
trypsin
Another pretreatment during G-banding aside from trypsin
Incubation of the chromosomes in hot-saline citrate or detergent/urea solution
Role of pretreatment methods in G-banding
Extract a characteristic subset of proteins from the chromosomes
Responsible for the banding and a reflection of the difference in the structure of the various chromosomal regions
Differential extraction
The ___ G-bands, which are the dark bands, correspond to the hydrophobic regions o the chromosomes and favors the formation of the ___-___ precipitate
Positive
thiazine-eosine
The negative G-bands, which are light bands, correspond to the ___ (more/less) hydrophobic regions of the chromosomes
less
__-banding reveals the GC-rich euchromatin and produces positive bands that correspond to the negative G-bands
R
__-banding reveals the AT-rich centromere
C
This banding technique is done by incubating the chromosomes in an ionic solution at a high temperature followed by Giemsa staining
R-banding
This banding technique involves acid treatment, hot saline incubation, and alkali treatment of chromosomes
C-banding
___-band is more resistant to extraction than the DNA in other regions of the chromosomes
C
Organic molecules that are capable of undergoing fluorescence
Fluorochromes
___ and ___ ___ can change the structure of a fluorochrome thus affecting its fluorescence
pH
ionic strength
T/F: Fluorescence staining does not require pretreatment process
T
In fluorochrome staining, the ___ wavelength shows how much energy is required to excite the fluorochrome while the ___ wavelength shows the energy of the photon emitted by the fluorochrome
Excitation
Emission
The emission wavelength is usually ___ (shorter/longer) than the excitation wavelength
longer
In fluorochrome staining, the ___ ___ or ___ shows the possibility of the excited fluorochrome to undergo fluorescence
quantum efficiency
yield (φ)
Formula to get the φ
of photon emitted / # of photon absorbed
The quantum efficiency of a fluorescence ___ (increases/decreases) as the size of the conjugated system increases
Increases
Identify the fluorochrome (2):
Binding mode: Intercalation
Mechanism of Banding: Differential fluorescence
Selectivity: AT
Quinacrine, Daunomycin
Identify the fluorochrome:
Binding mode: Minor groove
Mechanism of Banding: Differential binding
Selectivity: AT
Hoechst 33258, DAPI
Identify the fluorochrome:
Binding mode: Minor groove
Mechanism of Banding: Differential fluorescence
Selectivity: GC
Chromomycin A3
___ using fluorochromes occur due to the quenching of their fluorescence at certain regions of the chromosomes
Banding
___ ___ is responsible for banding using fluorochromes that have a binding specificity and attach only to certain regions of the chromosomes
Differential binding
An aminoacridine dye first used by Caspersson in 1970
Quinacrine mustard
The replacement for quinacrine mustard, which is a less toxic quinacrine compound
Quinacrine dihydrochloride
At ___ (high/low) dye/DNA ratio and ___ (high/low) ionic strength, quinacrine binds to DNA through intercalation
Low
High
Quinacrine has a ___ charge which is capable of intercalating with the ___ charged phosphate groups of the DNA
Positive
Negatively
T/F: Quinacrine is observed to bind randomly throughout the length of the chromosome
F (uniformly, not randomly)
T/F: Quinacrine does not fluoresce brightly in the AT-rich centromere
F
An anthracycline antibiotic dye
Daunomycin
Daunomycin stain results to the unwinding of the helix by how many degrees?
12
T/F: Daunomycin cannot form H-bonds with the DNA base pairs
F (it can because it has hydrogen donor and acceptor)
T/F: There is no base preference in the DNA binding of daunomycin
T
A fluorochrome that has a very high quantum yield of approximately 0.92
4’6-diamidino-2-phenylindol (DAPI)
T/F: DAPI is strongly fluorescent in water and is greatly influenced upon binding to the chromosome because it is shielded from the solvent
F (it is weakly fluorescent)
T/F: The fluorescence of DAPI does not fade easily compared with other fluorochromes
T
At low DAPI/DNA ratio, DAPI binds to the ___ ___ of AT-rich sequences while at high ratio, ___ in the banding mode of DAPI is observed
Minor groove
Heterogeneity
A bi-benzimidazole derivative stain that binds to the minor groove of the DNA, specifically to the AT-rich regions
Hoechst 33258
T/F: The mechanism of binding of the Hoechst 33258 with DNA is very similar to that of DAPI
T
A technique used to induce banding with fluorochromes that bind to fluoresce uniformly throughout the chromosome
Counterstaining
Two mechanisms responsible for the formation or enhancement of the bands
- electronic energy transfer
- direct binding competition
A process where the counterstain absorbs the fluorescence of the primary stain
Electron energy transfer
For this process to occur, there must be a spectral overlap of the fluorescence emission of the primary stain and the absorption of the counterstain
Electron energy transfer
This involves the selective displacement of the primary stain by the counterstain
Direct binding competition
T/F: The primary stain and the counterstain should have different binding modes
F (same binding mode)
Identify the counterstain:
Primary stain: DAPI
Type of bands: C
Distamycin A
Identify the counterstain:
Primary stain: Chromomycin A3
Type of bands: R
Methyl green
Identify the counterstain:
Primary stain: DAPI
Type of bands: G
Chromomycin A3
Actinomycin D
Stains with AT specificity (3)
DAPI
Distamycin A (DA)
Methyl Green
Stains with GC specificity (2)
Chromomycin A3
Actinomycin D (AMD)
The use of ___ ___ as stains for chromosomes is particularly important when studying the chromosomes with electron microscopy or CDI
heavy metals
How do heavy metal stains improve the visualization of chromosomes?
Enhances the signal obtained in electron microscopy
A well known DNA-metallointercalater that is nonfluorescent involving a metal-centered electronic transition
Platinum complexes
[Pt(terp)Cl]+
A heavy metal stain that is nonfluorescent because its d-d excited states are short-lived and easily subjected to radiationless deactivation
Platinum complexes
[Pt(terp)Cl]+
Enhancing the fluorescence of this metal complex is advantageous because the use of a fluorescent heavy metal stain allows the chromosome specimen to be imaged using both fluorescence microscopy, and electron microscopy or CDI
Platinum complexes
[Pt(terp)Cl]+
Heavy metal stains (2) that are widely used in studying biological samples with electron microscopy and can be used for both positive and negative staining
Uranyl acetate (UO2(CH3COO)2) and Osmium tetroxide (OsO4)
A radioactive substance that is known to especially increase the contrast of nucleic acids
Uranyl acetate (UO2(CH3COO)2)
Ratio of uranyl ion and phosphate group attachment
1:2
The treatment of DNA with uranyl acetate increases the dry weight of the DNA by a factor of __ and prevents the extraction of the DNA from its ___ shell
2
Histone
T/F: Uranyl acetate is known to interact with carboxyl groups, which may stain the proteins of other cellular components
T
Uranyl acetate have ___ (stronger/weaker) affinity for phosphate groups than carboxyl groups
Stronger
Uranyl acetate is commonly used in double staining with ___ ___
Lead citrate
Widely used as a heavy metal stain in electron microscopy for biological sample due to its property as a strong oxidant
Osmium tetroxide
