ASCP SMB Exam; Molecular Techniques-
QUESTIONS & ANSWERS VERIFIED 100%
CORRECT
Q-banding
The visualization technique of ____ when chromosomes are stained with the fluorescent dye
quinacrine or quinacrine mustard. It may be used to ID Y chromosome in interphase.
G-banding
chr treated with the chemical dye Giemsa, differing based on chr treatment before staining
mild treatment yields pattern comparable to fluorescent dyes
trypsin (or other proteolytic agents) treatment mapped structural aberrations
most commonly used staining method for chr
Fuelgen staining after treatment with DNase I
High-resolution banding
Chromosome banding using chr before they reach maximal metaphase condensation (prophase
or prometaphase chromosomes), which are more extended than metaphase chromosomes and
thus yield more bands and greater resolution.
R-banding
,pattern opposite of G-banding produced by harsher treatment before Giemsa staining
C-banding
centromere staining
may be associated with heterochromatin seqaround centromeres
euchromatin may not be stained that much
Nucleolar Organizing Region Staining
using silver nitrate to highlight constricted regions/stalks on the acrocentric chromosomes
4',6-diamidino-2-phenylindole (DAPI)
binds to surface grooves of dsDNA and fluoresces blue under UV light
used to visualize chromsomes as well as whole nuclei
Karyotyping
direct observation of metaphase chromosome structure by arranging metaphase chromosomes
according to size
collect living cells and grow them in culture in a lab for 48-72 hours
cell division is stimulated by mitogen then arrested in metaphase - this yields chr spread when
cell nuclei are disrupted
,can detect translocations
Interphase FISH
does not require culturing of cells
fixed cells are exposed to a probe (60-200kb DNA frag attached covalently to fluorescent mol
bound probe visualized under fluorescent microscope and are designed to be complementary to a
particular chr or locus so that imagine will correlate with the state of that chr or locus
ie. a probe unique to any region on chr 22 should yield an image of 2 signals, 1 for each copy of
chr 22 in somatic cell nucleus
use multiple different colored signals to detect translocations or other rearrangements
- normal nucleus would have 2 of each signal
- translocated chr would combine 2 signals resulting in loss of 1 of each signal in nucleus (=3
colors)
Dual Fusion Probes
sensitivity for detection of translocations increased
- mixtures of 2 single probes, each labeled with a different dye
- designed to bind to regions spanning breakpoint of both translocation partners
- translocation will be observed as signal from both the translocation junction and reciprocal of
the translocation junction
, Break-apart Probes
FISH probes designed to detect translocations where there are multiple possible partners
bind intact chr flanking breakpoint: 2 probes separate when a translocation occurs
Centromeric Probes (CEN Probe)
designed to hybridize to highly repetitive alpha satellite sequences surrounding centromeres
detect aneuploidy of any chr
Telomeric Probes
designed to bind telomeres and detect chr str abnomalities ie. cryptic translocations or small
deletions
Interphase FISH pros/cons
Pros:
1. cells do not require culture
2. sensitivity higher than that of metaphase procedures
Cons:
1. inability to ID chr changes other than those at the specific binding region(s) of the probe(s)
Karyotyping can detect any chr change that causes change in size, # or banding pattern within
the sensitivity limits
QUESTIONS & ANSWERS VERIFIED 100%
CORRECT
Q-banding
The visualization technique of ____ when chromosomes are stained with the fluorescent dye
quinacrine or quinacrine mustard. It may be used to ID Y chromosome in interphase.
G-banding
chr treated with the chemical dye Giemsa, differing based on chr treatment before staining
mild treatment yields pattern comparable to fluorescent dyes
trypsin (or other proteolytic agents) treatment mapped structural aberrations
most commonly used staining method for chr
Fuelgen staining after treatment with DNase I
High-resolution banding
Chromosome banding using chr before they reach maximal metaphase condensation (prophase
or prometaphase chromosomes), which are more extended than metaphase chromosomes and
thus yield more bands and greater resolution.
R-banding
,pattern opposite of G-banding produced by harsher treatment before Giemsa staining
C-banding
centromere staining
may be associated with heterochromatin seqaround centromeres
euchromatin may not be stained that much
Nucleolar Organizing Region Staining
using silver nitrate to highlight constricted regions/stalks on the acrocentric chromosomes
4',6-diamidino-2-phenylindole (DAPI)
binds to surface grooves of dsDNA and fluoresces blue under UV light
used to visualize chromsomes as well as whole nuclei
Karyotyping
direct observation of metaphase chromosome structure by arranging metaphase chromosomes
according to size
collect living cells and grow them in culture in a lab for 48-72 hours
cell division is stimulated by mitogen then arrested in metaphase - this yields chr spread when
cell nuclei are disrupted
,can detect translocations
Interphase FISH
does not require culturing of cells
fixed cells are exposed to a probe (60-200kb DNA frag attached covalently to fluorescent mol
bound probe visualized under fluorescent microscope and are designed to be complementary to a
particular chr or locus so that imagine will correlate with the state of that chr or locus
ie. a probe unique to any region on chr 22 should yield an image of 2 signals, 1 for each copy of
chr 22 in somatic cell nucleus
use multiple different colored signals to detect translocations or other rearrangements
- normal nucleus would have 2 of each signal
- translocated chr would combine 2 signals resulting in loss of 1 of each signal in nucleus (=3
colors)
Dual Fusion Probes
sensitivity for detection of translocations increased
- mixtures of 2 single probes, each labeled with a different dye
- designed to bind to regions spanning breakpoint of both translocation partners
- translocation will be observed as signal from both the translocation junction and reciprocal of
the translocation junction
, Break-apart Probes
FISH probes designed to detect translocations where there are multiple possible partners
bind intact chr flanking breakpoint: 2 probes separate when a translocation occurs
Centromeric Probes (CEN Probe)
designed to hybridize to highly repetitive alpha satellite sequences surrounding centromeres
detect aneuploidy of any chr
Telomeric Probes
designed to bind telomeres and detect chr str abnomalities ie. cryptic translocations or small
deletions
Interphase FISH pros/cons
Pros:
1. cells do not require culture
2. sensitivity higher than that of metaphase procedures
Cons:
1. inability to ID chr changes other than those at the specific binding region(s) of the probe(s)
Karyotyping can detect any chr change that causes change in size, # or banding pattern within
the sensitivity limits
