U8: FISH Flashcards
FISH is under what branch of cytogeneyics?
Molecular
This technique is used because it has a fast turn around time.
FISH
FISH stands for?
Fluorescence In-Situ Hybridization (FISH)
This is a cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complimentary to its sequence.
FISH
This is useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes.
FISH
What disease can be detected in using FISH?
Leukemias
The earliest kind of karyotype staining is?
Fluorescent staining (Quinacrine mustard)
Fluorescence involves the use of what?
excitation light (most likely UV as it is energetic)
T/F: You may use other lights in the spectrum of visible light for fluorescence.
T
Electrons will jump an e____ l_____ and when the excitation light is no longer present, electron will go down to its r____ s_____ to release photons.
energy level, resting state
These are small strips of single stranded DNA, complementary to the sites that we want to examine in the chromosome.
Probes
The DNA that is bound by the probe is called?
Target chromosome
T/F: Small refers to a thousand limit in bases.
T
Number of bases for FISH
200-400 bases
This refers to native DNA, which is DNA extracted from the patient.
Target chromosome
This is artificial DNA, made in laboratories or biotechnology companies. The strips of DNA are created in order to target the native DNA.
Probe
In what direction is the probe running and is arresting what sequence?
3’ to 5’, complementary
If you have just 1 probe to isolate a certain DNA portion (seen in locus-specific FISH), it is called?
Detection
This refers to when the entire chromosome is tagged with different colors of probes or automation (whole chromosome FISH)
DNA Mapping
FISH is applied to provide s____ l_____ of genes on chromosomes
specific localization
R_____ d_____ of trisomies and microdeletions is acquired using specific probes.
Rapid diagnosis
Used to check the cause of t_____, m____ s_____, etc.
trisomies, microdeletion syndromes
T/F: FISH can detect 1 base
F ; 200-400
This is hybridization of a fluorescent probe to a n____ D____.
native DNA
The native DNA of the patient or hybridization happens on a?
slide preparation
This allows for visualization of target DNA sequences of clinical interests.
slide preparation
Choice of technique for c_____-e_____ molecular testing is FISH.
cost-effective
S_____ m____ using FISH can be performed on a variety of specimen with only a basic requirement
Sequencing methods
What is the basic requirement in FISH for sequencing methods?
The DNA should be undegraded, or preserved.
T/F: In molecular techniques, it is okay if you only have the fragment of DNA.
T
T/F: DNA must be intact in FISH.
T
T/F: Any specimen that can be used in chromosome studies which evaluate metaphase cells can also be used for FISH.
T
You can also do FISH in i_____ chromosomes.
interphase
Other term for the target DNA.
Template
dito daw ginagawa yung probe > release ng complementary sequence
This is to separate DNA strands and allow probe access to target DNA.
Denature
H____ together to bind probe to target DNA.
Hybridize
A____ probe signals using a fluorescent microscope.
Analyze
What is denatured in the process because it is double stranded?
Target/Template/Native DNA
Denaturation involves the use of h_____ and denaturing chemicals like F______.
heat, Formamide
This means that in a target sequence or on the site, you can attach a probe.
in situ
This is performed in a glass slide, once you denature the DNA, the probe will bind on site.
In situ hybridization
T/F: Hybridization is when the original pair of DNA is still present.
F ; dapat native DNA and artificial DNA
Analyzing is visualizing the regions via f_____.
fluorescence
Only a portion of the chromosome will light up, the rest will be what?
Counterstained
Examples of Counterstains
DAPI, Propidium Iodide
This refers to dividing cells, and cells are thick.
Metaphase
These cells are the following: blood, bone marrow, skin fibroblast
Metaphase Cells
Metaphase Cells used in post-natal
- Blood
- Bone marrow
- Skin fibroblast
These cells in infants are the following:
Chorionic villi, amniocytes, tumors
Metaphase cells in infants:
- Chorionic villi
- Amniocytes
- Tumors
These are non-dividing cells, in the quiescent stage of cell cycle.
Interphase Cells
They can be used for screening.
Interphase Cells
They can come from direct preparations: uncultured cells from blood, bone marrow, cytospins.
Interphase Cells
Where are bone marrow specimens from?
Flat bones in the body
These are bladder cells from bladder cancer. Usually used for fluids, and collected by cytocentrifugation from abnormal cells.
Cytospins
FORMATIVE: Speed for cytospin
maximum of 1-3 minutes
Interphase Cells
Uncultured cells are from?
- Blood
- Bone marrow
- Cytospins
Interphase Cells
Smears are made from?
- Blood
- Buccal cells
- Bone marrows
They can come from direct preparations: smears made from blood, buccal cells, bone marrow
Interphase Cells
Interphase Cells
Paraffin Embedded Tissue Sections:
- Tumors
- Products of conceptions
Abnormal tissues are preserved in what?
Formalin Fixed Paraffin Embedded (FFPE) Tissues
FFPE stands for?
Formalin Fixed Paraffin Embedded
This is the gold standard and routinely done on cultured cells.
Metaphase FISH
Gold standard because it is the most rigorous process
This allows direct visualization of chromosomes and exact position of signals.
Metaphase FISH
This is useful in detection of structural changes in the genome.
Metaphase FISH
This may also be done on uncultured specimens.
Interphase FISH
This is advantaegous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies.
Interphase FISH
The major disadvantage of ______ FISH is the inability to d_____ u____ structural changes.
Interphase, detect unknown
T/F: We light up the whole chromosome in Interphase FISH.
T
What is detected more in pre-natal diagnosis?
Aneuploidies
T/F: Metaphase FISH is used for de novo mutations.
T
but with modifications
T/F: You report results in Interphase FISH as positive or negative since in this technique, you already have the disease in mind.
T
Samples for Metaphase FISH
- Amniocytes
- Chorionic villous cells
- Lymphocytes
- Cells from bone marrow aspirates or solid tumors or cytospin
- Fibroblasts (skin fibroblasts)
Samples for Interphase FISH
- Amniocytes
- Peripheral blood smears
- Bone marrow aspirate smear or direct harvest
Interphase FISH
Used for ploidy analysis during prenatal studies
Amnicoytes
Interphase FISH
Used for ploidy analysis in newborns
Peripheral blood smears
Interphase FISH
Used for translocation or copy number analysis in cancer studies
Bone marrow aspirate smear or direct harvest
These are complementary sequences
This is an important tool to visualize copy number changes detected by other methods (microarrays, CGH)
Metaphase FISH
This is a primary screening tool for duplications and deletions.
Comparative Genomic Hybridization (CGH)
This is used for the direct detection of where the duplications and deletions happens.
pre-test in CGH and for solid tumors
What does Metaphase FISH detect after testing for CGH?
specific regions where we see detectable changes
M___ are used for CGH.
Microarrays
Interphase FISH offers s___ d____ t____ t____, a faster screening process for certain assays because you don’t have to culture cells.
same day turnaround time
These are complementary DNA sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores.
Probe
R___ and a____ are also complementary sequences to DNA.
RNA and analogs
The size of probes ranges from?
20 to 1000 bases (1 megabase)
Usual size range for FISH Probes
200 to 400 bases
Two kinds of FISH Labelling
- Direct Labeling
- Indirect Labeling
This refers to when fluorophores are directly attached to the probe, and is less sensitive.
Direct Labeling
Examples of probes used in Direct Labeling
FITC, Rhodamine, Cyanine
T/F: Fluorescent dye may attach to intercalated DNA, depending on its staining properties.
T
Probes are usually on the e___ of DNA.
ends
This refers to when chemical conjugation of the nucleic acid with a non-fluorescent molecule that can bind fluorescent material after hybridization.
Indirect Labelling
Examples of probes used in Direct Labelling
Biotin, Digoxigenin
Indirect Labelling
Partner molecules of Biotin
Avidin, Streptavidin
T/F: In Biotin, the partner molecule will have the fluorescent dye.
T
Indirect Labelling
Biotin have very strong c_____ interactions.
covalent
Indirect Labelling
Biotin is derived from e___ y____. Avidin is found in e___ w___ if not, produced artificially.
egg yolk, egg white
Indirect Labelling
These two molecules have a very strong covalent interaction with each other.
Biotin, Avidin
This refers to unbound fluorescent dyes that might confuse stains.
Background
For better specificity of t_____, secondary reactions are used in Indirect Labelling.
tagging
FISH Process
Specimen processing/harvesting
- Pretreatments to harden chromatin
- Dehydration
FISH Process
Denature DNA
Break Hydrogen bonds to form single stranded DNA
FISH Process
Hybridize with fluorescently labeled DNA probe
Reannealing for several hours
This refers to breaking the Hydrogen bonds.
Denaturation
T/F: There is more heat if there is more G-C bonds.
T
This refers to the DNA sticking together with the probes. This also refers to closing up of the DNA double strand.
Reannealing
Pretreatments require several changes of?
Alcohol
FORMATIVE: What percentage of alcohol is used to dehydrate chromatin?
70-80-90 (ascending order series)
B___ increases temperatures which is not advisable in FISH.
Baking
This is another term for the pretreatments to harden chromatin in room temperature.
aging process
Pre-soaking in s____ s___ can help c____ and prepare the chromatin.
salt solutions, condense
T/F: Target DNA and Probe DNA can be denatured separately or co-denatured.
T
To reanneal, you have to _____ the temperature.
lower
T/F: Temperatures for reannealing are given by the manufacturers.
T
Range of Reannealing
4 to 20 hours
The rate at which strands reanneal is dependent upon d_____ c__________.
DNA concentration
The rate at which strands reanneal is dependent on the following factors:
- DNA concentration
- Size of probe
- Degree of similarity between probe and target DNA
S______ of probe is a determinant of how long it reanneals with DNA.
Specificity
T/F: Both probe DNA and native DNA concentration can be used to determine rate of reannealing.
T
Step after staining
Washing off excess unbound probe
This refers to excess and unbound probe that may still fluoresce.
Background Flourescence / Background Signals
Background Flourescence contributes to?
noise
Background Flourescence decreases the s_______ of the process.
sensitivity
Reagents in Washing
Buffer, Wash solution, Wash buffer
What are the usual washing buffers?
Detergents with a certain pH (technology-specific)
This refers to the strictness of conditions defined how much the probe will bind and how much will be washed.
Stringency
Stringency can be modified by the following:
- Temperature
- Concentration of Formamide or Salt solution
What step is done for better visualization?
Counterstain
Counterstains in FISH
- Propidium Iodide (PI)
- DAPI (4’6-diamidino-2-phenylindole)
Color of Propidium Iodide (PI)
Red
Color of DAPI (4’6-diamidino-2-phenylindole)
Blue
Visualization is done under?
Fluorescence or Darkfield microscope
Choice of kilobases
2-400
The size of the t__ will affect how small the p____ will be
target, probe
T/F: Highly repeated targets will appear bright even with small sequences.
T
paulit-ulit didikit sa region
T/F: The bigger the probe, the brighter the fluorescence.
T
Most commonly used FISH probes
- Whole Chromosome Paints (WCPs)
- Chromosome Enumeration Probes (CEPs)
- Locus-specific identifiers (LSIs)
T/F: WCPs can cover both the whole chromosome or just the arm.
T
These comprises libraries of multiple overlapping chromosomes or chromosomal region-specific probes which covers the length of the entire chromosome or just the arm.
Whole Chromosome Paints (WCPs)
This means multiple probes in multiple sites of the chromosome.
Libraries
If the libraries are small, what do we call the method?
Shotgun method
This refers to coloring the entire chromosome or region-specific, leaving no space in between.
Overlapping
Probes are usually created via?
PCR
WCPs are useful in the following:
- studying marker chromosomes
- translocations
- aneuploidy in metaphase cells
These are known as centromeric probes.
Chromosome Enumeration Probes (CEPs)
CEPs apply to both?
metaphase and interphase FISH
CEPs target what DNA sequences?
satellite DNA or alpha-satellite DNA (highly repetitive DNA)
CEPs are also known as?
alpha-satellite probes
Highly repetitive DNA reside in what regions?
heterochromatic
CEPs target what region?
centromeric regions
CEPs are often used as?
control probes (check if you have or don’t have)
T/F: CEPs do not give off large bright signals.
F
CEPs are useful in?
fast detection of aneuploidies
T/F: LSIs can be used for centromeric regions.
T
This is used for more gene/region-specific, single-copy, or unique sequence probe.
Locus-specific identifiers (LSIs)
LSIs span region and average of what range in size?
200-300 kb
LSIs are useful for:
- Deletions
- Duplications
- Rearrangements
- Amplifications
LSIs can also be used in f____-t____ probe strategies.
fusion-type
This probe binds to a particular region of a chromosome, and used when only a small portion of a gene is isolated.
Locus Specific probe
Locus specific probes are used to determine:
- which chromosome the gene is located
- how many copies of the gene exist within a genome
This is designed cover a gene of interest, done on cultured cells.
Single Color FISH Probe
This is designed to cover any 2 genes, and allows simultaneous detection of two to three regions in one FISH assay.
Dual Color FISH Probe
This is used to identify specific chromosome rearrangements, typically in regions with recurring breakpoints. This involved 2 chromosomes.
Dual Fusion FISH Probe Strategies
Dual Fusion FISH Probe Strategies
The presence of yellow fluorescence would mean that a f___ had occured.
fusion
Gene fusions are usually seen in?
leukemias and cancers
T/F: Dual fusion is better for detecting what we already assume what is there.
T
This is used to detect chromosomal rearrangements, wherein the fusion is located within one chromosome.
Break Apart (BAP) Fish Probe Strategies
What samples are used in BAP?
FFPE samples (Formalin fixed, paraffin embedded)
BAP is useful for rearrangements with what patterns?
multiple known patterns
BAP is useful for detecting the translocation of what genes?
promiscuous
T/F: BAP cannot be used for de novo mutations.
F ; better for de novo
This is generated from repetitive sequences found in the middle of each chromosome.
Alphoid / Centromeric Repeat Probe
This is used to determine whether individual has the correct number of chromosomes, aneuploidies or trisomy.
Alphoid / Centromeric Repeat Probe
This is locus-specific to the subtelomere region of the chromosome.
Subtelomeric probe
This is useful in detection of subtelomere deletions and rearrangements.
Subtelomeric probe
Telemore deletions indicate signs of?
cancer
T/F: If most of your subtelomeric regions light up, you do not have telomeric regions and seen in cancerous states.
T
This is comprised of different combinations of fluorophore-labeled probes specific for chromosomes 13, 18, 21, X and/or Y.
Pre-natal FISH Probe
Pre-natal FISH probe targets what chromosomes?
13, 18, 21, X and/or Y
T/F: Pre-natal FISH Probes are locus-specific.
T
Pre-natal FISH probe is used for screening, as early as?
24 hours or earlier
What cells are used for Pre-natal FISH probe?
interphase
Minimum TAT for Karyotyping
3 days
Pre-natal FISH probe uses what sample?
regardless of what sample but usually chorionic villi, amniotic fluid, bone marrow
T/F: Pre-natal FISH only targets the usual aneuploidies.
T
Single-Probe cell scoring is used for?
interphase FISH, detecting aneuploidy
deletion in c15 (15q11-13)
Prader-Willi
duplication in chromosome?
11
For what disorders is the MYB probe used?
B cell disorders
What probe is used for B cell disorders?
MYB Probe
MYB probe is used for?
Chronic Lymphocytic Leukemia (CLL)
This is a specialized form of WCPs.
Multiplex FISH (M-FISH)
This technique views a karyotype with each chromosome painted with a different color.
Multiplex FISH (M-FISH)
T/F: Multiplex FISH (M-FISH) is automated.
T
T/F: In M-FISH, you can start with the fluorescence FISH in which the tags will still be given other colors afterwards.
T
This is used to create a distinct computer-generated false color for each chromosomes.
Ratio-labeled probes
This is useful for complex rearrangements, such as those seen in neoplastic disorders and solid tumors.
Multiplex FISH (M-FISH)
This means being stained separately.
Multiplex
Multiplex FISH (M-FISH) can be used for pre-B or B lymphocytes
Acute Lymphocytic Leukemia (ALL)
Multiplex FISH (M-FISH)
This is done and shown by the computer or thru automation.
Blended colors, pseudocolors
This uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes.
Multiple Banding (mBand) analysis
Estimated resolution for mBand analysis
550 bands
In mBand, pseudocolors are produced where?
within chromosome
different bands in entire single chromosome
mBand is advantageous for the following:
*determination of breakpoints
* intrachromosomal rearrangements
* inversions
This is a technique that allows chromosomes to be stretched out or elongated, where in probes are applied and physically ordered.
Fiber FISH
First step in Fiber FISH
removal of clumping
interphase cell - sirain ang histone, scaffolds to stretch dna
1 chromosome is equal to a
thin spread
This yields more precise information as to the localization of specific DNA probe on the chromosome. Commonly used for research.
Fiber FISH
Fiber FISH has been used in?
telomeric regions
PRINS stand for?
Primed In Situ Labelling (PRINS)
This is essentially described as PCR on a slide.
Primed In Situ Labelling (PRINS)
3 steps in PRINS
- Denaturation (cycling temperatures in PCR unlike in FISH na maintained)
- Hybridization
- Annealing
PRINS can differentiate hybridization with alpha satellite sequences for chromosomes?
13 and 21
T/F: In PRINS, you create your probe in the slide itself.
T
This is used to identify material of unknown origin.
Reverse FISH
Reverse FISH
Unknown DNA used for karyotyping or band of interest can be acquired from?
crime scene
Steps in Reverse FISH
- Karyotype (G-banding)
- Take band of interest
- PCR Amplification and Fluorescent Labelling
- Hybridize with chromosomes of suspected criminal
Reverse FISH can also be used in?
DNA Profiling
