MT6315 LESSON 8 FISH & CGH Flashcards
(187 cards)
1
Q
FISH stands for?
A
Fluorescence In situ Hybridization
2
Q
FISH is a cryogenic technique that uses what to do what?
A
Fluorescent probes to bind specifically to a part of chromosomes complementary to its sequence
3
Q
The fluorescent probes use what kind of light?
A
Excitation light
4
Q
Small strips of single stranded DNA complementary to the sites being examined
A
Probes
5
Q
How many bases are usually seen in the probes?
A
~200-400
6
Q
FISH is useful in detecting and mapping what?
A
The presence or absence of particular DNA sequences within chromosomes
7
Q
What is the DNA bound to the probe called?
A
Target DNA or native DNA from the patient
8
Q
Probes are (artificial/native)
A
Artificial
9
Q
FISH is applied to provide what?
A
specific localization of genes on chromosomes
10
Q
What is acquired using specific probes?
A
Rapid diagnosis of trisomies and microdeletions
11
Q
FISH is also used to check the cause of?
A
trisomies, microdeletion syndromes
12
Q
(Native/Artificial DNA) is on the slide prep to allow for visualization
A
Native
13
Q
T or F: Karyotyping and PCR are more cost-effective than FISH technique in detecting disease
A
F, FISH is more cost-effective since culturing is no longer needed thus lowering costs
14
Q
Sequencing by FISH can be performed in what condition of the DNA?
A
Preserved/ Undegraded Metaphase DNA (should being intact)
15
Q
What binds to the target DNA?
A
Fluorescently labelled probe DNA
16
Q
Target DNA is also called t______
A
template
17
Q
Process of separating the DNA strands and to allow the probe to access target DNA
A
Denaturation
18
Q
How is denaturation usually done?
A
Heat or chemical means via Formamide
19
Q
What is denatured, native or artificial DNA?
A
Native/ Target
20
Q
T or F: FISH is possible in interphase chromosomes
A
T
21
Q
Process of binding together the probe to the target DNA
A
Hybridize/ Hybridization
22
Q
Probe signals are analyzed using?
A
A fluorescent microscope
23
Q
What are the 2 specimen types for FISH?
A
Metaphase FISH
Interphase FISH
24
Q
METAPHASE OR INTERPHASE FISH: Gold standard and routinely done
A
Metaphase
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METAPHASE OR INTERPHASE FISH: Done on cultured cells
Metaphase
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METAPHASE OR INTERPHASE FISH: Allows direct visualization of chromosomes and exact position of signals
Metaphase
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METAPHASE OR INTERPHASE FISH: Useful in the detection of structural changes in the genome
Metaphase
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METAPHASE OR INTERPHASE FISH: Can be modified to be used in de novo mutations
Metaphase
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METAPHASE OR INTERPHASE FISH: May also be done on uncultured specimens
Interphase
30
METAPHASE OR INTERPHASE FISH: Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies
Interphase
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METAPHASE OR INTERPHASE FISH: Beneficial in the study of samples with a low mitotic index such as most solid tumors
Interphase
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METAPHASE OR INTERPHASE FISH: Major disadvantage is the inability to detect unknown structural chromosomal changes.
Interphase
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METAPHASE OR INTERPHASE FISH: Probe is already made with the disease in mind
Interphase
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METAPHASE OR INTERPHASE FISH: Whole chromosome is of interest
Interphase
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METAPHASE OR INTERPHASE FISH: Not suitable for de novo mutation
Interphase
36
METAPHASE OR INTERPHASE FISH: Offers opportunity for same day turn around time
Interphase
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METAPHASE OR INTERPHASE FISH: May be used in karyotyping to visualize the exact position of the fusion
Metaphase
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Why is metaphase FISH the gold standard?
More rigorous and the best time to see the components of the chromosome
39
Considered a "pre-test" for duplications, deletions
CGH
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Samples that can be used for Metaphase FISH
1. Amniocytes
2. Chorionic villous cells
3. Lymphocytes
4. Cells from bone marrow aspirates or solid tumors
5. Fibroblasts
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Samples that can be used for Interphase FISH
Amniocytes – for ploidy analysis during prenatal studies
Peripheral blood smears – for ploidy analysis in newborns
Bone marrow aspirate smear or direct harvest – translocation or copy number analysis in cancer studies
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Complementary sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores
FISH Probes
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What is the size range for the FISH probes?
20-1000 base pairs (1000 base pairs = 1 megabase)
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2 types of labelling in FISH probes?
Direct and Indirect Labelling
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DIRECT OR INDIRECT LABELLING: Fluorophores are directly attached to the probe
Direct
46
DIRECT OR INDIRECT LABELLING: Drawback is that is it less sensitive
Direct
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DIRECT OR INDIRECT LABELLING: FITC, Rhodamine
Direct
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DIRECT OR INDIRECT LABELLING: Cyanines
Direct
49
DIRECT OR INDIRECT LABELLING: Fluorophores usually attached at the end of the DNA
Direct
50
DIRECT OR INDIRECT LABELLING: Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization
Indirect
51
DIRECT OR INDIRECT LABELLING: Biotin and Digoxigenin
Indirect
52
Unbound fluorescent dyes are also known as?
Background dyes
53
Biotin is derived from?
Egg yolk
54
Biotin's partner molecule is usually?
Avidin which has the fluorescent dye
55
Have very strong covalent action in nature
Biotin and Avidin
56
Which is attached to the dye, avidin or biotin?
Avidin
57
Which is attached to the probe, avidin or biotin?
Biotin
58
What are the different types of FISH probes?
Locus Specific Probe
Alphoid or Centromeric repeat probe
Subtelomeric Probe
Whole Chromosome Probe
Pre-natal FISH Probe
59
WHAT KIND OF FISH PROBE: Binds to a particular region of a chromosome
Locus specific
60
Locus specific probe is only used when?
only a small portion of a gene is isolated;
to determine on which chromosome the gene is located, or how many copies of a gene exist within a particular genome
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Locus specific identifiers are more ____ specific
Gene/region
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The locus specific probes span the specific region and average what range of kb in size?
200-300 kb
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Locus specific identifies are useful diagnostic tools to detect?
Deletions
Duplications
Rearrangements
Amplifications
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Locus specific identifies can also be used in?
Fusion-type probe strategies
65
2 types of Color FISH probes in Locus-specific probes?
Single color
Dual color
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SINGLE OR DUAL COLOR FISH PROBE: Designed to cover a gene of interest
Single
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SINGLE OR DUAL COLOR FISH PROBE: Designed to cover any 2 genes for the detection of any aberrations.
Dual
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Dual color FISH probe allows simultaneous detection of?
numerical abnormalities of two to three regions in one FISH assay.
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SINGLE OR DUAL COLOR FISH PROBE: Creates a color that is a mixture of the previous colors which signifies a gene fusion
Dual
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Dual fusion FISH strategies are probes that span what?
Specific regions of interest with recurring breakpoints
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Dual fusion FISH strategies are used to identify what?
Chromosome rearrangements involving 2 chromosomes
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WHAT KIND OF FISH PROBE: Generated from repetitive sequences found in the middle of each chromosome
ALPHOID/CENTROMERIC REPEAT
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WHAT KIND OF FISH PROBE: Used to determine whether an individual has the correct number of chromosomes or if there is aneuploidy in the patient’s genome
ALPHOID/CENTROMERIC REPEAT
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Alpha-satellite probes are also known as?
Chromosome Enumeration probes
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Alpha-satellite probes bind to?
Highly repetitive DNA
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Alpha-satellite probes are most often used as what kind of probes?
Control probes
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Most alpha-satellite probes give out what to detect aneuploidies?
Large, bright signal
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WHAT KIND OF FISH PROBE: Specific to the subtelomere region of chromosome
Subtelomere Probe
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WHAT KIND OF FISH PROBE: Useful in the detection of subtelomere deletions and
rearrangements
Subtelomere Probe
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A kind of deletion that is important in cancer detection
Subtelomere deletions
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WHAT KIND OF FISH PROBE: Collection of smaller probes that bind to the whole length of chromosome
Whole Chromosome Probes
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Whole Chromosome Probes are useful in the examination of?
chromosomal aberrations
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Hybridize to the unique sequences which cover the length of an entire chromosome or chromosome arm
Whole chromosome paints
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Whole chromosome paints are useful in studying what?
Marker chromosomes, translocations and aneuploidy in metaphase cells
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Are whole chromosome paints useful in Interphase cells?
No
86
T or F: Whole chromosome paints are useful in detecting insertions
T
87
WHAT KIND OF FISH PROBE: Comprise of different combinations of fluorophore- labeled probes
Pre-Natal FISH Probes
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Pre-Natal FISH Probes are specific for what chromosomes?
chromosomes 13, 18, 21, X, and/or Y
89
T or F: Pre-Natal FISH Probes provide a shorter TAT than karyotyping
T
90
Steps in FISH?
1. Probe and target DNAs are denatured using high temperature incubation in a formamide/ salt solution.
2. Probe sequences hybridize to the complementary target sequences, and nonspecific binding is eliminated via stringent washing.
3. The probe hybridization is detected with fluorescence microscopy
91
What are some applications of FISH?
1. Detection and characterization of chromosome abnormalities
2. Detection and analysis of prenatal chromosomal abnormalities
3. Study of chromosomal abnormalities associated with cancer
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a technique that uses DNA from the cells of interest, rather than using a standard karyotype, for chromosomal analysis.
Comparative Genomic Hybridization (CGH) on Metaphase Cells
93
This can be very useful, especially in some cancers when only DNA is available rather than any growing cells.
Comparative Genomic Hybridization (CGH) on Metaphase Cells
94
This technology has been used successfully for clinical analysis, particularly with cases that have a low (or no) mitotic index.
Comparative Genomic Hybridization (CGH) on Metaphase Cells
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CGH is NOT useful for detecting what?
balanced rearrangements
96
A technique that allows the investigator to view a karyotype so that each chromosome is “painted” with a different color
Multiplex FISH (M-FISH)
97
Used to create a distinct computer-generated false color for each chromosome
Ratio-labeled probes
98
Useful for complex rearrangements, such as those seen in neoplastic disorders and solid tumors
Multiplex FISH (M-FISH)
99
Uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes
Muticolor Banding (mBAND) analysis
100
In mBAND, a computer program analyzes metaphase chromosome data and produces what?
pseudocolored, banded karyotype with an estimated resolution of 550 bands, regardless of chromosome length
101
Advantageous for the determination of breakpoints and the analysis of intrachromosomal rearrangements and can be particularly useful in preparations with shorter chromosomes.
Muticolor Banding (mBAND) analysis
102
A technique that is almost entirely used for research
Fiber Fish
103
It allows the chromosomes to be stretched out and elongated
Fiber Fish
104
The probes are applied and can be physically ordered on the fibers
Fiber Fish
105
Provides a much higher spatial resolution and allows for correct orientation and placement of probes and for precise mapping of probes
Fiber Fish
106
Essentially PCR on a slide
Primed In Situ Labeling (PRINS)
107
In PRINS, primers of interest are ________ on a slide and then subjected to cycles of __________ that are used to incorporate labeled nucleotides. The labels are then detected _________, or labeled nucleotides are _________________ during the reaction
hybridized
denaturation, reannealing, and elongation
fluorescently
incorporated
108
PRINS can differentiate hybridization with the alpha satellite sequences for chromosomes _____ and _____ , something that cannot be done with traditional FISH
13 and 21
109
Used to identify material of unknown origin
Reverse FISH
110
In reverse FISH, unidentified material, such as a marker chromosome or duplication, is ____________ off of a slide after ___________
flow sorted or microdissected
G-banding
111
The DNA from this material is extracted, PCR- amplified and labeled with a fluorochrome. This is then used as a probe and hybridized to normal or patient metaphase chromosomes to identify the origin of the unknown material
Reverse FISH
112
Template is the one with the probe, not the nature DNA
Reverse FISH
113
Dividing specimen for FISH testing
Metaphase cells
114
Non-dividing specimen for FISH testing
Interphase cells
115
Direct preparation samples from interphase cells include?
Uncultured cells from blood, bone marrow and cytospins
Smears made from blood, buccal cells, bone marrow
116
Paraffin embedded tissue sections under interphase cells include?
Tumors
Products of conceptions
117
What are FFPE preserved tissues?
Formalin Fixed Paraffin Embedded Tissues
118
What step in the FISH process includes the treatments to harden chromatin and dehydration?
Specimen processing/ harvesting
119
T or F: FISH specimen includes baking at high temperature similar to that of G banding
F, does NOT include baking
120
What will happen if the sample for FISH was baked at high temperatures?
The chromatin will dry our and will result in poor or low hybridization process
121
What temperature is the FISH process usually set at?
37C
122
Denaturing of DNA includes breaking what?
Hydrogen bonds to form single stranded DNA
123
The denaturation temperature is lowered using what?
Formamide
124
Hybridization with fluorescently labelled DNA probes includes what process which lasts for several hours?
Reannealing
125
Incubation period for reannealing is usually how long?
4-20hrs
126
Temperature has to (lower/heighten) for reannealing
Lower
127
What needs to be washed off after staining?
Excess unbound probe / background
128
What happens if the excess bound probes are not washed off?
There will be noise and this is decrease the specificity of the process
129
What solution is effective is removing the unbound DNA?
Stringent Solution
130
What is used to visualize nuclear boundaries and the morphology?
Counterstain
131
What are examples of counterstains to visualize nuclear boundaries and morphology?
Propidium iodide and DAPI
132
In this probe strategy, the probes will flank the specific region of interest
Break apart (BAP) FISH probe strategy
133
BAP strategy is used to detect what?
Chromosomal rearrangements where there are multiple known partners (promiscuous genes)
134
BAP strategy is useful in what type of samples?
FFPE
135
What are some clinical applications of FISH?
Additional interpretation of abnormal karyotypes
Detecting diagnostic abnormalities
Providing prognostic information in some cancer types
Abnormal FISH patterns may serve as a monitor of disease or response to therapy
136
Alterations of the DNA of a genome that results in the cell having an abnormal number of copies of one or more section of the DNA
Copy Number Variations (CNVs)
137
On certain chromosomes, CNVs result in the large regions of the chromosomes being _____ OR _______
Deleted (fewer than normal) or duplicated (more than normal)
138
What can contribute to tumorigenesis?
Amplifications and deletions
139
What is the most common change seen in malignancies?
Amplifications
140
Provides an approach to associate an aberration with a disease phenotype and localizing critical genes
Detection and mapping
141
Affected by localizing the biomarkers that would indicate the phenotype of the disease
Prognosis and therapeutics
142
What are the other uses of CGH?
Resistance and susceptibility to disease
Mental retardation, developmental delay and seizures
Dysmorphic features and multiple congenital anomalies
Schizophrenia and autism
143
Molecular cytogenetic method for the analysis of CNVs in the DNA content of a given subject's DNA, often in tumor cells
Comparative Genomic Hybridization or Chromosomal Microarray Analysis (CMA)
144
CGH/CMA was first described by?
Kallioniemi et al 1993
145
What are the 2 types of techniques in CGH?
Chromosomal technique
Genomic technique
146
In CGH, how are metaphase chromosomes made into single stranded?
Alkaline treatment done on the disease and normal chromosome (should be from the same person)
147
In CGH, the DNA from the subject tissue and normal tissue are labelled with?
Different tags
148
For array or matrix CGH, the DNA is _______ to _______ chromosomes
Hybridized
Metaphase
149
What can be detected and used for identifying abnormal regions in the genome?
Regional differences in the fluorescence ratio of gains and losses vs control DNA
150
What is applied for detecting all genomic imbalances?
CGH (special FISH technique using dual probes)
151
The basics of the CGH technique comprises what?
The comparison of total genomic DNA of the given sample DNA with total genomic DNA of normal cells
152
T or F: Identical amount of both tumor and normal DNAs are labelled with the same fluorescent dyes in CGH
F, labelled with 2 different fluorescent dyes
153
The mixture of the tumor and normal DNAs in CGH is added and hybridized to a?
Lymphocyte metaphase slide
154
What devices/equipments are used for evaluation in CGH?
Fluorescent microscope equipped with a CCD camera
155
What is a CCD device?
Charged couple device - a capture device that can count and interpret signals
156
CGH is used to determine what?
copy number alterations of genome in cancer and those cells whose karyotype is hard or impossible to prepare or analyze
157
T or F: CGH will detect only unbalanced chromosomal changes
T
158
T or F: CGH can detect structural chromosome aberrations such as balanced reciprocal translocations or inversions
F, CANNOT be detected, as they do not change the copy number.
159
CGH is _____________ of two differentially labeled genomic DNAs (eg. tumor and normal) to human metaphase chromosome spreads.
based onco-hybridization
160
CGH is based on the ____________ onto ____________, which usually have been prepared from ____________ of a healthy donor.
co-hybridization of differentially labelled test and reference DNA
metaphase spreads
peripheral blood lymphocytes
161
In the CGH process, the __________ of the two labels along the chromosomes then reflect _____________ in the test genome relative to the _____________.
signal intensity ratios
DNA copy number changes
reference genome
162
The resolution for the signal intensity ratio of the 2 labels for CGH is limited to about?
3-10Mb
163
In CGH, after extraction of test DNA (i.e. from a tumor sample) and normal DNA (i.e. from peripheral blood), the samples are differentially labeled with?
discernable fluorochromes
164
What are examples of discernable fluorochromes used for labelling test and normal DNA?
tumor DNA with FITC [green] and control DNA with TRITC [red]
165
In CGH, the genomes are combined with an excess of what and hybridized to?
Cot 1 DNA and hybridized to metaphase chromosomes
166
What is a common problem in assays?
Background hybridization due to repetitive DNA sequences
167
What blocks repetitive DNA sequences and prevents non specific hybridization?
Cot-1 DNA blocking reagent
168
Images of metaphase spreads are then acquired with ___________ and __________ to capture the FITC and TRITC fluorescence
CCD camera and fluorochrome- specific optical filter sets
169
Differences in fluorescence intensity values between tumor and control DNA represent?
gains and losses of specific chromosomes or chromosomal regions
170
A gain of a chromosomal region in the test sample would result in?
an increased intensity of green fluorescence
171
A loss within a chromosomal region in the tumor would be indicated by?
a shift towards red intensities.
172
What measures fluorescence intensity values along the length of the chromosomes and translates the ratios into chromosome profiles?
CGH analysis software
173
The ratio of green to red fluorescence values is used to quantitate what?
genetic imbalances in tumor samples.
174
Who is the first person to define the karyotype as the phenotypic appearance of the somatic chromosomes in contrast to their genic contents?
Gregorio Levitsky (1931)
175
A long or short term technique to understand the various molecular, cellular and organic functions
Tissue culture
176
Simplest form of metaphase chromosome preparation by using lectin as a mitigen like phytohemagglutinin, concanavilin A, pokeweed mitogen etc.
Short time lymphocyte culture
177
Short time lymphocyte culture uses what as a mitigen which is similar to what other mitogens?
Lectin which is similar to phytohemagglutinin, concanavilin A, pokeweed mitogen etc.
178
Used to detect chromosomal abnormality and rearrangements
Metaphase chromosome
179
What are the types of karyotypes?
Asymmetric and Symmetric
180
Which between the types of karyotyping show large difference between smaller and larger chromosome in a set?
Asymmetric
181
Which between the types of karyotyping have more acrocentric chromosomes?
Asymmetric
182
Which between the types of karyotyping show lesser difference between smaller and larger chromosomes in a set?
Symmetric
183
Which between the types of karyotyping have more metacentric chromosomes?
Symmetric
184
Facultative chromatins
Euchromatin
185
Allows to judge the changes in smaller pieces of the chromosomes and related abnormalities which are not visible in analysis
Banding
186
Prenatal diagnosis for chromosomal abnormalities, fetal infections and sex determination
Amniocentesis
187
Done by obstetricians 10-14 weeks of pregnancy by collecting a small sample of placenta either through a thin needle through the abdomen or a thin tube entering the vagina (similar to that of Pap's smear)
Chorionic Villus Sampling
