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2S CYTOGEN > U8: FISH > Flashcards

U8: FISH Flashcards

1
Q

FISH is under what branch of cytogeneyics?

A

Molecular

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2
Q

This technique is used because it has a fast turn around time.

A

FISH

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3
Q

FISH stands for?

A

Fluorescence In-Situ Hybridization (FISH)

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4
Q

This is a cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complimentary to its sequence.

A

FISH

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5
Q

This is useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes.

A

FISH

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6
Q

What disease can be detected in using FISH?

A

Leukemias

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7
Q

The earliest kind of karyotype staining is?

A

Fluorescent staining (Quinacrine mustard)

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8
Q

Fluorescence involves the use of what?

A

excitation light (most likely UV as it is energetic)

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9
Q

T/F: You may use other lights in the spectrum of visible light for fluorescence.

A

T

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10
Q

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.

A

energy level, resting state

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11
Q

These are small strips of single stranded DNA, complementary to the sites that we want to examine in the chromosome.

A

Probes

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12
Q

The DNA that is bound by the probe is called?

A

Target chromosome

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13
Q

T/F: Small refers to a thousand limit in bases.

A

T

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14
Q

Number of bases for FISH

A

200-400 bases

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15
Q

This refers to native DNA, which is DNA extracted from the patient.

A

Target chromosome

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16
Q

This is artificial DNA, made in laboratories or biotechnology companies. The strips of DNA are created in order to target the native DNA.

A

Probe

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17
Q

In what direction is the probe running and is arresting what sequence?

A

3’ to 5’, complementary

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18
Q

If you have just 1 probe to isolate a certain DNA portion (seen in locus-specific FISH), it is called?

A

Detection

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19
Q

This refers to when the entire chromosome is tagged with different colors of probes or automation (whole chromosome FISH)

A

DNA Mapping

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20
Q

FISH is applied to provide s____ l_____ of genes on chromosomes

A

specific localization

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21
Q

R_____ d_____ of trisomies and microdeletions is acquired using specific probes.

A

Rapid diagnosis

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22
Q

Used to check the cause of t_____, m____ s_____, etc.

A

trisomies, microdeletion syndromes

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23
Q

T/F: FISH can detect 1 base

A

F ; 200-400

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24
Q

This is hybridization of a fluorescent probe to a n____ D____.

A

native DNA

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25
Q

The native DNA of the patient or hybridization happens on a?

A

slide preparation

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26
Q

This allows for visualization of target DNA sequences of clinical interests.

A

slide preparation

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27
Q

Choice of technique for c_____-e_____ molecular testing is FISH.

A

cost-effective

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28
Q

S_____ m____ using FISH can be performed on a variety of specimen with only a basic requirement

A

Sequencing methods

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29
Q

What is the basic requirement in FISH for sequencing methods?

A

The DNA should be undegraded, or preserved.

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30
Q

T/F: In molecular techniques, it is okay if you only have the fragment of DNA.

A

T

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31
Q

T/F: DNA must be intact in FISH.

A

T

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32
Q

T/F: Any specimen that can be used in chromosome studies which evaluate metaphase cells can also be used for FISH.

A

T

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33
Q

You can also do FISH in i_____ chromosomes.

A

interphase

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34
Q

Other term for the target DNA.

A

Template

dito daw ginagawa yung probe > release ng complementary sequence

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35
Q

This is to separate DNA strands and allow probe access to target DNA.

A

Denature

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36
Q

H____ together to bind probe to target DNA.

A

Hybridize

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37
Q

A____ probe signals using a fluorescent microscope.

A

Analyze

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38
Q

What is denatured in the process because it is double stranded?

A

Target/Template/Native DNA

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39
Q

Denaturation involves the use of h_____ and denaturing chemicals like F______.

A

heat, Formamide

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40
Q

This means that in a target sequence or on the site, you can attach a probe.

A

in situ

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41
Q

This is performed in a glass slide, once you denature the DNA, the probe will bind on site.

A

In situ hybridization

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42
Q

T/F: Hybridization is when the original pair of DNA is still present.

A

F ; dapat native DNA and artificial DNA

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43
Q

Analyzing is visualizing the regions via f_____.

A

fluorescence

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44
Q

Only a portion of the chromosome will light up, the rest will be what?

A

Counterstained

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45
Q

Examples of Counterstains

A

DAPI, Propidium Iodide

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46
Q

This refers to dividing cells, and cells are thick.

A

Metaphase

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47
Q

These cells are the following: blood, bone marrow, skin fibroblast

A

Metaphase Cells

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48
Q

Metaphase Cells used in post-natal

A
  • Blood
  • Bone marrow
  • Skin fibroblast
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49
Q

These cells in infants are the following:

A

Chorionic villi, amniocytes, tumors

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50
Q

Metaphase cells in infants:

A
  • Chorionic villi
  • Amniocytes
  • Tumors
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51
Q

These are non-dividing cells, in the quiescent stage of cell cycle.

A

Interphase Cells

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52
Q

They can be used for screening.

A

Interphase Cells

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53
Q

They can come from direct preparations: uncultured cells from blood, bone marrow, cytospins.

A

Interphase Cells

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54
Q

Where are bone marrow specimens from?

A

Flat bones in the body

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55
Q

These are bladder cells from bladder cancer. Usually used for fluids, and collected by cytocentrifugation from abnormal cells.

A

Cytospins

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56
Q

FORMATIVE: Speed for cytospin

A

maximum of 1-3 minutes

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57
Q

Interphase Cells

Uncultured cells are from?

A
  • Blood
  • Bone marrow
  • Cytospins
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58
Q

Interphase Cells

Smears are made from?

A
  • Blood
  • Buccal cells
  • Bone marrows
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59
Q

They can come from direct preparations: smears made from blood, buccal cells, bone marrow

A

Interphase Cells

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60
Q

Interphase Cells

Paraffin Embedded Tissue Sections:

A
  • Tumors
  • Products of conceptions
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61
Q

Abnormal tissues are preserved in what?

A

Formalin Fixed Paraffin Embedded (FFPE) Tissues

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62
Q

FFPE stands for?

A

Formalin Fixed Paraffin Embedded

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63
Q

This is the gold standard and routinely done on cultured cells.

A

Metaphase FISH

Gold standard because it is the most rigorous process

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64
Q

This allows direct visualization of chromosomes and exact position of signals.

A

Metaphase FISH

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65
Q

This is useful in detection of structural changes in the genome.

A

Metaphase FISH

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66
Q

This may also be done on uncultured specimens.

A

Interphase FISH

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67
Q

This is advantaegous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies.

A

Interphase FISH

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68
Q

The major disadvantage of ______ FISH is the inability to d_____ u____ structural changes.

A

Interphase, detect unknown

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69
Q

T/F: We light up the whole chromosome in Interphase FISH.

A

T

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70
Q

What is detected more in pre-natal diagnosis?

A

Aneuploidies

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71
Q

T/F: Metaphase FISH is used for de novo mutations.

A

T

but with modifications

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72
Q

T/F: You report results in Interphase FISH as positive or negative since in this technique, you already have the disease in mind.

A

T

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73
Q

Samples for Metaphase FISH

A
  • Amniocytes
  • Chorionic villous cells
  • Lymphocytes
  • Cells from bone marrow aspirates or solid tumors or cytospin
  • Fibroblasts (skin fibroblasts)
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74
Q

Samples for Interphase FISH

A
  • Amniocytes
  • Peripheral blood smears
  • Bone marrow aspirate smear or direct harvest
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75
Q

Interphase FISH

Used for ploidy analysis during prenatal studies

A

Amnicoytes

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76
Q

Interphase FISH

Used for ploidy analysis in newborns

A

Peripheral blood smears

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77
Q

Interphase FISH

Used for translocation or copy number analysis in cancer studies

A

Bone marrow aspirate smear or direct harvest

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78
Q

These are complementary sequences

A
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79
Q
A
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80
Q

This is an important tool to visualize copy number changes detected by other methods (microarrays, CGH)

A

Metaphase FISH

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81
Q

This is a primary screening tool for duplications and deletions.

A

Comparative Genomic Hybridization (CGH)

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82
Q

This is used for the direct detection of where the duplications and deletions happens.

A

pre-test in CGH and for solid tumors

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83
Q

What does Metaphase FISH detect after testing for CGH?

A

specific regions where we see detectable changes

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84
Q

M___ are used for CGH.

A

Microarrays

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85
Q

Interphase FISH offers s___ d____ t____ t____, a faster screening process for certain assays because you don’t have to culture cells.

A

same day turnaround time

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86
Q

These are complementary DNA sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores.

A

Probe

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87
Q

R___ and a____ are also complementary sequences to DNA.

A

RNA and analogs

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88
Q

The size of probes ranges from?

A

20 to 1000 bases (1 megabase)

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89
Q

Usual size range for FISH Probes

A

200 to 400 bases

90
Q

Two kinds of FISH Labelling

A
  • Direct Labeling
  • Indirect Labeling
91
Q

This refers to when fluorophores are directly attached to the probe, and is less sensitive.

A

Direct Labeling

92
Q

Examples of probes used in Direct Labeling

A

FITC, Rhodamine, Cyanine

93
Q

T/F: Fluorescent dye may attach to intercalated DNA, depending on its staining properties.

A

T

94
Q

Probes are usually on the e___ of DNA.

A

ends

95
Q

This refers to when chemical conjugation of the nucleic acid with a non-fluorescent molecule that can bind fluorescent material after hybridization.

A

Indirect Labelling

96
Q

Examples of probes used in Direct Labelling

A

Biotin, Digoxigenin

97
Q

Indirect Labelling

Partner molecules of Biotin

A

Avidin, Streptavidin

98
Q

T/F: In Biotin, the partner molecule will have the fluorescent dye.

A

T

99
Q

Indirect Labelling

Biotin have very strong c_____ interactions.

A

covalent

100
Q

Indirect Labelling

Biotin is derived from e___ y____. Avidin is found in e___ w___ if not, produced artificially.

A

egg yolk, egg white

101
Q

Indirect Labelling

These two molecules have a very strong covalent interaction with each other.

A

Biotin, Avidin

102
Q

This refers to unbound fluorescent dyes that might confuse stains.

A

Background

103
Q

For better specificity of t_____, secondary reactions are used in Indirect Labelling.

A

tagging

104
Q

FISH Process

Specimen processing/harvesting

A
  • Pretreatments to harden chromatin
  • Dehydration
105
Q

FISH Process

Denature DNA

A

Break Hydrogen bonds to form single stranded DNA

106
Q

FISH Process

Hybridize with fluorescently labeled DNA probe

A

Reannealing for several hours

107
Q

This refers to breaking the Hydrogen bonds.

A

Denaturation

108
Q

T/F: There is more heat if there is more G-C bonds.

A

T

109
Q

This refers to the DNA sticking together with the probes. This also refers to closing up of the DNA double strand.

A

Reannealing

110
Q

Pretreatments require several changes of?

A

Alcohol

111
Q

FORMATIVE: What percentage of alcohol is used to dehydrate chromatin?

A

70-80-90 (ascending order series)

112
Q

B___ increases temperatures which is not advisable in FISH.

A

Baking

113
Q

This is another term for the pretreatments to harden chromatin in room temperature.

A

aging process

114
Q

Pre-soaking in s____ s___ can help c____ and prepare the chromatin.

A

salt solutions, condense

115
Q

T/F: Target DNA and Probe DNA can be denatured separately or co-denatured.

A

T

116
Q

To reanneal, you have to _____ the temperature.

A

lower

117
Q

T/F: Temperatures for reannealing are given by the manufacturers.

A

T

118
Q

Range of Reannealing

A

4 to 20 hours

119
Q

The rate at which strands reanneal is dependent upon d_____ c__________.

A

DNA concentration

120
Q

The rate at which strands reanneal is dependent on the following factors:

A
  • DNA concentration
  • Size of probe
  • Degree of similarity between probe and target DNA
121
Q

S______ of probe is a determinant of how long it reanneals with DNA.

A

Specificity

122
Q

T/F: Both probe DNA and native DNA concentration can be used to determine rate of reannealing.

A

T

123
Q

Step after staining

A

Washing off excess unbound probe

124
Q

This refers to excess and unbound probe that may still fluoresce.

A

Background Flourescence / Background Signals

125
Q

Background Flourescence contributes to?

A

noise

126
Q

Background Flourescence decreases the s_______ of the process.

A

sensitivity

127
Q

Reagents in Washing

A

Buffer, Wash solution, Wash buffer

128
Q

What are the usual washing buffers?

A

Detergents with a certain pH (technology-specific)

129
Q

This refers to the strictness of conditions defined how much the probe will bind and how much will be washed.

A

Stringency

130
Q

Stringency can be modified by the following:

A
  • Temperature
  • Concentration of Formamide or Salt solution
131
Q

What step is done for better visualization?

A

Counterstain

132
Q

Counterstains in FISH

A
  • Propidium Iodide (PI)
  • DAPI (4’6-diamidino-2-phenylindole)
133
Q

Color of Propidium Iodide (PI)

A

Red

134
Q

Color of DAPI (4’6-diamidino-2-phenylindole)

A

Blue

135
Q

Visualization is done under?

A

Fluorescence or Darkfield microscope

136
Q

Choice of kilobases

A

2-400

137
Q

The size of the t__ will affect how small the p____ will be

A

target, probe

138
Q

T/F: Highly repeated targets will appear bright even with small sequences.

A

T

paulit-ulit didikit sa region

139
Q

T/F: The bigger the probe, the brighter the fluorescence.

A

T

140
Q

Most commonly used FISH probes

A
  • Whole Chromosome Paints (WCPs)
  • Chromosome Enumeration Probes (CEPs)
  • Locus-specific identifiers (LSIs)
141
Q

T/F: WCPs can cover both the whole chromosome or just the arm.

A

T

142
Q

These comprises libraries of multiple overlapping chromosomes or chromosomal region-specific probes which covers the length of the entire chromosome or just the arm.

A

Whole Chromosome Paints (WCPs)

143
Q

This means multiple probes in multiple sites of the chromosome.

A

Libraries

144
Q

If the libraries are small, what do we call the method?

A

Shotgun method

145
Q

This refers to coloring the entire chromosome or region-specific, leaving no space in between.

A

Overlapping

146
Q

Probes are usually created via?

A

PCR

147
Q

WCPs are useful in the following:

A
  • studying marker chromosomes
  • translocations
  • aneuploidy in metaphase cells
148
Q

These are known as centromeric probes.

A

Chromosome Enumeration Probes (CEPs)

149
Q

CEPs apply to both?

A

metaphase and interphase FISH

150
Q

CEPs target what DNA sequences?

A

satellite DNA or alpha-satellite DNA (highly repetitive DNA)

151
Q

CEPs are also known as?

A

alpha-satellite probes

152
Q

Highly repetitive DNA reside in what regions?

A

heterochromatic

153
Q

CEPs target what region?

A

centromeric regions

154
Q

CEPs are often used as?

A

control probes (check if you have or don’t have)

155
Q

T/F: CEPs do not give off large bright signals.

A

F

156
Q

CEPs are useful in?

A

fast detection of aneuploidies

157
Q

T/F: LSIs can be used for centromeric regions.

A

T

158
Q

This is used for more gene/region-specific, single-copy, or unique sequence probe.

A

Locus-specific identifiers (LSIs)

159
Q

LSIs span region and average of what range in size?

A

200-300 kb

160
Q

LSIs are useful for:

A
  • Deletions
  • Duplications
  • Rearrangements
  • Amplifications
161
Q

LSIs can also be used in f____-t____ probe strategies.

A

fusion-type

162
Q

This probe binds to a particular region of a chromosome, and used when only a small portion of a gene is isolated.

A

Locus Specific probe

163
Q

Locus specific probes are used to determine:

A
  • which chromosome the gene is located
  • how many copies of the gene exist within a genome
164
Q

This is designed cover a gene of interest, done on cultured cells.

A

Single Color FISH Probe

165
Q

This is designed to cover any 2 genes, and allows simultaneous detection of two to three regions in one FISH assay.

A

Dual Color FISH Probe

166
Q

This is used to identify specific chromosome rearrangements, typically in regions with recurring breakpoints. This involved 2 chromosomes.

A

Dual Fusion FISH Probe Strategies

167
Q

Dual Fusion FISH Probe Strategies

The presence of yellow fluorescence would mean that a f___ had occured.

A

fusion

168
Q

Gene fusions are usually seen in?

A

leukemias and cancers

169
Q

T/F: Dual fusion is better for detecting what we already assume what is there.

A

T

170
Q

This is used to detect chromosomal rearrangements, wherein the fusion is located within one chromosome.

A

Break Apart (BAP) Fish Probe Strategies

171
Q

What samples are used in BAP?

A

FFPE samples (Formalin fixed, paraffin embedded)

172
Q

BAP is useful for rearrangements with what patterns?

A

multiple known patterns

173
Q

BAP is useful for detecting the translocation of what genes?

A

promiscuous

174
Q

T/F: BAP cannot be used for de novo mutations.

A

F ; better for de novo

175
Q

This is generated from repetitive sequences found in the middle of each chromosome.

A

Alphoid / Centromeric Repeat Probe

176
Q

This is used to determine whether individual has the correct number of chromosomes, aneuploidies or trisomy.

A

Alphoid / Centromeric Repeat Probe

177
Q

This is locus-specific to the subtelomere region of the chromosome.

A

Subtelomeric probe

178
Q

This is useful in detection of subtelomere deletions and rearrangements.

A

Subtelomeric probe

179
Q

Telemore deletions indicate signs of?

A

cancer

180
Q

T/F: If most of your subtelomeric regions light up, you do not have telomeric regions and seen in cancerous states.

A

T

181
Q

This is comprised of different combinations of fluorophore-labeled probes specific for chromosomes 13, 18, 21, X and/or Y.

A

Pre-natal FISH Probe

182
Q

Pre-natal FISH probe targets what chromosomes?

A

13, 18, 21, X and/or Y

183
Q

T/F: Pre-natal FISH Probes are locus-specific.

A

T

184
Q

Pre-natal FISH probe is used for screening, as early as?

A

24 hours or earlier

185
Q

What cells are used for Pre-natal FISH probe?

A

interphase

186
Q

Minimum TAT for Karyotyping

A

3 days

187
Q

Pre-natal FISH probe uses what sample?

A

regardless of what sample but usually chorionic villi, amniotic fluid, bone marrow

188
Q

T/F: Pre-natal FISH only targets the usual aneuploidies.

A

T

189
Q

Single-Probe cell scoring is used for?

A

interphase FISH, detecting aneuploidy

190
Q

deletion in c15 (15q11-13)

A

Prader-Willi

191
Q

duplication in chromosome?

A

11

192
Q

For what disorders is the MYB probe used?

A

B cell disorders

193
Q

What probe is used for B cell disorders?

A

MYB Probe

194
Q

MYB probe is used for?

A

Chronic Lymphocytic Leukemia (CLL)

195
Q

This is a specialized form of WCPs.

A

Multiplex FISH (M-FISH)

196
Q

This technique views a karyotype with each chromosome painted with a different color.

A

Multiplex FISH (M-FISH)

197
Q

T/F: Multiplex FISH (M-FISH) is automated.

A

T

198
Q

T/F: In M-FISH, you can start with the fluorescence FISH in which the tags will still be given other colors afterwards.

A

T

199
Q

This is used to create a distinct computer-generated false color for each chromosomes.

A

Ratio-labeled probes

200
Q

This is useful for complex rearrangements, such as those seen in neoplastic disorders and solid tumors.

A

Multiplex FISH (M-FISH)

201
Q

This means being stained separately.

A

Multiplex

202
Q

Multiplex FISH (M-FISH) can be used for pre-B or B lymphocytes

A

Acute Lymphocytic Leukemia (ALL)

203
Q

Multiplex FISH (M-FISH)

This is done and shown by the computer or thru automation.

A

Blended colors, pseudocolors

204
Q

This uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes.

A

Multiple Banding (mBand) analysis

205
Q

Estimated resolution for mBand analysis

A

550 bands

206
Q

In mBand, pseudocolors are produced where?

A

within chromosome

different bands in entire single chromosome

207
Q

mBand is advantageous for the following:

A

*determination of breakpoints
* intrachromosomal rearrangements
* inversions

208
Q

This is a technique that allows chromosomes to be stretched out or elongated, where in probes are applied and physically ordered.

A

Fiber FISH

209
Q

First step in Fiber FISH

A

removal of clumping

interphase cell - sirain ang histone, scaffolds to stretch dna

210
Q

1 chromosome is equal to a

A

thin spread

211
Q

This yields more precise information as to the localization of specific DNA probe on the chromosome. Commonly used for research.

A

Fiber FISH

212
Q

Fiber FISH has been used in?

A

telomeric regions

213
Q

PRINS stand for?

A

Primed In Situ Labelling (PRINS)

214
Q

This is essentially described as PCR on a slide.

A

Primed In Situ Labelling (PRINS)

215
Q

3 steps in PRINS

A
  • Denaturation (cycling temperatures in PCR unlike in FISH na maintained)
  • Hybridization
  • Annealing
216
Q

PRINS can differentiate hybridization with alpha satellite sequences for chromosomes?

A

13 and 21

217
Q

T/F: In PRINS, you create your probe in the slide itself.

A

T

218
Q

This is used to identify material of unknown origin.

A

Reverse FISH

219
Q

Reverse FISH

Unknown DNA used for karyotyping or band of interest can be acquired from?

A

crime scene

220
Q

Steps in Reverse FISH

A
  1. Karyotype (G-banding)
  2. Take band of interest
  3. PCR Amplification and Fluorescent Labelling
  4. Hybridize with chromosomes of suspected criminal
221
Q

Reverse FISH can also be used in?

A

DNA Profiling

2S CYTOGEN (15 decks)

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