U8: FISH Flashcards by maki 🐈‍⬛

FISH is under what branch of cytogeneyics?

Molecular

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

FISH

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FISH stands for?

Fluorescence In-Situ Hybridization (FISH)

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

FISH

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

FISH

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What disease can be detected in using FISH?

Leukemias

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The earliest kind of karyotype staining is?

Fluorescent staining (Quinacrine mustard)

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Fluorescence involves the use of what?

excitation light (most likely UV as it is energetic)

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

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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

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

Probes

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The DNA that is bound by the probe is called?

Target chromosome

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

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Number of bases for FISH

200-400 bases

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

Target chromosome

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

Probe

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

3’ to 5’, complementary

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

Detection

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

DNA Mapping

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FISH is applied to provide s____ l_____ of genes on chromosomes

specific localization

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

Rapid diagnosis

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

trisomies, microdeletion syndromes

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T/F: FISH can detect 1 base

F ; 200-400

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

native DNA

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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/F: DNA must be intact in FISH.

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

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.

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.

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.

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.

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.

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.

To reanneal, you have to _____ the temperature.

lower

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

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.

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.

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.

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.

CEPs are useful in?

fast detection of aneuploidies

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

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.

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.

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.

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.

Single-Probe cell scoring is used for?

interphase FISH, detecting aneuploidy

deletion in c15 (15q11-13)

Prader-Willi

duplication in chromosome?

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

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.

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

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

Reverse FISH can also be used in?

DNA Profiling

U8: FISH Flashcards

(221 cards)