UNIT 8.2 FISH Flashcards
A cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complementary to its sequence
Fluorescence In-Situ Hybridization (FISH)
Hybridizing two complementary sequences such as
probe and target sequence
small sequence of nucleotides that are complementary to a DNA sequence
probes
Useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes
Fluorescence In-Situ Hybridization (FISH)
Detects and localizes specific DNA sequences using fluorescently labeled complementary DNA probes.
Fluorescence In-Situ Hybridization (FISH)
Detection is through?
fluorescence
T OR F
No fluorescence means that no hybridization occurred, therefore the target sequence is present
FALSE
target sequence should be absent
Rapid diagnosis of ___ and ___ is acquired using specific probes
trisomies and microdeletions
To separate DNA strands and
allow probe access to target
DNA
Denature
the process wherein the DNA uncoils and strands separate.
Denaturation
Together to bind probe to target
DNA
Hybridize
Probe signals using a
fluorescent microscope
Analyze
Metaphase or Interphase
Gold standard and routinely done
Metaphase FISH
Metaphase or Interphase
Done on cultured cells
Metaphase FISH
Metaphase or Interphase
Allows direct visualization of chromosomes and exact position of signals
Metaphase FISH
Metaphase or Interphase
useful in the detection of structural changes in the genome; for molecular analysis
Metaphase FISH
Metaphase or Interphase
May also be done on
uncultured specimens
Interphase FISH
Metaphase or Interphase
Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies; detection of genetic abnormalities
Interphase FISH
Metaphase or Interphase
beneficial in the study of samples with a low mitotic index, such as most solid tumors
Interphase FISH
Metaphase or Interphase
Major disadvantage is the inability to detect unknown structural chromosomal changes
Interphase FISH
samples for Interphase FISH
- Amniocytes
- Peripheral blood smears
- Bone marrow aspirate smear or direct harvest
samples for Metaphase FISH
- Amniocytes
- Chorionic villous cells
- Lymphocytes
- Cells from bone marrow aspirates or solid tumors fibroblasts
translocation or copy number analysis in cancer studies
Bone marrow aspirate smear or direct harvest
For ploidy analysis in newborns
Peripheral blood smears
Found within the brain.
Produces CSF
Chorionic villous cells
For ploidy analysis during prenatal studies
Amniocytes in Interphase FISH
Cells from amniotic fluid
Amniocytes in Metaphase FISH
Complementary sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores
FISH Probes
Complementary binding of base pairs
(A-T, G-C)
Size ranges from ___ to ___ base pairs
20 to 1000
T OR F
The longer the base pairs, the less specific, and the less expensive
FALSE
should be more specific and more expensive
- Fluorophores are directly attached to the probe
- Less sensitive
Direct Labeling
examples of direct labeling
- Fluorescent isothiocyanate (FITC)
- Rhodamine
- Cyanines
■ Most commonly used label
■ Apple green signal
Fluorescent isothiocyanate (FITC)
Reddish signal
Rhodamine
Bluish signal
Cyanines
More expensive but is preferred because the materials used add up to the sensitivity of the probe
Indirect Labeling
Major disadvantage is that the process is much more tedious due to additional steps
Indirect Labeling
Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization
Indirect Labeling
examples of indirect labeling
- Biotin
- Digoxigenin
- Antibodies
Commonly used due to having many sources
Biotin
Biotin is an essential nutrient of
hair
Biotin is complementary to Avidin which is acquired from ___
eggs
T OR F
What is bound to the probe is fluorophore and the Biotin is attached to Avidin
FALSE
fluorophore = Avidin
probe = Biotin
Avidin and Biotin will bind, forming a complex with the ____ attached
fluorophore
Binds to a particular region of a chromosome
Locus Specific Probe
Used when only a small portion of a gene is isolated and want to determine in which chromosome the gene is located, or how many copies of a gene exist within a particular genome
Locus Specific Probe
Single or Dual Color FISH Probe
Designed to cover a gene of interest
Single Color FISH Probe
Single or Dual Color FISH Probe
Uses one probe and one fluorophore specific to an area
Single Color FISH Probe
Single or Dual Color FISH Probe
Designed to cover any 2 genes for the detection of any aberrations.
Dual Color FISH Probe
Single or Dual Color FISH Probe
Allows simultaneous detection of numerical abnormalities of two to three regions in one FISH assay.
Dual Color FISH Probe
Single or Dual Color FISH Probe
Uses two probes labeled with two different fluorophores
Dual Color FISH Probe
Generated from repetitive sequences found in the middle of each chromosome
Alphoid or Centromeric Repeat
Used to determine whether on individual has the correct number of chromosomes or if there is aneuploidy in the patient’s genome
Alphoid or Centromeric Repeat
Targets centromere and is designed to be complementary to it
Alphoid or Centromeric Repeat
Specific to the subtelomere region of chromosome
Subtelomeric Probe
Useful in the detection of subtelomere deletions and rearrangements
Subtelomeric Probe
If the reason behind down syndrome cannot be identified, this is used.
Subtelomeric Probe
Collection of smaller probes that bind to the whole length of chromosome
Whole Chromosome Probe
Smaller probes are specific to an individual locus in a chromosome
WHOLE CHROMOSOME PROBE
Tag the whole chromo by collecting different small probes, not just a one long probe
Whole Chromosome Probe
T or F
Whole chromosome probe is useful in the examination of chromosomal aberrations
TRUE
Specific to chromosomes to determine if the fetus would develop common anueploidies/trisomies
Pre-natal FISH Probe
Comprised of different combinations of fluorophore-labeled probes specific for chromosomes 13 (Patau), 18 (Edward), 21 (Down), X (Klinefelter), and/or Y (Turner)
Pre-natal FISH Probe
Types of fish probes
- alphoid or centromeric repeat
- subtelomeric probe
- locus specific probe
- whole chromosome probe
- pre-natal fish probe
first step in FISH
- Probe and target DNAs are denatured using high temperature incubation in a formamide/ salt solution.
second step in FISH
- Probe sequences hybridize to the complementary target sequences, and nonspecific binding is eliminated via stringent washing.
most critical step as it removes probes that are unbound to the DNA.
Stringent washing (2nd step)
T or F (steps in fish)
Probes will continue to fluoresce whether they are bound or unbound.
TRUE
T or F (steps in fish)
Binding is a signal for fluorescence.
FALSE
it is NOT a signal
too much / too little results to what?
false negative/false positive
third step in FISH
The probe hybridization is detected with fluorescence microscopy
The third or last step in FISH is a fragile procedure because we are dealing with what?
small samples such as 5 microliters
- The nucleus should be separate and the boundaries easily seen
- No overlapping
Cell scoring in FISH
______ might be due to a twisted nuclei or one on top of another
Overlapping
A ______ would either be directly next to
each other or have a very small space between them
split signal
APPLICATION OF FISH
- Detection and characterization of chromosome
- Detection and analysis of prenatal chromosomal abnormalities
- Study of chromosomal abnormalities associated with cancer
A technique that uses DNA from the cells of interest, rather than using a standard karyotype, for chromosomal analysis.
CGH on metaphase cells
CGH on metaphase cells can be very useful, especially in some ____ when only DNA is available rather than any growing cells.
cancers
CGH on metaphase cells has been used successfully for clinical analysis, particularly with cases that have a ____
low (or no) mitotic index.
CGH on metaphase cells is not useful for detecting _____
balanced rearrangements
T or F
CGH on metaphase cells has low sensitivity and specificity
FALSE
should be high sensitivity
A technique that allows the investigator to view a karyotype so that each chromosome is “painted” with a different color
Multiplex FISH (M-FISH)
MULTIPLEX FISH (M-FISH)
____ are used to create a distinct computer-generated false color for each chromosome
Ratio-labeled probes (invisible colors detected by computer)
MULTIPLEX FISH (M-FISH)
Useful for complex rearrangements, such as
those seen in ___ and ____
neoplastic disorders and solid tumors
Uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes.
Multicolor banding (mBAND) analysis
MULTICOLOR BANDING (mBAND) ANALYSIS
A computer program analyzes metaphase chromosome data and produces a ______
pseudo colored
MULTICOLOR BANDING (mBAND) ANALYSIS
banded karyotype with an estimated resolution of _____, regardless of chromosome length.
550 bands
Advantageous for the determination of breakpoints and the analysis of intrachromosomal rearrangements, and can be particularly useful in preparations with shorter chromosomes.
Multicolor banding (mBAND) analysis
Multicolor banding
Region-specific probes labeled with different
_______ and ________ produces a definable number of colored bands per chromosome, regardless of chromosome length.
partial chromosome paints (PCP) and computer false color (MetaSystems’ mBAND)
A technique that is almost entirely used for research
Fiber FISH
T or F
fiber fish allows the chromosomes to be stretched out and elongated
TRUE
This provides a much higher spatial resolution and allows for correct orientation and placement of probes and for precise mapping of probes
Fiber FISH
Tries to extend chromosome; chromosome stretched to a string and bound with a probe
Fiber FISH
Essentially PCR on a slide.
All procedures are done on that slide.
Primed In Situ Labeling (PRINS)
PRIMED IN SITU LABELING (PRINS)
Primers of interest are hybridized on a slide and then subjected to cycles of ___, ____ and _____ that are used to incorporate labeled nucleotides. The labels are
then detected fluorescently or labeled nucleotides are incorporated during the reaction
- denaturation, reannealing, and elongation
PRINS can differentiate hybridization with the alpha satellite sequences for chromosomes ___ and ___, something that cannot be done with traditional FISH
13 and 21
PRIMED IN SITU LABELING (PRINS)
Metaphase chromosomes are subjected to PRINS with _______ specific for chromosomes X, 11, and 17
______ staining is seen at the centromeres of these chromosomes
- alpha satellite oligonucleotides
- Bright yellow fluorescein
Used to identify material of unknown origin
Reverse FISH
REVERSE FISH
This unidentified material, such as a marker chromosome or duplication, is ____ or _____ of a slide after G-banding. 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
flow sorted or microdissected
T or F
in reverse fish, if binding occurs, then the unknown sequence is found
TRUE
what are the specialized and evolving technologies
- CGH on metaphase cells
- multiplex FISH (M-FISH)
- multicolor banding (mBAND) analysis
- fiber FISH
- primed in situ labeling (PRINS)
- reverse FISH
