MT6315 LESSON 8 FISH & CGH Flashcards
FISH stands for?
Fluorescence In situ Hybridization
FISH is a cryogenic technique that uses what to do what?
Fluorescent probes to bind specifically to a part of chromosomes complementary to its sequence
The fluorescent probes use what kind of light?
Excitation light
Small strips of single stranded DNA complementary to the sites being examined
Probes
How many bases are usually seen in the probes?
~200-400
FISH is useful in detecting and mapping what?
The presence or absence of particular DNA sequences within chromosomes
What is the DNA bound to the probe called?
Target DNA or native DNA from the patient
Probes are (artificial/native)
Artificial
FISH is applied to provide what?
specific localization of genes on chromosomes
What is acquired using specific probes?
Rapid diagnosis of trisomies and microdeletions
FISH is also used to check the cause of?
trisomies, microdeletion syndromes
(Native/Artificial DNA) is on the slide prep to allow for visualization
Native
T or F: Karyotyping and PCR are more cost-effective than FISH technique in detecting disease
F, FISH is more cost-effective since culturing is no longer needed thus lowering costs
Sequencing by FISH can be performed in what condition of the DNA?
Preserved/ Undegraded Metaphase DNA (should being intact)
What binds to the target DNA?
Fluorescently labelled probe DNA
Target DNA is also called t______
template
Process of separating the DNA strands and to allow the probe to access target DNA
Denaturation
How is denaturation usually done?
Heat or chemical means via Formamide
What is denatured, native or artificial DNA?
Native/ Target
T or F: FISH is possible in interphase chromosomes
T
Process of binding together the probe to the target DNA
Hybridize/ Hybridization
Probe signals are analyzed using?
A fluorescent microscope
What are the 2 specimen types for FISH?
Metaphase FISH
Interphase FISH
METAPHASE OR INTERPHASE FISH: Gold standard and routinely done
Metaphase
METAPHASE OR INTERPHASE FISH: Done on cultured cells
Metaphase
METAPHASE OR INTERPHASE FISH: Allows direct visualization of chromosomes and exact position of signals
Metaphase
METAPHASE OR INTERPHASE FISH: Useful in the detection of structural changes in the genome
Metaphase
METAPHASE OR INTERPHASE FISH: Can be modified to be used in de novo mutations
Metaphase
METAPHASE OR INTERPHASE FISH: May also be done on uncultured specimens
Interphase
METAPHASE OR INTERPHASE FISH: Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies
Interphase
METAPHASE OR INTERPHASE FISH: Beneficial in the study of samples with a low mitotic index such as most solid tumors
Interphase
METAPHASE OR INTERPHASE FISH: Major disadvantage is the inability to detect unknown structural chromosomal changes.
Interphase
METAPHASE OR INTERPHASE FISH: Probe is already made with the disease in mind
Interphase
METAPHASE OR INTERPHASE FISH: Whole chromosome is of interest
Interphase
METAPHASE OR INTERPHASE FISH: Not suitable for de novo mutation
Interphase
METAPHASE OR INTERPHASE FISH: Offers opportunity for same day turn around time
Interphase
METAPHASE OR INTERPHASE FISH: May be used in karyotyping to visualize the exact position of the fusion
Metaphase
Why is metaphase FISH the gold standard?
More rigorous and the best time to see the components of the chromosome
Considered a “pre-test” for duplications, deletions
CGH
Samples that can be used for Metaphase FISH
- Amniocytes
- Chorionic villous cells
- Lymphocytes
- Cells from bone marrow aspirates or solid tumors
- Fibroblasts
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
Complementary sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores
FISH Probes
What is the size range for the FISH probes?
20-1000 base pairs (1000 base pairs = 1 megabase)
2 types of labelling in FISH probes?
Direct and Indirect Labelling
DIRECT OR INDIRECT LABELLING: Fluorophores are directly attached to the probe
Direct
DIRECT OR INDIRECT LABELLING: Drawback is that is it less sensitive
Direct
DIRECT OR INDIRECT LABELLING: FITC, Rhodamine
Direct
DIRECT OR INDIRECT LABELLING: Cyanines
Direct
DIRECT OR INDIRECT LABELLING: Fluorophores usually attached at the end of the DNA
Direct
DIRECT OR INDIRECT LABELLING: Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization
Indirect
DIRECT OR INDIRECT LABELLING: Biotin and Digoxigenin
Indirect
Unbound fluorescent dyes are also known as?
Background dyes
Biotin is derived from?
Egg yolk
Biotin’s partner molecule is usually?
Avidin which has the fluorescent dye
Have very strong covalent action in nature
Biotin and Avidin
Which is attached to the dye, avidin or biotin?
Avidin
Which is attached to the probe, avidin or biotin?
Biotin
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
WHAT KIND OF FISH PROBE: Binds to a particular region of a chromosome
Locus specific
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
Locus specific identifiers are more ____ specific
Gene/region
The locus specific probes span the specific region and average what range of kb in size?
200-300 kb
Locus specific identifies are useful diagnostic tools to detect?
Deletions
Duplications
Rearrangements
Amplifications
Locus specific identifies can also be used in?
Fusion-type probe strategies
2 types of Color FISH probes in Locus-specific probes?
Single color
Dual color
SINGLE OR DUAL COLOR FISH PROBE: Designed to cover a gene of interest
Single
SINGLE OR DUAL COLOR FISH PROBE: Designed to cover any 2 genes for the detection of any aberrations.
Dual
Dual color FISH probe allows simultaneous detection of?
numerical abnormalities of two to three regions in one FISH assay.
SINGLE OR DUAL COLOR FISH PROBE: Creates a color that is a mixture of the previous colors which signifies a gene fusion
Dual
Dual fusion FISH strategies are probes that span what?
Specific regions of interest with recurring breakpoints
Dual fusion FISH strategies are used to identify what?
Chromosome rearrangements involving 2 chromosomes
WHAT KIND OF FISH PROBE: Generated from repetitive sequences found in the middle of each chromosome
ALPHOID/CENTROMERIC REPEAT
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
Alpha-satellite probes are also known as?
Chromosome Enumeration probes