FISH-Fluorescent in situ hybridization

FISH-Fluorescent in situ hybridization

• FISH (Fluorescent in situ hybridization) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. 
• It uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. 
• Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosome. FISH is often used for finding specific features in DNA. 
• These features can be used in genetic counseling, medicine, and species identification.
• Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use in the Human Genome Project. 
• The size of the human genome is so large, compared to the length that could be sequenced directly, that it was necessary to divide the genome into fragments. 
• The fragments were added into a framework that made it possible to use bacteria to replicate the fragments. 
• The fragments were put into order by analyzing size-exclusion separation of enzymatically-digested fragments.
•  Clonal populations of bacteria, each population maintaining a single artificial chromosome, are stored in various laboratories around the world. 
• The artificial chromosomes (BAC) can be grown, extracted, and labeled, in any lab. These fragments are on the order of 100 thousand base-pairs, and are the basis for most FISH probes.

Preparation and Hybridization Process

• Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.
• First, a probe is constructed. 
• The probe must be large enough to hybridize specifically with its target but not too large to impede the hybridization process. 
• The probe is tagged directly with fluorophores, with targets for antibodies or with biotin. Tagging can be done in various ways, for example nick translation and PCR using tagged nucleotides.
• Then, an interphase or metaphase chromosome preparation is produced. 
• The chromosomes are firmly attached to a substrate, usually glass. 
• Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample. 
• The probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. 
• everal wash steps remove all un-hybridized or partially-hybridized probes. 
• The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images.
• If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. 
• Florescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. 
• Fluorescently-tagged antibodies or streptavidin are bound to the dye molecule. 
• These secondary components are selected so that they have a strong signal.

Variations on Probes and Analysis

• FISH is a very general technique. 
• It is often arbitrarily divided into more specific categories based on application, however each category is similar in that, in a chemical sense, the technique is the same; hybridization is the common denominator. 
• The differences between the various FISH techniques are usually due to the construction and content of the fluorescently-labeled DNA probe. 
• The size, overlap, colour, and mixture of the probes make possible all FISH techniques.
• Probe size is important because longer probes hybridize more specifically than shorter probes. 
• The overlap defines the resolution of detectable features. 
• If the goal of an experiment is to detect the breakpoint of a translocation, then the overlap of the probes — the degree to which one DNA sequence is contained in the adjacent probes — defines the minimum window in which the breakpoint occurs.
• The mixture of probes determines the type of feature the probe can detect. 
• Probes that hybridize along an entire chromosome are used to count the number of a certain chromosome, show translocations, or identify extra-chromosomal fragments of chromatin. 
• This is often called “whole-chromosome painting.” If every possible probe is used, every chromosome, (in essence the whole genome) would be marked fluorescently, which would not be particularly useful for determining features of individual sequences. 
• A mixture of smaller probes can be created that are specific to a particular region (locus) of DNA; these mixtures are used to detect deletion mutations. 
• When combined with a specific colour, a locus-specific probe mixture is used to detect very specific translocations. 
• Special locus-specific probe mixtures are often used to count chromosomes, by binding to the centromeric regions of chromosomes, which are unique enough to identify each chromosome (with the exception of Chromosome 13, 14 21, 22.)
• Because modern microscopes can detect a range of colours in fluorescent dyes each human chromosome can be identified (M-FISH) using whole-chromosome probe mixtures and a variety of colours. 
• There are currently twice as many chromosomes than fluorescent dye colours. 
• However, ratios of probe mixtures can be used to create additional colours. 
• As with comparative genomic hybridization, the probe mixture for the secondary colours is created by mixing the correct ratio of two sets of differently-labeled probes for the same chromosome. 
• Differently coloured probes can be used for the detection of translocations. 
• Several techniques exploit the resolution limitations of microscopes to resolve spatial distributions of dye below a few hundred nanometers. 
• Colours that are adjacent appear to overlap, and a secondary colour is observed.
• In reciprocal translocations, where both breakpoints are known, locus-specific probes are made for it and part of the region one either side of breakpoint. 
• In normal cells, two colours will be visible; in diseased cells such as those found in BCR/ABL translocations, the two dye colours overlap, and a third colour is observed. 
• This technique is known as double-fusion FISH or D-FISH. 
• In translocations where only one of the breakpoints is known or constant, locus-specific probes are made for one side of the breakpoint and the other intact chromosome. 
• In normal cells, the secondary colour is observed, but only the primary colour is observed when the translocation occurs. 
• This technique is known as “break-apart FISH”.

Medical applications

• Often parents of children with a developmental delay want to know more about their child’s conditions before choosing to have another child. 
• These concerns can be addressed by analysis of the parents’ and child’s DNA. 
• In cases where the child’s developmental delay is not understood, the cause of it can be determined using FISH and cytogenetic techniques. 
• Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome.
• In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate remission of a disease, such as cancer. Treatment can then be specifically tailored.
•  A traditional exam involving metaphase chromosome analysis is often unable to identify features that distinguish one disease from another, due to subtle chromosomal features; FISH can elucidate these differences. 
• FISH can also be used to detect diseased cells more easily than standard Cytogenetic methods, which require dividing cells and requires labor and time-intensive manual preparation and analysis of the slides by a technologist. 
• FISH, on the other hand, does not require living cells and can be quantified automatically, a computer counts the fluorescent dots present. 
• However, a trained technologist is required to distinguish subtle differences in banding patterns on bent and twisted metaphase chromosomes.

Species identification

• FISH is often used in clinical studies. 
• If a patient is infected with a suspected pathogen, bacteria, from the patient’s tissues or fluids, are typically grown on agar to determine the identity of the pathogen. 
• Many bacteria, however, even well-known species, do not grow well under laboratory conditions. 
• FISH can be used to detect directly the presence of the suspect on small samples of patient’s tissue.
• FISH can also be to used compare the genomes of two biological species, to deduce evolutionary relationships. 
• A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region.
• FISH is widely used in the field of microbial ecology, to identify microorganisms. Bio-films, for example, are composed of complex (often) multi-species bacterial organizations. 
• Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. 
• Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm.

1. What is the full form of FISH?
   a) Fluorescence In Standard Hybridization
   b) Fluorescent In Situ Hybridization
   c) Fluorescence Internal Sequence Hybridization
   d) Fluorescent Internal Signal Hybridization
   Ans: b
2. FISH is used primarily for:
   a) Protein synthesis
   b) Carbohydrate digestion
   c) DNA sequence localization
   d) Lipid metabolism
   Ans: c
3. The FISH technique uses:
   a) Radioactive labels
   b) Fluorescent probes
   c) Enzyme-linked antibodies
   d) Alkaline phosphatase
   Ans: b
4. FISH is a type of:
   a) Biochemical assay
   b) Cytogenetic technique
   c) Enzymatic reaction
   d) Electrophoresis method
   Ans: b
5. Which of the following microscopes is used to observe FISH results?
   a) Light microscope
   b) Transmission electron microscope
   c) Fluorescence microscope
   d) Phase contrast microscope
   Ans: c
6. FISH technique can be applied on:
   a) Proteins only
   b) RNA only
   c) DNA on chromosomes
   d) Lipids
   Ans: c
7. Which of the following is commonly used in FISH probes?
   a) Alkaline phosphatase
   b) Biotin
   c) Peroxidase
   d) Trypsin
   Ans: b
8. What type of chromosomes are required for metaphase FISH?
   a) Relaxed
   b) Condensed
   c) Circular
   d) Acetylated
   Ans: b
9. The initial step of FISH includes:
   a) Washing
   b) Probe hybridization
   c) Fixation and denaturation
   d) Fluorescent detection
   Ans: c
10. Which fluorescent molecule is commonly used in FISH?
    a) Cy3
    b) FITC
    c) DAPI
    d) All of the above
    Ans: d

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11. FISH detects chromosomal abnormalities such as:
    a) Phosphorylation
    b) Translocations and deletions
    c) Ubiquitination
    d) Replication errors
    Ans: b
12. A commonly used dye to visualize nuclei in FISH is:
    a) Sudan black
    b) DAPI
    c) Coomassie blue
    d) Safranin
    Ans: b
13. In FISH, the fluorescent probe binds by:
    a) Covalent bonding
    b) Ionic bonding
    c) Hydrogen bonding
    d) Van der Waals forces
    Ans: c
14. What is BAC in FISH?
    a) Basic Antibody Complex
    b) Bacterial Artificial Chromosome
    c) Binding Amplified Chromosome
    d) Binary Active Complex
    Ans: b
15. What is the size of DNA fragments typically used in FISH probes?
    a) 10–50 bp
    b) 100–1000 bp
    c) ~100,000 bp
    d) 1 million bp
    Ans: c
16. Which probe technique is used for counting chromosomes?
    a) D-FISH
    b) Whole chromosome painting
    c) Break-apart FISH
    d) Southern blotting
    Ans: b
17. Which chromosomal regions are targeted by centromeric probes in FISH?
    a) Telomeres
    b) Introns
    c) Exons
    d) Centromeres
    Ans: d
18. Which of the following diseases is detected using FISH?
    a) Malaria
    b) Tuberculosis
    c) Prader-Willi syndrome
    d) Diabetes
    Ans: c
19. In FISH, signal amplification may be necessary due to:**
    a) High signal strength
    b) Weak fluorescence
    c) No probe binding
    d) Excess noise
    Ans: b
20. Which software assists in analyzing FISH signals?
    a) SPSS
    b) GraphPad
    c) ImageJ
    d) Notepad++
    Ans: c

21. In FISH, hybridization occurs between:
    a) Protein and probe
    b) Lipid and dye
    c) DNA and probe
    d) RNA and antibody
    Ans: c
22. What is the incubation time during hybridization in FISH?
    a) 1 hour
    b) 3 hours
    c) ~12 hours
    d) 24 hours
    Ans: c
23. Which of the following is not an advantage of FISH?
    a) High specificity
    b) Can use non-dividing cells
    c) Needs radioactive materials
    d) Automated detection possible
    Ans: c
24. Which technique is used to localize a gene in the nucleus using FISH?
    a) Northern blotting
    b) D-FISH
    c) PCR
    d) RIA
    Ans: b
25. Which FISH variant is used to identify a specific translocation fusion?
    a) Break-apart FISH
    b) D-FISH (Double Fusion FISH)
    c) Sandwich ELISA
    d) Microarray
    Ans: b
26. Which type of FISH probe is used to detect deletions?
    a) Whole-chromosome
    b) Locus-specific
    c) Metaphase probes
    d) Telomeric probes
    Ans: b
27. Why is whole-chromosome painting not ideal for detailed mutation study?
    a) It is expensive
    b) It uses RNA
    c) It labels the whole chromosome, hiding specific features
    d) It does not fluoresce
    Ans: c
28. Break-apart FISH shows what result in the presence of a translocation?
    a) Two merged signals
    b) No signal
    c) One color disappears
    d) Single color shifts location
    Ans: c
29. Which type of bacteria is often used to replicate FISH probes?
    a) E. coli
    b) Streptococcus
    c) Mycobacterium
    d) Bacillus
    Ans: a
30. Which sequence is commonly targeted in bacterial FISH?
    a) tRNA
    b) 16S rRNA
    c) 5S rRNA
    d) 28S rRNA
    Ans: b

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31. What is “zoo blot”?
    a) RNA profiling method
    b) Protein hybridization test
    c) Cross-species hybridization technique
    d) In vitro culture method
    Ans: c
32. Which dye is used for multicolor FISH (M-FISH)?
    a) Rhodamine only
    b) FITC only
    c) Multiple dyes in combination
    d) No dye is needed
    Ans: c
33. Which is not a medical application of FISH?
    a) HIV diagnosis
    b) Leukemia detection
    c) Chromosome counting
    d) Blood glucose estimation
    Ans: d
34. Which chromosomes cannot be uniquely identified by centromeric probes in FISH?
    a) 1 and 2
    b) 13, 14, 21, 22
    c) 17 and 18
    d) X and Y
    Ans: b
35. Which of the following probes provides highest resolution?
    a) Whole-chromosome
    b) Shorter, locus-specific probes
    c) Telomeric probes
    d) Ribosomal probes
    Ans: b
36. What is a limitation of traditional metaphase cytogenetics?
    a) High cost
    b) Requires living, dividing cells
    c) Fluorescence interference
    d) Cannot count chromosomes
    Ans: b
37. Which chromosome abnormality is detected by FISH in CML (Chronic Myeloid Leukemia)?
    a) BCR-ABL fusion (t9:22)
    b) t(14:18)
    c) trisomy 21
    d) Turner syndrome
    Ans: a
38. Which is not required in FISH procedure?
    a) Probe
    b) Fluorescent dye
    c) Hybridization
    d) PCR amplification
    Ans: d
39. What is used to block repetitive DNA sequences in FISH?
    a) RNase
    b) Blocking DNA
    c) Restriction enzymes
    d) Agarose
    Ans: b
40. How are FISH results visualized?
    a) UV-Vis spectrophotometer
    b) Electrophoresis
    c) Fluorescence microscope
    d) Centrifuge
    Ans: c

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41. Why are wash steps used after hybridization in FISH?
    a) To enhance fluorescence
    b) To remove unbound or partially bound probes
    c) To fix chromosomes
    d) To digest proteins
    Ans: b
42. What does a yellow signal in dual-color FISH indicate?
    a) No binding
    b) Overlap of red and green dyes
    c) Chromosomal deletion
    d) Loss of signal
    Ans: b
43. What is “biofilm” in bacterial ecology?
    a) Antibiotic compound
    b) Bacterial cell wall
    c) Complex, multi-species microbial layer
    d) Plasmid DNA
    Ans: c
44. Which field uses FISH for species identification?
    a) Organic chemistry
    b) Microbial ecology
    c) Plant physiology
    d) Enzymology
    Ans: b
45. Which component can amplify weak fluorescent signal?
    a) FITC only
    b) Streptavidin-biotin system
    c) SDS
    d) Trypsin
    Ans: b
46. In double fusion FISH, how many colors indicate fusion?
    a) 1
    b) 2
    c) 3
    d) 4
    Ans: c
47. What is the key hybridization step in FISH?
    a) Probe replication
    b) DNA-RNA binding
    c) Probe-DNA binding
    d) Chromosome division
    Ans: c
48. Which chromosomal disorder is confirmed by FISH in prenatal diagnosis?
    a) Marfan syndrome
    b) Turner syndrome
    c) Down syndrome
    d) Hemophilia
    Ans: c
49. What allows FISH to be more rapid than conventional karyotyping?
    a) No microscope needed
    b) DNA amplification
    c) Use of fixed cells and automated imaging
    d) Colorless probes
    Ans: c
50. Which organisms’ 16S rRNA is commonly used for FISH-based identification?
    a) Fungi
    b) Protozoa
    c) Bacteria
    d) Virus
    Ans: c