Wallagga University

Institute of Health Sciences

Medical laboratory Student 1st year 2nd Sinister
Molecular Biology
( Fluorescent in situ Hybridization )
Group Assignment

FISH techniques
Done By : Medical Laboratory Students Group 8
1. Muzamil Mohammed ..................... WU 1500707
2. Nuran Adam .......... ............... ..........WU 1500710
3. Nasradin Aliyi .......... .................. .....WU 1500708
4. Nuguse Tasama .......... .....................WU 1500
5. Magarsa Bogale .......... .......................WU 1500706

Submitted to : Mr Kinfu
 Fluorescent in situ hybridization
Outlines:-
 Introduction  Probe designing
 History  Procedure
 Objectives  Interpretation of FISH results
 Diagnostic applications of FISH  Advantages
 Types of Fish  Limitations
 Principle  Conclusions
 Protocol  Reference
 Types of probes
 Introduction
Fluorescent in situ hybridization is a molecular cytogenetic, physical mapping
technique that uses the application of fluorescent probes to bind to the DNA or
RNA sequence provided to the complementary sequence on the template. This
technique is used as a tag to map and sequence original templates. FISH has also
been employed for whole genome sequencing and mapping projects. FISH has
made it very convenient to find the absence, presence or malfunctioning of a gene
present in DNA.
 History and Progression of FISH
FISH (Fluorescence in situ Hybridization) techniques were developed in the late
1970s and early 1980s as a powerful tool for studying the structure and function of
chromosomes. Here is a brief history of the development of FISH techniques:
 The objectives of FISH include:
1. Visualizing specific DNA sequences: FISH allows researchers to observe the
presence and location of specific DNA sequences within cells or tissues.
2. Studying chromosomal aberrations: FISH can be used to identify chromosomal
abnormalities, such as deletions, duplications, translocations, or inversions.
3. Examining gene expression patterns: FISH can provide insights into the spatial
distribution of RNA transcripts within cells or tissues, allowing researchers to study
gene expression levels and patterns.
4. Investigating genomic organization: FISH can help researchers understand the
organization and arrangement of DNA sequences within chromosomes or nuclei.

Diagnostic applications of FISH:

They are commonly used in
I. clinical genetics for the detection of chromosomal abnormalities, such as
aneuploidies , genetic alterations, such as gene amplifications or translocations
II. Multiple diagnostic kits are available for diagnosis and prognosis of cancer
including cervical, bladder, multiple myeloma, chronic lymphocytic leukemia
and granulocytic leukemia.
III. Chromosomal structural and numerical abnormalities such as trisomy X,
turner’s syndrome, XYY syndrome and DiGeorge syndrome.
 Types of FISH
The most common type of FISH are
1. DNA FISH:
This is the most common type of FISH,
 This technique uses fluorescently labeled DNA probes that bind to
complementary DNA sequences.
 used to detect and visualize specific DNA sequences.
 It can be used to detect specific genomic regions, chromosomal abnormalities,
and gene amplifications.

2. RNA FISH:
 involves the use of fluorescently labeled RNA probes to detect and localize
specific RNA molecules within cells or tissues.
 uses fluorescently labeled RNA probes that hybridize to specific RNA molecules.
 It enables the visualization and quantification of gene expression levels within
cells.
 Principle
The basic principle is the hybridization of nucleic acid probe with the target DNA of
either metaphase chromosomes or interphase cells. The DNA probe is tagged with
the florescent marker or dye.
After the Denaturation the single-stranded florescent-tagged DNA probe then
allow to hybridize with equating target single-stranded DNA and this can be
visualized by using appropriate screening systems.
 FISH PROTOCOLS
 FISH technique is process of renaturation and denaturation of DNA strands for
several purposes, which helps to hybridized DNA single strand into double
stranded DNA.
 This technique includes four different protocols;
1. Denaturation
 This is done either by heat or alkaline method to apart double-stranded
DNA into single stranded form.
 It is a prerequisite for hybridization for the formation of new hydrogen
bonds between the probe and target DNA.
2. Hybridization
 is the formation of dual between the two complementary nucleotide
sequences either between RNA-RNA , DNA-DNA and DNA-RNA. After the
Denaturation _both target DNA and florescent tagged Probe can hybridize.
3. Detection
 The detection step is principally determined by the procedure and type of
probe labelling used .
4. Visualization
 signals done using different imaging systems with appropriate filters such as
Charge-coupled device (CCD) cameras, epifluorescence microscope and 2D-
 FISH analysis Zeiss microscope supplied with the sense’s cooled CCD cameras
are used .

PROBES IN FISH
 Probes are small pieces of DNA or RNA that are designed to bind to a specific
target sequence of DNA or RNA. They are used, to detect and visualize specific
genetic material.
 it is complementary to the DNA strand which it is going to bind.
 are specific sequences of DNA that play role in identification or quantification of
DNA sequence in sample which bind to it.
 It detects any abnormality occurs in the sequence of genes this could be absence
or presence of sequences.
Probes are choosing according to the target sample like DNA and RNA sequences.
 DNA probes
 are preferably used in FISH because probes are directly hybridized with
chromosomes and having large sensitivity as compare to RNA probes because
hybridization of these probes is not as much strong as RNA probes with mRNA.
 RNA probes
 RNA probes are also termed as riboprobes.
 These probes detect the gene expression and mRNA presence.
Types of probes
There are mainly three types of probes.
1. Whole chromosome painting probes
2. Repetitive sequence probes
3. Locus specific probes
 Centromere probes  Whole chromosome
 which hybridized with alpha and  Collection of probes that bed to the
beta satellite probe sequences whole length of chromosome
present on the centromere of  Multiple probe labels are used
chromosomes.
 Hybride along the length of the
 Generated from repetitive chromosome.
sequences found in centromeres
 useful for examining both large-
 Centromere regions are stained and small-scale chromosomal
brighter abnormalities which have complex
rearrangement.
 Telomere
 Specific for telomeres  Locus specific probes
 is Pan-telomeric probe which  used for detection of any
hybridized with human abnormality like
chromosomal end repeated  deletion, inversion or
sequence (TTAGGG) or
 Translocation probes
 Specific to the 200 kb focus at the
end of specific chromosome.  Gene detection & localization
probes
 Gene amplification probes
 LABELLING OF PROBES OR ( PROBE DESIGNING )
 Probe designing is the process of creating a small piece of DNA or RNA that is
complementary to a specific target sequence of DNA or RNA.
 The designed probe is to detect and visualize specific genetic material.
 The probe is labeled with fluorophore compounds (a fluorescent) or radioactive
marker for visualization purposes. W/c is done by PCR and nick translation.
 Fluorophore compounds which tagged with probe are digoxigenin, biotin and
fluorescein.
There are two methods of probe labelling :

 DIRECT  INDIRECT
Reporter molecules. A hapten..
1. Enzyme, 1. Biotin,
2. Radioisotope or 2. Digoxigenin, or
3. Fluorescent marker are directly 3. Fluorescent are attached to the
attached to the DNA or RNA. probe and detected by a labeled
binding protein (typically an antibody).
Specimen Sources:
 Samples can be collected from the Peripheral Blood/ Bone Marrow and Paraffin
Embedded Tissue (FFPE).

TYPES OF SAMPLE:
 In FISH technique three different samples are used:
I. Formalin Paraffin Embedded Tissue (FFPE)
II. Unfixed Tissue (frozen tumor cells)
III. Metaphase spread ( Chromosomes from Bone Marrow)

PROCEDURE
 For Formalin Paraffin Embedded Tissue (FFPE) and Unfixed Tissue (frozen tumor
cells) FISH protocols are same but for metaphase spread are different
 Procedure consists of following steps:
I. FISH Protocol
II. Hybridization
III. Washing
IV. Mounting / Counter staining
 V. ImageAnalysis
Interpretation of FISH results:-
 The interpretation of FISH results involves analyzing the fluorescence
signals emitted by the labeled probes.
 A normal result shows a specific pattern of fluorescence signals
corresponding to the expected number and location of target sequences.
 Abnormal results may indicate chromosomal abnormalities, such as
deletions, duplications, or rearrangements , translocations etc.
 The interpretation of FISH results requires expertise in cytogenetics and
knowledge of normal and abnormal patterns.
 Example:-
a. FISH done on paraffin embedded tissue sections of malignant melanoma
using probe
 => for Cyclin D3 gene locus orange-signals at 11q13 and
 => for green-color centromeric region of chromosome 13.
 b. The FISH done on nuclei of bone marrow that contains translocation
t(11;19)(q23;p13) via duplex color separate-probe.
 Yellow —signals which is a fusion of Green–red show a normal cell.
 The individual red and green signals shows presence of the translocations.

Storage:
 The slides for a few days to months also be stored at –20 °C or –70 °C in the dark.
 ADVANTAGES:
 They provide high-resolution visualization of specific DNA sequences on
chromosomes, allowing for the detection of small genetic alterations.
 FISH can be performed on a variety of sample types, including cells, tissue
sections, and metaphase spreads.
 It is a relatively quick and cost-effective technique compared to other
cytogenetic methods.
 FISH also allows for the simultaneous visualization of multiple target sequences
using multicolor FISH techniques.
LIMITATIONS:
 It requires specific probes for each target sequence of interest, which can be
time-consuming and expensive to develop.
 FISH results are subjective and require expertise in interpretation.
 The technique may have limitations in detecting certain chromosomal
rearrangements or complex genetic alterations.
 FISH is also limited by the resolution of fluorescence microscopy, which may not
allow for the detection of very small genetic alterations.
 CONCLUSION
 FISH being the molecular cytogenetic technique is mainly used for the
chromosome analyses for diagnostic based and research purposes.
 Protocol steps include denaturation, hybridization, detection and visualization
techniques.
 Three types of samples can be prepared including Formalin Paraffin Embedded
Tissue (FFPE), unfixed tissue and Metaphase Chromosomes taken from bone
marrow.
 The advances with synthetic oligo probes will significantly expand the
applications of FISH specially in non-model plant species.
 The technique seems to have great future impacts on live cell imaging and
medical diagnostics specially in different types of cancer. The use of cytometric
algorithms with multi-color fluorescence has allowed the computerized FISH-
protocol.
 REFERENCES

 Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., & Gray, J.
(1988). Fluorescence in situ hybridization with human chromosome-specific
libraries: detection of trisomy
 21 and translocations of chromosome 4. Proceedings of the National Academy of
Sciences, 85(23), 9138-9142.
 Kearney, L. (2006). Multiplex-FISH (M-FISH): technique, developments and
applications. Cytogenetic and genome research, 114(3-4), 189-198.
 Arrigucci, R., Bushkin, Y., Radford, F., Lakehal, K., Vir, P., Pine, R., Martin, D.,
Sugarman, J., Zhao, Y., Yap, G. S., Lardizabal, A. A., Tyagi, S., & Gennaro, M. L. (2017).
FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single
cells using fluorescence in situ hybridization and flow cytometry. Nature
protocols, 12(6), 1245–1260. https://doi.org/10.1038/nprot.2017.039
 Ourliac-Garnier, I., & Londoño-Vallejo, A. (2011). Telomere length analysis by
quantitative fluorescent in situ hybridization (Q-FISH). Methods in molecular
biology (Clifton, N.J.), 735, 21–31. https://doi.org/10.1007/978-1-61779-092-8_3
 Coco-Martin, J. M., & Begg, A. C. (1997). Detection of radiation-induced
chromosome aberrations using fluorescence in situ hybridization in drug-induced
premature chromosome condensations of tumour cell lines with different
radiosensitivities. International journal of radiation biology, 71(3), 265–273.