“By hybridizing the fluorescently labeled probe to the complementary DNA sequence, the position of DNA sequence can directly locate on a chromosome. “
The molecular techniques alike the PCR and DNA sequencing is employed for the detection of mutations at the molecular level, for example, single nucleotide polymorphism. On the other side, the cytogenetic techniques are used for the detection of chromosomal anomalies or abnormalities.
Using conventional cytogenetic methods like karyotyping chromosomal abnormalities can be encountered. The karyotyping method relies on the banding techniques to find out any aberrations. For example, the GTG banding is used in the detection of numerical chromosomal anomalies while NOR banding is used for the detection of trisomy 21.
The molecular cytogenetic technique facilitates several benefits over the traditional cytogenetic method. one of them is the precision. In the present article, we are going to learn about one of the molecular cytogenetic techniques; Fluorescent in situ hybridization.
Read our previous article on cytogenetics: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray].
The overall idea of FISH:
The FISH method is based on the phenomenon of the denaturation and renaturation of DNA duplex. The DNA is a stable duplex, under normal conditions hydrogen bonding between two strands (two between adenine and thymine; and three between cytosine and guanine) makes it stable.
Although the duplex can be denatured using physical agents such as heat or chemicals when conditions are favorable, DNA becomes renatured.
When DNA on the chromosome are denatured, the fluorescently labeled complementary DNA probes hybridize with it and emits fluorescence, consequently. The hybridization is visualized under the fluorescent microscope.
The technique is an advanced version of the cytogenetic analysis used in gene mapping, identification of major deletion or copy number variations, disease diagnosis, medicines, and species identification.
What is Fluorescent in situ hybridization?
In the FISH, “The nucleotide sequence can be mapped or detected using the fluorescent probes complementary to the sequence located on chromosome in a cell under the fluorescent microscope from the biological specimen.”
The conventional karyotyping method is tedious and time-consuming, required a higher degree of expertise to interpret the results.
The chance of assay failure is higher in the conventional karyotyping method, Although numerical chromosomal abnormalities can be accurately assessed, it is hard to find minor copy number variations.
FISH is a ‘molecular cytogenetic technique‘ in which using the molecular probes, any type of chromosomal abnormalities can be encountered precisely by hybridization. The karyotyping takes at least 3 to 4 days to complete the entire process while the FISH method is rapid, one can get results within a day.
The interpretation part required one fold lesser expertise.
- Fluorescence: light emitted by a molecule
- In situ: on the original place or on the appropriate location (in our case, in a cell on the glass slide).
- Hybridization: here, binding of two complementary strands.
Principle of FISH:
A higher degree of a sequence-complementary DNA or RNA probe is hybridized on a chromosome, in a cell using chemical treatments.
The probes are fluorescently labeled, once it finds its complementary sequence (which is a sequence of our interest), it emits the fluorescent which we can observe under a microscope. A fluorescence microscope is needed though.
The unbound probes are washed off to avoid unwanted signals from the site of hybridization. The technique was developed in the year 1980. The whole principle is graphically explained here,
Process of FISH:
There are two major elements required in a conventional FISH assay: the probe and the target sequence.
Select the target sequence:
In the very first step, before doing any wet lab work we have to select the sequence or the portion of a chromosome we wish to study.
The FISH is capable of detecting the fragments of DNA more than few thousands of base pairs. The selected target might be any duplication, deletion, translocation, or any disease-related genes or DNA sequence or a portion of the chromosome.
Designing the probe:
Once the target sequence is selected, based on the data of the sequence we wish to study, the probe is designed. The probe is a single-stranded sequence of DNA or RNA complementary to our gene of interest.
The polynucleotide chain which we are utilizing as a probe is then labeled with the fluorophores. Generally, the direct labeling method is used for probe generation, in which the fluorophores are directly attached to the nucleotides.
The tagging of fluorophores on the nucleotide sequence can be done using either the nick translation method or PCR. Read our article on preparing probes: DNA Probes: Labelling, Types And Uses.
Types of probes used in the FISH:
For various applications in various varients of FISH different types of probes are used. Centromere probes, locus-specific probe, whole chromosome probe and telomere probes are some of the examples of it.
Whole chromosome probe:
The whole chromosome probes are used for the multi-color FISH and spectral karyotyping. It is a mixture of probes that binds to the entire chromosome length and thus different chromosomes are colored or labeled with different colored probes.
Each probe is derived from a single type of chromosome and binds to that particular chromosome only. It is used in the chromosomal rearrangement studies.
Locus specific probe:
The locus-specific probes are used in the study of a particular gene or DNA sequence of interest. It is used in the identification of the location of a gene of interest on a chromosome. The locus-specific probes are used to determine the location of the isolated gene and to quantify that gene within a genome.
Any chromosomal anomalies like deletion or duplication of the gene of our interest can be encountered using the locus-specific probe. Multiple locus-specific probes are used for the detection of multiple DNA sequences on different chromosomes.
Alphoid or centromeric probes:
These probes are also known as repeat sequence probe or alphoid-centromeric probes. Repetitive DNA sequences are present in the centromeric or telomeric regions of the chromosomes and that is the base to develop these probes. It is used in the analysis of numerical chromosomal anomalies like monosomy, disomy and trisomy.
Take a look at the figure to know how different probes work.
Examples of some commercially available probes for different FISH assays are given below,
|Probe name||Used for||Location|
Note: different probes are nowadays available for different chromosomal anomalies, probe designing is not required for performing any FISH experiment.
Sample preparation for FISH:
Both fixed or unfixed samples can be used in FISH. Paraffin-embedded tissues, FFPE tissues, tumor cells, cell culture, or chromosomal suspension are some common sample types used to do this.
In the next step, the cells are permeabilized using the chemical treatment for allowing the probe to enter the cells and hybridize on chromosomes.
Interestingly, for performing the fiber-FISH, the chromatin fibers are extracted and stretched on a slide using the fluid-flow method. Fibre-FISH allows greater resolution.
In the next step, the sample is denatured.
The DNA is a double helical structure and more stable. For making hybridization possible, we need to denature the sample so that our probe can bind to the DNA sequence. A higher temperature or denaturing agents are used for generating single-stranded target DNA for hybridization.
The DNA is denatured using heat or alkaline agents.
Interphase or metaphase chromosomes are the best choice for performing FISH efficiently. After the sample is prepared, the probe mixture is applied on the surface of the glass slide having the sample. Along with it, short DNA fragments are added to block the repetitive DNA hybridization with the probe.
Once hybridization completes, we need to wash the slide in order to wash off partially and incompletely hybridized probes. A ready to use wash buffer is recommended to wash the slide. Wash the slide as instructed by the manufacture.
In the next step, the slide is incubated for 12 hours for hybridization to occur. After that, the results are analyzed under the fluorescent microscope.
Results and interpretation:
Scientists visualize the slide under the fluorescent microscope, if the probe hybridized properly on its complementary sequence, it emits two fluorescent signals. The signals are clearly observed, however, expertise is required to interpret the results.
The brief overview of the whole process is explained hypothetically here in the figure.
Related article: Genetics Basics: A Beginners Guide To Learn Genetics.
A general protocol for FISH:
Materials and instruments:
Fluorescent dye or fluorescent-labeled probe complementary to our sequence of interest, sample specimen, fluorescent microscope, alkaline agent, SSC buffer, 10mM HCl, hybridization solution, ethanol, coverslip, slide, heating block, humid chamber and incubator.
- Prepare the slide with the cell suspension.
- Incubate the slide with 200µl RNAse solution for 1 hr at room temperature.
- Wash the slide with 2X SSC for 4 to 5 minutes, followed by 10mM HCL rinsing.
- Add 200µl pepsin and incubate for 8 to 10 minutes at room temperature.
- Rinse slide with distilled water and then with 2x SSC.
- Place the slide in the paraformaldehyde solution for 5 to 10 minutes for fixing and wash it with the same SSC buffer.
- Now perform dehydration to dry the slide with 70%, 80%, and 90% of ethanol each for 2 minutes.
- Place the slide for some time to air dry. Our slide is ready for hybridization.
- Add 30µl of hybridization solution on a slide, heat it at 65 to 70°C for 10 minutes and cool it by placing it on ice.
- Cover the slide with a coverslip and again heat it 65 to 70°C for 5 minutes for denaturation.
- Place the slide at room temperature for the hybridization of probe and DNA.
- Place it in a humidity chamber overnight at 37°C.
- Next morning, wash the slide with SSC buffer (2X) and then wash with 0.1X SSC buffer at 40°C and remove the coverslip.
- Give a final wash to slide with 2X SSC buffer at 40°C and block it in a blocking buffer for 30 minutes.
- Add antibody to the slide and incubate it for an hour and with it with 2X SSC buffer.
- If needed repeat the washing step followed by DAPI counterstain.
- Air-dry the slide and visualize under the fluorescent microscope.
Note: the protocol may vary lab to lab, minor modifications are required to achieve good FISH results. The protocol is originally adopted from Sigma-Aldrich.
Different types of FISH:
Depending upon the requirement of the researcher, different variations of the native FISH are available nowadays.
The M-FISH is known as multicolor FISH uses different colored probes for different chromosomes. Broadly, it is used in the characterization of different chromosomes and numerical chromosomal abnormalities.
Known as quantitative FISH used for the quantification of the genetic material hybridized by the probe. The fluorescent intensity is measured for quantification thus it is used in the study of telomere and aging, cancer and gene expression.
Flowcytometry FISH is a variation of FISH used for the quantification purpose which measures fluorescent emitted from every single cell is detected and measured.
A computation tool used for the prediction of the outcome of the FISH experiment is called electronic FISH. It is a BLAST-based program that utilizes the sequence information for in silico estimation of the hybridization process.
Using the chromatin fiber or DNA fiber, the high-resolution gene mapping can be done using the Fiber-FISH method. The present modification even permits the identification of the DNA fiber less than 1000bp.
Thus it is widely used in the gaps and overlap fragment analysis, assessment of duplication and other copy number variation detection which can not be detected by the conventional FISH method.
Using the salts or solvents, the chromatin fibers are released and fixed on the glass slide, instead of the whole chromosomes. The probe hybridization is done directly on the glass slide containing the chromatin fibers.
The ACM-FISH is a variation of the multicolor FISH especially designed for the sperm cells.
The ACM stands for alpha (centromere), classical and midi satellites of chromosome 1 for detection of duplication and deletion on the chromosome. It is used for the detection of the role of the chromosomal damage in the development of infertility in males.
The method is a type of RNA-FISH used to study the neurons associated with abnormal cognitive behavior. Cellular compartment analysis of temporal(cat) is abbreviated as catFISH.
Microfluidics- associated FISH uses microfluidics for improving the performance of the FISH. It increases the hybridization efficiency as well as decreases the time consumption thus it is used in the bread cancer for detection of the HER2 gene mutation.
COMBO-FISH stands for combinatorial oligonucleotide FISH used for the detection of homopurine or homopyrimidine region of the genome. The homopurine and homopyrimidines cover approximately 2% of the human genome.
The probes are designed to hybridize on these regions of the genome and create a triple helical structure with it by binding with the DNA duplex.
This unusual triplet structure is located using the fluorescent signals having the homopurines or homopyrimidines, this information is used for the 3-D (three-dimensional) study of the human genome.
One of the major advantages of COMBO-FISH is that we do not need to denature the sample prior to hybridization, thus, reduce the complexity in the FISH assay.
Single-molecule RNA-FISH is employed for the quantification of gene expression from the tissue sample, using the hybridization method. Here, instead of DNA, RNA is used as a target for probe hybridization.
Note: a special type of FISH applied for the temporary gene expression pattern within cells by using the RNA as the template for the FISH.
Advantages of FISH:
Cell culture process is not needed for performing FISH which is one of the most important advantages of it. In the conventional karyotyping method, scientists must have to culture chromosomes and arrest them on metaphase, however, cell culturing has several limitations.
The cell culture takes more time, approximately 3 to 4 days and the chance of contamination is higher as well. Contrary, the fluorescence in situ hybridization method is rapid and the chance of contamination is negligible.
Another advantage of FISH is it allows the analysis of the nondividing cells such as solid tumor cells. If cells are not dividing, we can not culture it using the conventional karyotyping method.
The karyotyping method is entirely based on the chromosome banding thus it is restricted, using multiple probes in FISH multiple hybridization sites have been analyzed using different fluorophores. Not only metaphase but also the interphase chromosomes can also be used in FISH in order to achieve higher resolution.
The cross-reactivity rate of the FISH probes is also very low.
It has a higher signal-to-noise ratio.
Paraffin-embedded, frozen tissue and cultured cells are used for FISH analysis, besides this, both types of cells (non-dividing as well as dividing cells) are applicable in FISH.
Applications of FISH:
Identification of specific chromosomal abnormality especially structural as well as numerical; chromosomal deletion, duplication, and translocation can be detected using the fluorescence in situ hybridization.
By using the variation like SKY and M-FISH, new non-random genetic abnormalities can be identified as well.
Spectral karyotyping or multicolor FISH is a modification of native FISH in which using the different colored sequence-specific probe each chromosome can be painted thus, chromosomal rearrangement can be encountered precisely.
However, smaller deletions and/or duplications can not be identified using the Spectral karyotyping because of the restricted resolution.
It is used in gene and genetic mapping. We have covered an article on gene mapping. Read it here: A Brief Introduction to “Gene Mapping”.
Using the FISH, marker chromosomes can be identified and characterized.
Also, a specific breakpoint at where the translocation occurs can be determined.
One of the most fascinating applications of quantitative FISH is in the monitoring of disease progression.
If the conventional karyotyping fails to find out any abnormalities, using the interphase FISH (which provides higher resolution), those type of abnormalities can be identified.
Moreover, the FISH is used in the study of three-dimensional chromosomal structure and organization.
It is practiced for monitoring the success of transplantation, for example, bone marrow transplantation.
The fluorescence in situ hybridization technique is capable of detecting larger copy number variation efficiently. Scientists are now applying different variations of FISH for different cytogenetic applications. One of them is the comparative genomic hybridization. CGH used for quantitative detection of copy number variations.
Although FISH is a highly versatile and rapid method, one should learn the basic chromosome preparation and karyotyping methods to boost their knowledge and expertise.
I strongly recommended learning basic karyotyping. Read our series of articles on cytogenetics:
- A Karyotyping Protocol For Peripheral Blood Lymphocyte Culture.
- What Is The Role Of RPMI 1640 In Karyotyping?
- Role Of L-Glutamine In Karyotyping.
- Emanuela V & Joanna M. “FISH glossary: an overview of the fluorescence in situ hybridization technique.” Biotechniques. 2018; 45: 385-409.
- Ratan ZA, Zaman SB, Mehta V, Haidere MF, Runa NJ, Akter N. Application of Fluorescence In Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science. Cureus. 2017;9(6):e1325. Published 2017 Jun 9.
- O’Connor, C. (2008) Fluorescence in situ hybridization (FISH). Nature Education 1(1):171 (link).