IJCRR

IJCRR - 13(18), September, 2021

Pages: 129-136

Date of Publication: 26-Sep-2021

Fluorescence in-situ Hybridization: Technology to Detect Genetic Mutation

Author: Dave Kruti, Prajapati Mitali, Pathak Deepika, Alisagar Luluaa, Suthar Krutish

Category: Healthcare

Abstract:Introduction: Karyotyping is the traditional method to detect the genetic mutation. Perhaps it has some limitations like tedious, low resolutions of results and strenuous. The fluorescence in situ hybridization technique brings a new era in molecular biology which is based on the complementary Deoxyribonucleic acid or Deoxyribonucleic acid/Ribonucleic acid strands. It is a combined approach of molecular and cytological to study chromosome structure and function as well as to detect the specific genes sequences. Objectives: To explore more knowledge about applications and different types of FISH. Due to the accuracy and versatility of FISH, it is widely used in cancer research to detect the various types of mutations, diagnostic and research fields. In this article, we briefly introduced MFISH (Multicolour FISH), QFISH (QuantitativeFISH) and RNA FISH. Method: The first step is to make short sequences of single-stranded DNA that should match a portion of the gene that is of interest. Now the tissue should be fixed by different fixers. Before hybridization, probes and sample sequences are denatured. To detect hybridized probes, different techniques are used. Result: As probes are labelled with different reporters, they can be detected by conventional light microscopy, fluorescence microscopy, etc. Conclusion: FISH and its types have a wide range of applications in the diagnostic field, genetic research, biomarker research and personalized medicines.

Keywords: Genetic mutations, Fluorescence In Situ Hybridization, Probes, MFISH, RNA FISH, Q FISH

Full Text:

INTRODUCTION:

Human cells are estimated to contain approximately 10 double-strand breaks per cell cycle as estimated by the incidence of spontaneous sister chromatid exchanges by the DNA replication process which is remarkably accurate. If it is left unrepaired, such damage results in the loss of chromosomes and/or the induction of cell death. If imprecisely repaired, the damage leads to mutations and chromosomal rearrangements.¹

During the cell cycle, DNA double-strand breaks are readily mediated to alter the superhelical state in DNA by ubiquitous enzymes also known as topoisomerases. For this enzyme temporary cleavage is essential to carry out their primary cellular functions including their roles in DNA replication, transcription, segregation, the maintenance of genome stability and chromosome condensation. However, these enzymes can promote chromosomal aberrations by illegitimate recombination. Through disincorporation during DNA replication or exposure to exogenous mutagens such as ionizing radiation or endogenous mutagens, mutations can occur in the genomes of all dividing cells. The diversity of mutations(aberration) that can initiate human cancer. Intrageneric mutations highlighted are small nucleotide changes that may be inherited by the cancer progeny from the progenitor cell, resulting in either a loss-of-function of genes or a gain-of-function of genes.

Mutated genes are identified in cancer called oncogenesis. Cancers are caused by various types of mutations shown in fig.1.²

karyotyping is considered as the gold standard method which is used to confirm the presence or absence of mutations but has some considerable limitations such as low resolution of the cell-treated field, time-consuming and laborious cell cultivation, and in the detection of chromosome abnormalities which are less than 5 mb in length.FISH became a powerful technique that has high sensitivity and specificity as well as with the performance speed it has considered as a clinical laboratory tool.³

The objective of fluorescence In- situ hybridization is to determine the presence or absence of DNA or RNA sequences of interest and to localize these sequences to specific cells or chromosomal sites as well as small deletions and duplications. It offers the possibility to specifically mark individual chromosomes over their entire length or defined chromosome regions in meta- and interphase preparations.‘Chromosome painting competitive hybridization was one of the first applications of FISH using entire chromosome-specific libraries for chromosomes as probes and human genomic DNA as the competitor.⁴

MECHANISM OF FISH:

The main steps of FISH are the binding of fluorescently labelled target-specific nucleic acid probes to their complementary DNA or RNA sequences. Also the visualization of these probes within cells in the tissue of interest. The steps of FISH are as below.⁵

1. PREPARATION OF PROBE AND TARGET SEQUENCE:

Probe and a target Sequence are the basic elements of any FISH technique. The first step is to make short sequences of single-stranded DNA that should match a portion of the gene that is of interest. These are called probes. After cooling of strands, they will anneal with complementary nucleotides making bonds back together with their homologous partners. The higher the number of nucleotides in a probe, it will mostly attach with its homologous target sequences. The choice of the probe is the important thing that must take account in the FISH analysis. There is a wide range of probes available, from whole genomes to small cloned probes (1–10 kb).⁶ (Table:1)

Now, these probes are usually directly or indirectly labelled. 1. Directlylabelled: Fluorochromes are directly attached to probes.2. Indirectly Labelled: Here a hapten (such as biotin or digoxigenin) is used for attachment. In indirect labelling, for detection fluorescently labelled antibodies (such as streptavidin and anti-digoxigenin) are used. Direct labelling is faster than indirect labelling. But in indirect labelling, there is the advantage of signal amplification by using several layers of antibodies and because it is produced by indirect labelling brighter compared with background levels.

FIXATION OF TARGET SAMPLE:

The fixation methods are formalin-based, paraffin-based or freshly prepared frozen tissue. For adherence of tissue sections, a specially treated glass slide is used to ignore the loss of tissue during the hybridization process. For this purpose, various “adhesives'' are available like poly-l-lysine, gelatin chrome alum and aminopropyl-triethoxysilane. For fixation of metaphase chromosome spreads methanol/acetic acid is used. For sections like cryostat 4% formaldehyde (~30 min), Bouin’s fixative, or paraformaldehyde vapour fixation may be used. Most often, tissue specimens are regularly fixed in 10% buffered formalin, procedures overnight in an automatic tissue processor, and embedded in paraffin wax. The optimal fixation time is 8-12 hours. Sections are voided well and then air-dried at room temperature. After de-paraffinization, slides are put down in an alcohol-cleaned staining container of diethylpyrocarbonate water. The staining container is then put down in the heated water bath at 23-37°C. The formaldehyde-based fixatives are used before paraffin embedding to cover up nucleic acid sequences. Digestion enhances probe penetration by expanding cell permeability with minimal tissue degradation.

DENATURATION OF PROBE AND TARGET SEQUENCE:

Both the labelled probe and the target DNA are denatured. The annealing of complementary DNA sequences occurring because of the combining sequences of the denatured probe and target DNA. In indirect labelling, one extra step is required for visualization of the non-fluorescent hapten that requires an enzymatic or immunological detection system. Although, the FISH probes are selected according to the diseases or anomalies under the field of interest. Before hybridization, if both the target and the probe are double-stranded, they must be denatured to make them single-stranded and this can be attained by heat or alkali treatment.⁷

HYBRIDIZATION:

In the molecular hybridization process, a single-stranded target sequence is annealed to a complementary single-stranded probe to make a double-stranded hybrid. In the denaturation process,

Single-stranded target and probe sequences are incubated in a hybridization mixture, which supplies an optimal environment for re-annealing of single-stranded sequences. Hybridization is done after denaturation. During cooling, a complementary probe and target sequence forms hydrogen bonding of the two strands of nucleic acids. The probe must make stable hydrogen bonds with the target. At the same time heating the probe and target to high temperatures may increase the stability and sensitivity of detection. For this, care must be taken to fully control this step of the FISH procedure.

 DETECTION:

For the detection of hybridized probes, enzymatic reactions that generate a coloured precipitate at the site of hybridization is used. The most often used enzymes for this application are alkaline phosphatase (AP) or horseradish peroxidase (HRP). The radioactively labelled probes are detected by autoradiography. This detection procedure is based on the emission of fast electrons or beta-particles from the probe. Autoradiography for radioactive labels is reported to be more sensitive than the immune enzyme systems. After hybridization, fluorophores can be connected with nucleic acid probes by chemical conjugation to the nucleic acid or chemical conjugation of the nucleic acid with a non-fluorescent molecule that can attach fluorescent material. There are four common fluorophore classes-fluoresceins, rhodamines, cyanines, or coumarins used. For the detection of tumour morphology, conventional light microscopy and fluorescence microscope is also used.  For the hapten detection immunohistochemically by a fluorophore-tagged antibody against the hapten is used.⁸

TYPES:

The diversification of real FISH protocol forms different types of FISH. Here the Table.3 represents the types of FISH, mostly used probes by these types and their functions.

Among all these types, some types have major importance in the scientific field. These types are M FISH, Q FISH and RNA FISH.

MFISH (Multicolour FISH)

By using different colours, FISH contains the ability to find several regions or genes simultaneously. Multicolour FISH, which can locate many different probes on chromosomes simultaneously, is an important branch of FISH techniques. Using M-FISH to construct FISH karyotype has been applied in many plant species, for example, Lotus japonicus, Pinus sp., Picea abies and Silenelatifolia. Ribosomal RNA genes (rDNAs) belong to a highly conservative repeat sequence family with hundreds of copies and locate on one or more pairs of chromosomes. FISH mapping of rDNA on chromosomes can provide important clues for molecular markers of karyotype analysis, the evolution of karyotype and phylogenetics research.¹⁰

The power of M-FISH (and SKY) lies in its ability to resolve complex karyotypes and identify the origin of marker chromosomes as evidenced by its many applications in tumour diagnostics and research, evolutionary cytogenetics and the study of chemical and radiation-induced aberrations. Impressively, even quite complex karyotypes of individual cells within a nonclonal population can be resolved with a high degree of confidence. One should give more attention before choosing probes in FISH analysis. The pe of the chromosome. This type of probes are flow-sorted (or microdissected), amplified and labelled by an oligonucleotide polymerase chain reaction to generate a ‘paint’ that highlights the entire chromosome.¹¹

QFISH (Quantitative FISH)

Telomeres are nucleoprotein like structures, which cap and protect linear chromosome ends. They comprise ~1500 TTAGGG repeats, a single strand overhang and a complex of six shelters in proteins. Telomeres prevent chromosomes from degradation and fusions by preventing the ends from being falsely recognized as DNA double-stranded breaks. However, the intrinsic inability of the replicative machinery to complete replication of linear DNA leads to progressive telomere reduction upon cell division which imposes a limit on the number of times a cell can divide. 90% of cancer cells survive this crisis due to upregulating telomerase, the enzyme that lengthens telomeres. Still, in stem and progenitor cells, telomerase activity is crucial for tissue maintenance and regeneration.¹²

Telomeres may represent a powerful readout of oxidative stress, a potential biomarker of exposures and disease risk. Three common techniques are widely used by laboratories to measure telomere length. The telomere restriction fragment (TRF) analyzed by the southern blot is considered the gold standard. Although this technique only yields average telomere length, generally requires one to three micrograms of genomic DNA. The quantitative Polymerase chain reaction is another common method that requires very low amounts of DNA but yields a ratio of telomeric DNA content relative to a single-copy gene. Telomere length analyzed by quantitative fluorescence in situ hybridization (qFISH) on metaphase chromosomes provides the ability to examine the length and integrity of individual telomeres. Q-FISH allows for the evaluation of the telomere length heterogeneity within the cell population and distinguishing of critically short telomeres. Moreover, qFISH also offers the ability to assess telomeric aberrations like telomere fragility, losses and fusions, associated with any illness, hence allowing the recording of multiple telomere parameters in a single experiment.¹³

Q-FISH uses peptide nucleic acid (PNA) probes; they are synthetic molecules formed by DNA bases linked, to a backbone by peptide bonds. Telomeric PNA probes can be Supportive to the G-rich or to the C-rich telomeric DNA strand which provides the possibility for simultaneous application and two-colour, staining required for chromatid orientated FISH.¹⁴ In this context, Q-FISH is specifically relevant because it can identify and quantify critically short telomeres.

RNA FISH

RNA molecules have an extensive range of characters in the cell. RNAs control all features of cell metabolism and thus be important regulators of physiologic and disease activity. Genome is crowded with a huge amount of long noncoding RNAs (lncRNAs) that have been correlated with a large number of cellular activities like differentiation and the beginning of the disease. RNA fluorescence in situ hybridization (RNA FISH) is a technique that provides a convenient supplement to label RNA molecules in the cell by labelled nucleic acid probes hybridization with target RNA for the detection of RNA.

Transcripts are present either in the nucleus or in the cytoplasm. The method also called expression-FISH, has been used to examine the transcriptional action taken by endogenous genes, exogenous genes like those belonging to integrated viral genomes and transgenes. RNA-FISH has also been used in studying different functional aspects of genome organization and nuclear architecture, also as a prenatal diagnosis tool for myotonic dystrophy type.

Single-particle RNA FISH depends on pools of short, marked DNA oligonucleotides that can identify singular atoms of RNA in situ, by managing its affectability to distinguish even low abundance lncRNA. In addition, it is enabling quanti?cation of the number and area of each target molecule inside the cell. Specialists have utilized single-atom RNA FISH to recognize lncRNA with progress and in two cases, it has demonstrated basic to unravelling the system of activity. In an ongoing methodical investigation of the use of single-particle RNA FISH to lncRNA, researchers found that lncRNAs can introduce special difficulties regarding recognizing nonspeci?c background from valid signals.¹⁵

APPLICATIONS:

There are diverse applications on FISH based which are from different fields of investigation, including clinical genetics, evolutionary biology, neuroscience, cellular genomics,  toxicology, microbial ecology, reproductive medicine, comparative genomics, and chromosome biology. FISH consumes less time than other techniques so makes easy diagnosis and also increases the life span of people.

CONCLUSION:

Fish has been developed to the next level and is a powerful technique for detecting mutation and alternation in gene expression at the microscopic level. Moreover, the application of Fish lies in the diagnostic and research. The use of fish is a game-changing method for the diagnosis of diseases like cancer as where chromosomal abnormality detection became very crucial for further treatment and monitoring of disease. Novel FISH techniques and types like M FISH, Q FISH, and RNA FISH are introduced with their wide range of applications that have been discussed in genetic research. The single-molecule FISH technique in cell-based genetic diagnosis is expected to enhance the capacity of the spectrum of genetic defaults from chromosomal and genetic abnormalities. Also, this technique helps to develop biomarker research and personalized medicine.

Acknowledgement: Nil

Conflict of interest: Nil

Funding: Nil

Individual author’s contribution:-

Dave Kruti: Overall review and compilation

Prajapati Mitali: Methods and M-FISH

Pathak Deepika:Q-FISH

Alisagar Luluaa: R-FISH

Suthar Krutish: Application

References:

1. Pfeiffer P, Goedecke W, Obe G. Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis. 2000 Jul 1;15(4):289-302.

2. Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007 Mar;446(7132):153-8.

3. Hurd D. Delving deep into the genetic basis of disease. American Biotech Lab. 2010;28(3):22.

4. Gozzetti A, Le Beau MM. Fluorescence in situ hybridization: uses and limitations. In Seminars in Hematology. 2000; 37(4): 320-333). WB Saunders.

5. Elbassiouni FE, Salim EI, Zineldeen DH. The Effect of the Combination of Cetuximab (Erbitux®) and Sodium Butyrate on Mucous Secreting Cells during Rat Colon Carcinogenesis. Am J Biotech Lab. 2018 Jul 23;6(2):18.

6. Liehr T, Pellestor F. Molecular cytogenetics: the standard FISH and PRINS procedure. InFluorescence In Situ Hybridization (FISH)—Application Guide 2009:23-34. Springer.

7. Rittié L, Perbal B. Enzymes used in molecular biology: a useful guide. J Clin Sci. 2008 Jun 1;2(1-2):25-45.

8. Tenover FC. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin Microb Rev. 1988 Jan 1;1(1):82-101.

9. Karhu R, Ahlstedt-Soini M, Bittner M, Meltzer P, Trent JM, Isola JJ. Chromosome arm?specific multicolour FISH. Genes, Chrom Canc. 2001 Jan;30(1):105-9.

10. 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 Jun;9(6).

11. Berrieman HK, Ashman JN, Cowen ME, Greenman J, Lind MJ, Cawkwell L. Chromosomal analysis of non-small-cell lung cancer by multicolour fluorescent in situ hybridisation. Br. J. Cancer. 2004 Feb;90(4):900-5.

12. Kaul Z, Cesare AJ, Huschtscha LI, Neumann AA, Reddel RR. Five dysfunctional telomeres predict the onset of senescence in human cells. EMBO Rep. 2012 Jan;13(1):52-9.

13. Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability. Nature reviews Molecular cell biology. EMBO Rep. 2017 Mar;18(3):175-86.

14. Rai R, Multani AS, Chang S. Cytogenetic analysis of telomere dysfunction. InTelomeres and Telomerase 2017 (pp. 127-131). Humana Press, New York, NY.

15. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nature Rev Gen. 2004 Jul;5(7):522-31.

16. Narimatsu R, Patterson BK. High-throughput cervical cancer screening using intracellular human papillomavirus E6 and E7 mRNA quantification by flow cytometry. Am J Clin Path. 2005 May 1;123(5):716-23.