In situ hybridization uses complementary base sequences between single strands of nucleic acid molecules to complement and pair radioactive or non radioactive foreign nucleic acids (i.e. probes) with DNA or RNA to be tested on tissues, cells or chromosomes to synthesize nucleic acid hybridization molecules. The nucleic acid to be tested is detected in tissues A position on a cell or chromosome. Fluorescence in situ hybridization (FISH) is a non radioactive molecular cellular genetic technology developed on the basis of radioactive in situ hybridization. The in situ hybridization method using fluorescent markers instead of isotopic markers is a molecular diagnostic technology using Fluorescent-labeled DNA probes to detect specific DNA sequences in cells, tissues and tumors.

Figure 1. Fluorescent in situ hybridization (Hao, et al. 2013).

Unlike other techniques used to study chromosomes, FISH is flexible and does not need to be detected in dividing cells. Therefore, it can be used to detect whether genes or chromosomes are abnormal. It is usually used for genetic counseling, medical and variety identification, and DNA specific function research. Relying on advanced experimental platform and professional research team, CD Biosciences can provide comprehensive and customized fluorescence in situ hybridization services to help your scientific research work. If you have any needs, please feel free to contact us.

Principle of Fluorescence In Situ Hybridization

The basic components of FISH are a DNA probe and a target DNA sequence located by it. Before hybridization, DNA probes are labeled by a variety of techniques, such as random primer labeling, nick translation, and PCR amplification. The following two labeling methods are commonly used: direct labeling and indirect labeling (Fig. 2). For the direct labeling method, nucleotides that have been directly altered to contain a fluorophore are used; however, in indirect labeling, the probe is labeled with altered nucleotides with haptens. Thereafter, the labeled probe and the target DNA are denatured, and the denatured probe is paired with the target DNA during the annealing of the cDNA sequence. If the probe is indirectly labeled, an additional step is required to observe the non-fluorescent hapten using immunological or enzymatic detection systems. Therefore, directly labeled probes are faster, and indirect labeling provides sufficient signal amplification by using multiple antibody membranes.

Figure 2. Principle of Fluorescence in situ hybridization (Mohanty, et al. 2020).

Different Types of FISH and Their Functions

Type	Function
ACM-FISH	In sperm cell, structural and numerical chromosomal abnormalities can be detected
CARD-FISH	Amplification of signal which is  obtained by peroxidase activity
CAT-FISH	Expression of genes patterns in brain
CO-FISH	The orientation of tandem repeats in the centromeric regions of chromosomes
CB-FISH	The cytological analysis of micronucleation and aneuploidy
COD-FISH	Quantification of gene copy number and the protein amount
Comet-FISH	DNA damage
D-FISH	Detection of  BCR/ABL fusion in chronic myeloid leukemia (CML)
DBD-FISH	Any sites of DNA damage/breakage in the sample genome
Fiber-FISH	Mapping of genes and chromosomal regions on fibers of chromatin or DNA
Flow-FISH	Visualize and measure the length of telomere
Harlequin-FISH	Cell cycle-controlled chromosome analysis in human lymphocytes
Immuno-FISH	Both DNA and proteins can be analyzed in the same sample
M-FISH	Facilitating the analysis of complex chromosomal rearrangement
ML-FISH	Identifying multiple microdeletion syndromes in patients
PCC-FISH	Chromosome damage after irradiation
Q-FISH	Determining the repeated number of telomere on a specific chromosome
QD-FISH	Human metaphase chromosomes, human sperm cells, bacterial cells, and also to detect subcellular mRNA distribution in tissue sections
Raman-FISH	For characterizing of chromosomes and chromosome amplifications in cancer
RING-FISH	Identification of individual genes and detection of halo appearance from fluorescence signals at the bacterial cell at periphery
RNA-FISH	Allelic-specific expression in per cell basis
T-FISH	Mapping of gene loci and looking for specific transcripts in cells

CARD-FISH: catalyzed reporter deposition FISH; CAT-FISH: capture antibody targeted detection FISH; CB-FISH: cytochalasin B FISH; CO-FISH: cyotochrome orientation FISH; COD-FISH: chromosome orientation and direction FISH; D-FISH: dual color FISH; DBD-FISH: deoxyribonucleic acid breakage detection FISH; DNA: deoxyribonucleic acid; M-FISH: multiple spectral karyotyping FISH; ML-FISH: multilocus FISH; mRNA: messenger ribonucleic acid; PCC-FISH: premature chromosome condensation FISH; Q-FISH: quantitative FISH; QD-FISH: quantum dots FISH; RNA: ribonucleic acid; T-FISH: tissue FISH

Applications of Fluorescence In Situ Hybridization

• Detection of numerical and chromosomal abnormalities
• Monitoring the effects of therapy
• Detection of early relapse or minimal residual disease
• Identification of the lineages of neoplastic cells
• Examination of the karyotypic pattern of interphase cells, including nondividing or terminally differentiated cells
• Detection of gene deletions and gene amplification

1. Hao, Ming, et al. "Production of hexaploid triticale by a synthetic hexaploid wheat-rye hybrid method." Euphytica 193.3 (2013): 347-357.
2. Mohanty, Swati Sucharita, Chita Ranjan Sahoo, and Rabindra Nath Padhy. "Role of hormone receptors and HER2 as prospective molecular markers for breast cancer: An update." Genes & Diseases (2020).
3. Gozzetti, Alessandro, and Michelle M. Le Beau. "Fluorescence in situ hybridization: uses and limitations." Seminars in hematology. Vol. 37. No. 4. WB Saunders, 2000.
4. Ratan, Zubair Ahmed, et al. "Application of fluorescence in situ hybridization (FISH) technique for the detection of genetic aberration in medical science." Cureus 9.6 (2017).

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