Research Progress and Application of Fluorescence in Situ Hybridization

Fluorescence in situ hybridization (FISH) is the most convincing technique for locating specific DNA sequences, diagnosing genetic diseases, mapping genes and identifying new oncogenes or genetic aberrations that lead to various types of cancer. FISH involves the annealing of DNA or RNA probes attached to fluorescent reporter molecules to specific target sequences in the sample DNA, which can be tracked under a fluorescence microscope. This technology has recently been extended to enable simultaneous screening of whole genomes by multicolor whole chromosome probe techniques such as multiplex FISH or spectral karyotyping, or by array-based methods using comparative genomic hybridization.

Fluorescence in situ hybridization (Fish) began with the discovery that nucleic acids can be chemically modified to incorporate haptens, such as biotin or digoxigenin, which in turn can be labeled with fluorescently labeled reporter molecules such as avidin or antifungal protein. Digoxin) detection. Since then, probe preparation and labeling techniques have been modified and simplified. Nucleotides can now be directly fluorescently labeled and incorporated into FISH probes, eliminating the often laborious detection step.

Fluorescence in situ hybridization (FISH) can detect specific sites of specific DNA sequences in metaphase or interphase cells. Originally developed for mammalian chromosomes, FISH has been used to detect rRNA and repetitive DNA sequences in plant chromosomes, such as African antelope, barley, rice, Arabidopsis, Brassica, soybean.

Since the Human Genome Project involved the widespread recognition of FISH as a physical mapping technique to support large-scale nucleotide sequencing; it has become an important part of other biology and medicine such as clinical genetics, neuroscience, reproductive medicine, cytogenetics, and chromosome biology. More accessible and popular techniques in the field of study.

Due to the increased sensitivity, specificity, and resolution of this technique, the original FISH protocol has diversified into various notable procedures developed over the years. These improved techniques, along with advances in fluorescence microscopy and digital imaging, have contributed to a better understanding of the chemical and physical properties of nucleic acids and chromatin.

One of the most fascinating aspects of FISH technology is the ability to simultaneously identify multiple regions or genes using different colors. By labeling with different fluorophore combinations, entire chromosomes can be mapped in a single hybridization. The technique involves labeling each probe with a unique combination of five spectrally separable fluorescent dyes in a 1:1 ratio. Initially, these probes were used to detect 24 human chromosomes simultaneously, but have since been used to analyze specific chromosomal subregions, such as centromeres and hypocentromeres. M-FISH and SKY differ only in their method of distinguishing differentially labeled probes. SKY uses a CCD camera and Fourier transform spectroscopy.

PNAs are synthetic analogs of DNA in which the deoxyribose phosphate backbone is replaced by an uncharged peptide backbone. Due to this unique structural property, there is no electrostatic repulsion when PNA oligomers hybridize to complementary DNA or RNA sequences. PNA-DNA and PNA-RNA duplexes become more stable than native homologous or heteroduplexes. FISH with a PNA probe was used for the first time to measure individual telomere length on chromosomes in metaphase.

FISH is now an important tool for gene mapping and characterization of chromosomal aberrations. Because the target DNA remains intact, unlike molecular genetic analysis, information about the position of probes relative to chromosomal bands or other hybridization probes is directly obtained. Chromosomal aberrations on specific chromosomes or chromosomal regions can be easily defined using differentially labeled probes. Diseases diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, Cri-du-Chat syndrome, velocardiofacial syndrome, and Down syndrome. Analysis of chromosomes 21, X, and Y can identify at-risk individuals with oligospermia.

In medicine, FISH can be used for the diagnosis, prognosis assessment, and remission assessment of diseases such as cancer. FISH can detect diseased cells more easily than standard cytogenetic methods. High-resolution FISH mapping and sequencing of probes relative to each other can be performed on released chromatin fibers.

Cancer cytogenetics greatly benefits from FISH technology, and therefore clinical laboratories benefit from this technology as it is fast and can be performed on smears or cell cultures. Since it is often difficult to disseminate chromosomes from tumor cells, the use of interphase FISH directly on tumor samples (biopsies, sections, and archived paraffin-embedded material) can identify chromosomal aberrations without preparing interphase chromosomes. Digital chromosomal aberrations, chromosomal deletions, and translocations can all be identified in the interphase nucleus, providing important diagnostic and/or prognostic information.

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