Fluorescence in situ hybridization technique and its application

Issuing time:2018-10-23 00:00

Fluorescence in situ hybridization technique and its application

Abstract: Fluorescence in situ hybridization (FISH) is a useful tool in molecular cytogenetics, especially for the diagnosis of some chromosome number abnormalities and complex chromosome abnormalities, which bridge the gap between chromosome banding technique and molecular genetics. In this paper, the development history, probe preparation and clinical application of fluorescence in situ hybridization are reviewed briefly.

Key words: FISH; Fluorescence in situ hybridization; Clinical application; Prenatal diagnosis; tumor

Fluorescence in situ hybridization and applications

SUN Jingjing,YAN Shouqing

(College of Animal science and Veterinary Medicine,Jilin University,Changchun 130062,China)

Abstract: FISH is a powerful molecular cytogenetic technique which allows rapid detection of numerical and complex chromosome aberrations on interphase cells and metaphase spreads, bridging the gap between conventional chromosome banding analysis and molecular genetic DNA studies. This review gives a brief overview of the historical developments of FISH techniques and applications in clinic genetic diagnostics.

Key words: FISH; Fluorescence in situ hybridization; Clinical applications; Prenatal diagnosis; Tumor

DNA荧光原位杂交(fluorescence in situ hybridization,FISH)The technique is a method of displaying the position of DNA sequence in the nucleus or chromosome by nucleic acid probe hybridization using non-radioactive fluorescent material [1] This technique has been widely used in the fields of cytogenetics, tumor biology, gene mapping, gene amplification, prenatal diagnosis and mammalian chromosome evolution research, because of its rapidity, safety, high sensitivity and long-term storage of probes.

1. The emergence of FISH Technology

In 1969, Gall and Pardue used radioisotope labeled DNA probes to detect the success of rDNA in the nucleus of Xenopus laevis cells, in the same year, mouse satellite DNA was used as a template, and 3H-containing RNA was used as a probe to hybridize in situ with metaphase chromosome specimen, thus, we created the RNA-DNA isotope in situ hybridization technique [2], but it is not widely used.In 1974 Evans first combined chromosome banding with in situ hybridization to improve the accuracy of gene mapping.In 1981, Langer's first biotin-labeled nucleotide probe ( Bio-dUTP ) was successfully applied to chromosome in situ hybridization, non-radioactive in situ hybridization ( nonradioactive in situ hybridization ) was established  In  Situ  At this point, a technique for in situ hybridization of genes on cell sections using fluorescence-labeled probes was established.The technology was introduced to plants in 1985.In 1986, Cremer and licher demonstrated the feasibility of using fluorescence in situ hybridization (FISH) to detect aneuploidy in interphase nuclei.Thus, interphase cytogenetic studies have been opened up [3].

2. The principle of FISH

The principle of fluorescence in situ hybridization (FISH) is to label DNA probes with fluorescent dyes, such as biotin and trichosanthin, and then direct hybridization the labeled probes to chromosomes.Or DNA fiber sections, and then coupled with fluorescein molecule monoclonal antibodies and probe molecules specific binding, through fluorescence hybridization signal to detect DNA sequence in the dye.Orientation, qualitative and relative quantitative analysis of chromatids or DNA fibers.Compared with other in situ hybridization techniques, fluorescence in situ hybridization has many advantages, mainly embodied in: FISH does not need radioisotope as probe marker, so it is safe.The experiment period of FISH is short and the probe stability is high. FISH can enhance the signal of hybridization through many times of immuno-chemical reaction, so as to improve the sensitivity.The resolution of FISH is as high as 3-20 Mb;fISH can label different DNA probes with different modified nucleotide molecules and detect different probe molecules with different fluorescein molecules.Therefore, the localization of several DNA probes can be observed simultaneously on the same slice under the fluorescence microscope, and their relative positions and sequences can be obtained directly, thus greatly accelerating the study of the location of biological genomes and functional genes [4].

Development of 3 FISH technology

In the 1990s, FISH gradually formed the developing trend of fiber-FISH from monochromatic to polychromatic, from intermediate chromosome FISH to thick-line, and from intermediate chromosome FISH to fiber-FISH in the method, Sensitivity and resolution have also improved significantly.

3.1multicolor fluorescence in situ hybridization (M-FISH)

Multicolor in situ hybridization (M-FISH), "M" for "Multicolor", "Multiplex" and "Multitar get" respectively. The most important feature of M-FISH is that it can be done in a single FISH experiment with multiple tedious FISH experiments and many different gene localizations. Cremer et al. established a bicolor FISH technique using biotin and mercury or aminoacetylfluoresin (AFA) labeled probe. In 1990, Nederlof et al proposed to detect three or more target DNA sequences with three fluorescein to create a polychromatic FISH method. Five new techniques have been developed based on multicolor FISH: chromosome paint ing, comparative genomic hybridization, and comparative genomic hybridization. CGH, spectral karyotyping analysis (SKY), cross-species color banding (Rx-FISH), multi-color in situ bootable marker (MAI) multicolor primed in situ labeling, multicolor PRINS) [5] 。

3.2DNA fiber-FISH Fluorescence in Situ Hybridization

Wiegant et al. and Heng used chemical methods to linearize the chromosomes first, and Heng used the linearized chromosome DNA as the carrier to carry out FISH. The resolution of FISH was improved remarkably, which was the initial fiber-FISH. Parra and Windle (1993), Heiskanen et al. (1994), Florijn et al. (1995) The method of stretching DNA fiber was further improved, and the extension degree of DNA fiber reached 2.5-3.5kb/μm from 80kb/μm. This value is very close to the theoretical value of 2.97kb/μm for the B-DNA molecule in the DNA double helix model established by Watson-Crick, So we can say that the DNA fibers we're getting are essentially the bare double-helix DNA molecules. The key of fiber-FISH is how to prepare high-quality linear DNA fiber-FISH with high resolution and quantitative analysis, the template is not high, Only a small number of DNA molecules (<10) need to be analysed. The advantages of high sensitivity are such as fiber-FISH that it plays a very important role in chromosome mapping, gene recombination studies and clinical chromosome sequencing, due to the advantages of fiber-FISH [1] 。


Tissue Microarray Arrangement  Microarray)

Microarray can detect the expression of hundreds of genes in a single cell in a single experiment (including reduced and increased).Issue Array was able to detect the expression of one gene in hundreds of cells in one experiment.Issue Microarray was created by issue In the microarray region, 500-1000 individual tumor tissues are combined with tubular biopsies, and the biopsies are cut into more than 200 pieces for DNA,rNA probes.Single hybridization provides signals for all samples on a single slide, and later sections can be analyzed with other probes or antibodies.Multiple overlapping areas of the same tissue sample slice can form thousands of slices [6].The combination of tissue and cDNA microarray technology provides a powerful method for identifying genes in vivo, which can make an important assessment of molecular changes in cancer or other diseases.


Fluorescence Immunophenotype Analysis and Interphase Cytogenetics ( fluorescence immunophenotyping and interphase cytogenetics, FICTION)

FICTION is a combination of karyotype analysis and in situ hybridization, which can show the immunophenotype and certain genetic changes in a single tumor cell population at the same time. FICTION morphology related, can make a retrospective study of the archived material [7] FICTION diagnosis is rapid and reproducible. It is suitable for cytological samples of pre- and post-FISH samples of previous cells without the need for FISH analysis [8] FICTION can be used to analyze the pedigree of hematologic tumors to gain a better understanding of tumor pathology.

4 FISHApplication of the technology

FISH The technique has been widely used in cytogenetics, tumor biology, gene mapping, gene amplification, prenatal diagnosis and mammalian chromosome evolution research.


Detection of chromosome structural variation and aneuploidy

Fluorescence in situ hybridization ( FISH) simplifies the detection of chromosome structural variation. For example, aneuploidy of a plant chromosome may be caused by the abnormal behavior of the chromosome, i.e., the number and structural differences of the gametes of both parents, or the distant genetic relationship of the chromosome, etc. Deletion, addition or replacement of chromosomes can be more easily detected by in situ hybridization [9] 。


Detection of gene amplification and deletion

FISH spatial resolution and sensitivity make it possible for parents and amplified genes to be located in pest-resistant cells [10] The amplified genes were mainly located on the same chromosome arm, but far away from the original parent gene, and were often located independently of the telomere site. FISH analysis showed that cell fragmentation may be due to structural rearrangement of the amplified region in chromosomes. At the same time, FISH was used to locate the foreign gene position and copy number of transgenic plants. This method has been successfully used in tomato, tobacco, barley, wheat, rye and other crops. FISH can also be used to detect gene deletions associated with inherited diseases, such as aniridia, a rare genetic disorder of the iris, successfully detected in patients with aniridia disease.


Gene mapping

Fluorescence in situ hybridization ( FISH ) has been widely used in gene mapping. PP2Ac mutant lung cancer related gene was located in the chromosome region, and the characteristics of hybridization signal were recorded and analyzed under fluorescence microscope. As a result, hybridization signals were observed on chromosome 5q23-31 of normal human lymphocytes, and strong signals appeared on chromosome 5 and 7 of GLC-82 cells. Point mutation caused PP2Ac activity changes, resulting in gene translocation, resulting in the production of lung tumors[11]. FISH combined with biochemical, computer and recombinant DNA techniques showed that the DNA sequence was related to band type.FISH analysis of the distribution of coding genes in the human genome revealed that the genes were mainly concentrated in G + C (35% of the whole genome). ) the most abundant DNA segment. Fish technology provides an important means for the study of centromere structure. The chromosome telomere can be directly observed by FISH technique, which simplifies the study of the structure and function of chromosomes in the nucleus.


gene mapping

The location of DNA on the chromosome can be directly detected by FISH technique, which is the actual physical position of the gene on the chromosome. FISH has become an important tool for repeat sequence and polygene mapping because in situ hybridization is not influenced by intra-site variation and inter-locus copy number, The physical patterns of B-gliadin loci in barley chromosome 5 were obtained by fluorescence in situ hybridization, such as M. C. lark. Multicolor probe labeling provides a more convenient method for probe localization. If the two probes are in red and the third is in green, the position of the green sites is either outside or between the two, and the probe sequence can be determined. Thus, the probe is separated by a sequence of at least 20kbp, which can be located using FISH technology [3] 。


Prenatal diagnosis

FISH was first used for prenatal diagnosis in the United States. As early as 1993, FISH was approved by the American genetic association (ACMG) for prenatal diagnosis.The final diagnosis depends on karyotype analysis.And by 2000, in view of the high sensitivity and specificity of FISH technology, ACMG announced that FISH technology can be used for the diagnosis of common chromosome number abnormalities, and no longer just as an auxiliary means of prenatal diagnosis. In 2001, tepperberg et al.[12]the results of rapid prenatal diagnosis by FISH were compared with those of karyotype analysis.The false positive rate was 0.019%, and the false negative rate was 0.049%.FISH has a very high sensitivity and specificity for rapid prenatal diagnosis and can be used for the diagnosis of common chromosomal numerical abnormalities.Since then, FISH has been widely used for rapid prenatal diagnosis in some developed countries.

FISH is suitable for a variety of specimens, such as amniotic fluid cells, villus cells, fetal nucleated red blood cells and preimplantation embryonic cleavage cells, etc.[13]it applies not only to metaphase chromosomes, but also to all stages of interphase nuclei and cell cycle. The outstanding advantage of FISH is that the result can be obtained quickly, with a duration of 7 ~ 12. Compared with conventional chromosome banding analysis, FISH can be used in 24~ 48. H completed, reduce the waiting time for patients with positive chromosome disease screening test in anxiety, so that the clinician to make a diagnosis as soon as possible, to develop further treatment programs. Compared with radioactive in situ hybridization, FISH has no radioisotope contamination and no special safety protection, and the sensitivity is equivalent to that of isotope detection, it can completely replace radioactive in situ hybridization and become a new method of examination [14].


Chromosome RNA and genomic evolution

The major components of chromosomes include DNA and histones, in addition to nonhistones and RNA. Accurate mapping of the distribution of these substances in chromosomes is the key to the study of the advanced structures of chromosomes and the construction of chromosome models. The study of DNA and protein in chromosomes has been carried out deeply, but the nature, species, sources, distribution and biological functions of RNA in chromosomes are not well studied. Song Linsheng, et al [15] The cDNA of 5s rRNA and tRNA in Maize 18s, rRNA Labeled with Biotin as a Probe, In situ hybridization using ultrathin sections of the root tip of maize to confirm that there are rRNA or its precursor from the nucleolus in the chromosome of maize, rRNA, 5s rRNA or hnRNA from the nuclear matrix (nuclear heterogeneity RNA) The species and sources of RNA in chromosomes are revealed.


Blood tumor detection

Hematological malignancies are one of the ten most common tumors in China. With the further study of the pathogenesis of the disease, it is found that cytogenetics is of great significance for tumor classification, diagnosis, treatment and prediction of prognosis. Clinical FISH detection of hematological malignancies mainly focuses on the following aspects: ①Detection of fusion gene in heterotopic formation of chromosome①. ②Detection of gene deletion. The absence of some key genes may help us in the diagnosis and prognosis of tumors. ③Detection of minimal residual lesions. ④Monitoring of implantation status of heterosexual hematopoietic stem cell transplantation.


In solid tumors

FISH can quickly detect chromosomal abnormalities in almost any type of histiocyte, whether the tissue is fresh or some formaldehyde solution (formalin) fixed old groupwoven Specimen. FISH is widely accepted as an adjunctive diagnostic tool for solid tumors such as breast cancer, bladder cancer, cervical cancer, lung cancer, lymphoma, etc.Early diagnosis, efficacy detection, individualized treatment and prognosis judgment of[16].

In conclusion, the application of FISH technology in clinical application has been developed rapidly, and the sensitivity and specificity of FISH have been improved significantly, and more commercial probes are available. However, because the price of the instrument and probe is too expensive and the technical level of the operator is also high, FISH is limited to a certain extent  The clinical application of technology. In particular, in China, only some large hospitals have carried out FISH projects, and the detection scope is limited to blood tumors, breast cancer and prenatal diagnosis.Other testing projects carried out less. Therefore, in the future for a period of time, in order to make FISH more widely used, how to reduce costs, simplify the operation is still a difficult task for researchersquestions.


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