Method of fluorescent hybridization in situ (FISH) in the diagnosis of chromosomal diseases. Fluorescence hybridization Fluorescence hybridization

Fluorescence in situ hybridization, or the FISH method (fluorescence in situ hybridization - FISH), is a cytogenetic method that is used to detect and determine the position of a specific DNA sequence on metaphase chromosomes or in interphase nuclei in situ. In addition, FISH is used to detect specific mRNAs in a tissue sample. In the latter case, the FISH method makes it possible to establish spatiotemporal features of gene expression in cells and tissues.
The FISH method is used in preimplantation, prenatal and postnatal genetic diagnostics, in the diagnosis of oncological diseases, in retrospective biological dosimetry.

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hybridization method in situ* (in place, lat.) is based on the ability of DNA or RNA to form stable hybrid molecules with DNA / RNA probes directly on preparations of fixed chromosomes and interphase nuclei. Using this method, you can determine the exact location of almost any DNA or RNA sequence directly in the cell, cell nucleus or chromosomes.

For hybridization. in situ suitable cytological or histological preparations of cells of any tissues or organs, prepared according to standard methods. In a clinical cytogenetic laboratory, preparations of cultured peripheral blood lymphocytes, chorionic epithelial cytotrophoblast cells, cultured and uncultivated cells of amniotic fluid, various tissues from abortion material, as well as smears of buccal epithelium and blood cells are used.

hybridization method in situ is of particular importance for practical cytogenetics due to the development of a non-isotopic variant based on the use of probes labeled with non-radioactive modified nucleotides. Non-isotopic variants of hybridization on preparations (especially fluorescent ones) have a number of advantages compared to isotopic ones: high resolution, which is equal to the resolution of a microscope (0.1 - 0.2 μm), no need for statistical processing of results, speed and safety for health researchers

In addition, the combination of differently modified probes detected using different detection systems allows simultaneous determination of the location of two or more DNA sequences in one cell or on one metaphase plate. And the use of repetitive sequences labeled with fluorochromes as DNA probes reduces the procedure time to 7–9 hours (classic non-isotope hybridization takes two days, isotope variants from a week to a month), which is especially important for prenatal diagnosis. Usage FISH method in cytogenetic diagnostics, it allows identifying structural chromosomal rearrangements, establishing the nature of marker chromosomes, and analyzing numerical violations of the chromosome set, both on metaphase chromosomes and in interphase nuclei.

Principle of the FISH method

At the core FISH method lies the hybridization reaction between an artificially created DNA probe and its complementary nucleotide sequence of nuclear DNA. The DNA molecule consists of two helical nucleotide chains, and hybridization is possible only if the chains separate. To disconnect the nucleotide chains of DNA, denaturation is used (for subsequent hybridization, both the DNA in the nuclei of the sample under study and the DNA probe itself must be denatured). After denaturation, the DNA probe hybridizes to its complementary nucleotide sequence and can be detected using a fluorescent microscope.

Thus, the general form of the protocol for setting FISH can be presented in the following form:

1. Preparation of a histological or cytological preparation.
The preparation of a histological preparation is carried out according to the standard scheme: cutting, marking, wiring, pouring, microtomy, placing the cut on a glass slide and deparaffinization. When preparing a cytological preparation, special precipitating solutions and centrifugation are used, which makes it possible to obtain a concentrated cell suspension.

2. Pre-treatment (if necessary).
The preparation is processed by proteases to eliminate the presence of proteins that hinder hybridization.

3. Applying a DNA probe to the preparation and subsequent denaturation.
In order to denature the probe and sample DNA, they are treated with formamide and heated to a temperature of about 85-90°C.

4. Hybridization.
After denaturation, the drug is cooled to a certain temperature (37°C in the case of clinical studies) and incubated in a humid chamber for several hours (the duration of incubation is indicated in each specific protocol). Currently, automatic hybridizers are used for denaturation and hybridization.

5. Washing.
Once hybridization is complete, unbound probes must be washed out, which would otherwise create a background that makes it difficult to evaluate FISH results. For flushing, a solution containing citrate and sodium chloride (SSC) is usually used.

6. Counter-staining.
With the help of fluorescent dyes (DAPI - 4,6-diamidin-2-phenylindole; propidium iodide), all nuclear DNA is stained.

7. Analysis of the results using a fluorescent microscope. Routine operations (dewaxing, pretreatment, washing) can be automated.

* - The material was prepared on the basis of information from open sources.

In situ hybridization of nucleic acids The method is based on the possibility of forming double-stranded hybrids between labeled probes artificially created on the basis of single-stranded sequences (ribo- or deoxyribo-, oligo- or polynucleotide) and sequences complementary to them in targets - analyzed DNA or RNA molecules. To identify hybridization sites, a DNA probe (DNA probe) is labeled with a reporter group: ü a radioactive isotope, ü a fluorochrome, ü an enzyme that gives a stain or a luminescent product, ü a hapten with which the labeled body binds, etc.

By detecting a hybrid due to the presence of a reporter group in the probe, it is possible - to estimate the number of genes encoding a certain type of RNA, - to determine the proportion of non-transcribed DNA in the genome, - to establish the linkage of certain genes with each other - and their exact location on chromosomes. The molecular hybridization method has high sensitivity (a small amount of labeled probe (10 -15 and 10 -19 M) and, accordingly, its complementary sequence in the target can be detected) and analysis speed, which allows using this method both for research activities and for the diagnosis of hereditary and infectious diseases in medicine, veterinary medicine and crop production.

The emergence of new technologies in molecular cytogenetics, based mainly on in situ hybridization of nucleic acids, has significantly expanded the possibilities of chromosomal diagnostics.

Interphase cytogenetics: 1. Multicolor chromosome banding (MCB). 2. Fluorescent in situ hybridization (FISH). 3. Combination of FISH with other methods: -cytology + FISH; -histology + FISH; -immunophenotyping + FISH (FICTION); Metaphase cytogenetics: 1. Solid staining of chromosomes (Whole painting). 2. Comparative genomic hybridization (CGH). 3. Color change karyotyping (CCK). 4. Multicolor karyotyping: Spectral karyotyping (SKY); multicolor FISH (M - FISH, M - BAND).

FISH - fluorescence in situ hybridization - is a cytogenetic method used for detection and localization of specific DNA sequences on chromosomes, mRNA, etc. The technique is based on the hybridization of a fluorescently labeled DNA/RNA probe with a complementary DNA/RNA sequence. The label is detected using a fluorescent microscope. The FISH method was introduced over 30 years ago. It has become widespread as a method of physically mapping genes on chromosomes. Later, it was applied to other areas of research (in the fields of clinical genetics, reproductive medicine, toxicology, evolutionary biology, comparative and cellular genomics, and chromosomal biology). At the moment, FISH is mainly used to build physical and genetic maps of chromosomes, to detect structural rearrangements, translocations, microdeletions, gene amplifications in interphase and metaphase chromosomes. As a result of advances in science (better understanding of the chemical and physical properties of nucleic acids and chromatin), as well as the development of fluorescence microscopy and digital imaging, the method has been constantly improved (improvements in sensitivity, specificity, resolution) and many variations of this method have been developed.

1) Cells are dropped on to a glass slide causing chromosomes to spread 4) Chromosomes are counter stained using DAPI 2) Fluorescently labeled probe is placed on chromosomes and sealed. 3) The probe and chromosomes are denatured, hybridised then washed 5) Slide is viewed under a fluorescent microscope

The general view of the protocol for setting up FISH can be represented as follows: 1. Preparation of a histological or cytological specimen. The preparation of a histological preparation is carried out according to the standard scheme: cutting, marking, wiring, pouring, microtomy, placing the cut on a glass slide and deparaffinization. When preparing a cytological preparation, special precipitating solutions and centrifugation are used, which makes it possible to obtain a concentrated cell suspension. 2. Pre-treatment (if necessary). The preparation is processed by proteases to eliminate the presence of proteins that hinder hybridization. 3. Applying a DNA probe to the preparation and subsequent denaturation. In order to denature the probe and sample DNA, they are treated with formamide and heated to a temperature of about 85-900 C.

4. Hybridization. After denaturation, the drug is cooled to a certain temperature (370 C in the case of clinical studies) and incubated in a humid chamber for several hours (the duration of incubation is indicated in each specific protocol). Currently, automatic hybridizers are used for denaturation and hybridization. 5. Washing. Once hybridization is complete, unbound probes must be washed out, which would otherwise create a background that makes it difficult to evaluate FISH results. For flushing, a solution containing citrate and sodium chloride (SSC) is usually used. 6. Counter-staining. With the help of fluorescent dyes (DAPI - 4, 6-diamidin-2 phenylindole; propidium iodide), all nuclear DNA is stained. 7. Analysis of results using a fluorescence microscope Routine operations (dewaxing, pretreatment, washing) can be automated.

Examination of telomeric regions using a fluorescent microscope. The images obtained at the stage of interphase (nucleus) and metaphase (individual chromosomes) are combined. blue color - DAPI.

Advantages of FISH-method: 1. Possibility to study genetic material in interphase nuclei; 2. Obtaining objective results on a yes/no basis is a quantitative method; 3. Relatively simple interpretation of the results; high resolution. Disadvantages of the FISH method: 1. Fluorescent dyes quickly "fade"; microscope.

The FISH method is based on the hybridization reaction between a DNA probe created using special technologies, which is a nucleotide sequence of a limited size, and a complementary section of the nuclear DNA of the cytogenetic preparation under study. The DNA probe is the main element for setting up FISH and carries a "label", i.e. it contains nucleotides that are directly linked to a fluorochrome or a hapten for further visualization by antibodies carrying a fluorochrome. Unbound labeled DNA is washed off and then the hybridized DNA probe is detected using a fluorescent microscope.

DNA labeling is divided into direct and indirect. Direct labeling introduces reporter elements into DNA - fluorochromes (rhodamine, diethylaminocoumarin, Texas red, etc.). Indirect labeling method - a DNA sample is pre-conjugated with intermediate ligands (biotin, dioxygenin, 2, 4-dinitrophenol), the presence of which on a cytological preparation is then detected using fluorochromes. In this case, the sensitivity of the method greatly increases, since the ligand may contain several sites of interaction with the fluorochrome. In situ hybridization with a 32 P-labeled probe was first described in 1969. The development of non-radioactive systems for labeling and detecting DNA probes has made the in situ hybridization method safe, easy to perform, and less laborious in processing the results. Fluorescent dyes are soft and easy to use, can be stored indefinitely, give high resolution, and allow multiple DNA sequences to be examined simultaneously.

Probes: single-stranded/double-stranded DNA/RNA primary/secondary labeled probes. Probes are labeled: 1) directly: by inserting nucleotides to which a fluorochrome is attached (PCR or nick translation) 2) through a reporter molecule (ex: digoxigenin, biotin) to which fluorescently labeled antibodies are attached. To enhance the signal, you can use secondary, tertiary, etc. antibodies labeled with a fluorescent label. DNase nicks DNA Nick Translation DNA polymerase I adds new nucleotides to the 3' hydroxyl DNA polymerase I removes individual bases from the 5' end Chromosome imaging: using DAPI (2 mg/ml). 4', 6-diamidino-2-phenylindole; strong binding to A-T rich DNA regions; excitation with UV light; emission 461 nm (blue).

Currently, there are several main approaches that are widely used by modern molecular cytogenetics: Identification of the material of extended chromosome regions and whole chromosomes. Detection in the region of interest of a specific DNA sequence. Analysis of the imbalance of individual chromosomal regions. To identify the material of extended chromosomal regions and whole chromosomes, chromosome-specific and region-specific DNA probes (“painting probes”) are widely used.

Characteristics of various types of probes 1. Locus-specific probes (LSI - locus - specific identifiers that bind to certain regions of chromosomes.) These probes are used to identify the available short (non-repeating) sequence of isolated DNA, which is used to prepare a labeled probe and its subsequent hybridization with set of chromosomes. They are designed to detect diagnostically and prognostically significant chromosomal aberrations in various pathological conditions (monosomy and trisomy for individual chromosomes). The most widespread use of these samples was obtained in prenatal cytogenetic diagnostics, especially in studies of chorionic tissue cells and interphase nuclei of amniocytes.

2. DNA probes to telomeric regions of chromosomes (TEL telomericprobe) are designed to detect deletions and rearrangements affecting the terminal regions of chromosome arms. Such probes are specific for the p- or q-arms of chromosomes and are complementary to a region about 300 kb long. from the end of the chromosome. As a rule, DNA probes for short and long arms of chromosomes are associated with different fluorochromes, which makes it possible to detect both telomeric regions of the chromosome on one cytogenetic preparation.

3. centromere repeat probes, alpha or chromosomal numerators (CEP - centromere. Enumeration. Probe) are chromosome-specific DNA probes represented by sequences of tandem alpha and beta satellite repeats. These repeats are located predominantly in the centromeric or pericentromeric heterochromatin regions of chromosomes. With their help, each chromosome can be painted in a different color, which allows you to quickly determine the number of chromosomes and deviations from their normal number, complementary to individual sections of the chromosome, but as a whole covering its entire length. Using a library of such probes, one can "color" the entire chromosome and obtain a differential spectral karyotype of an individual. This type of analysis is used to analyze chromosomal aberrations, such as translocations, when a piece of one chromosome is transferred to the shoulder of another.

Applications FISH is widely used to solve the following clinical diagnostic tasks: 1. Perimplantation prenatal and diagnosis of chromosomal abnormalities. Used in in vitro fertilization (IVF) clinics and perinatal centers. Allows timely (before implantation of the embryo in the case of IVF or in the early stages of fetal development) to identify genetic disorders in the unborn child and take the necessary measures. 2. Oncohematology. Oncohematological diseases arise as a result of various chromosomal aberrations, therefore, appropriate CEP and LSI probes are used for their diagnosis. 3. Diagnosis of solid tumors. Currently, more and more causal relationships are being established between the development of specific cancers and specific changes in the genome. Therefore, when using appropriate DNA probes, it is possible to carry out oncological FISH diagnostics.

Interphase cytogenetics: Combination of FISH with other methods: - FISH immunophenotyping (FICTION); + FICTION (Fluorescence Immunophenotyping and Interfase Cytogenetics as a Tool for Investigation Of Neoplasms) - for the study of tumor cells. For this analysis, unstained smears of blood, bone marrow, or preparations of other tissues are used. Stage 1 - preparations are incubated with specific monoclonal antibodies, stage 2 - conjugation with fluorophores is carried out for subsequent visualization of the antigen-monoclonal antibody complex. Stage 3 - carry out in situ hybridization with DNA probes. Fluorophores that differ in color are used to detect monoclonal antibodies and DNA probes. The study of the preparation under a fluorescent microscope with the necessary set of filters allows simultaneous analysis of the immunophenotype of hybridization and signals in interphase nuclei.

Chromosomaldeletion of TNFAIP 3 in c. HL detected by interphase cytogenetics. FICTION analyzes of. representative c. HL cases combined. CD 30 expression(red) and FISH probes for TNFAIP 3 and chromosome 6 centromere (blue [b]). In the double-color assays in A–C and E and F, the TNFAIP 3 probe is labeled in green (g); in the triple-color assay applied in D, the TNFAIP 3 probe gives a g/orange colocalized (co) signal. Two different strategies are used to display double- and triple-color FISH assays in combination with CD 30 immunofluorescence; i. e. , double-color assays (A–C and E and F) are shown using a triple-color display, whereas a false multicolor display as obtained by Isis software is applied for the triple-color assay (D) to simultaneously show four colors ( ie , CD 30 [r], TNFAIP 3 [g] and orange, and chromosome 6 centromere [b]).

Digital recording of microimages has opened up the possibility of converting into pseudocolors not only combinations of fluorophores, but also their ratios, intensities

Multicolor staining of chromosomes (Muli. Color Banding - MSV) This method is not intended for a complete analysis of all chromosomes, but for a detailed analysis of a single chromosome. Locus-specific DNA probes are labeled with different fluorophores or combinations of fluorophores so that the signal level of each of the DNA probes varies in intensity. Overlapping intensity profiles of DNA probe signals provide variations in the ratios of fluorescence intensities of different fluorophores along the chromosome. Intensity ratios can be translated into pseudocolors, and thus each point of the image, and, consequently, each of the chromosomal loci, will have its own pseudocolor. This variant of the multicolor FISH method turned out to be highly effective in the analysis of not only interchromosomal but also intrachromosomal rearrangements in oncological diseases. However, for its successful application, it is necessary to first determine the chromosome that will be investigated. This method of molecular cytogenetics is suitable for the analysis of karyotype disorders associated with certain chromosomes.

To date, sets of DNA probes have been created that provide MCV for all human chromosomes on chromosome 5; multicolor banding of chromosome 5; idiogram of chromosome 5; fluorochromes used for MSV).

Metaphase cytogenetics, which historically arose earlier than interphase cytogenetics, makes it possible to determine a wide range of chromosomal disorders, but this requires that the cells under study be at the metaphase stage of meiosis.

Multicolor karyotyping The analysis is carried out using DNA probes of continuous staining to the arms or whole chromosomes (WCP probes - whole chromosome paint). Such probes hybridize with numerous short DNA sequences located along the entire length of the chromosome. Thus, when viewed under a fluorescent microscope, the chromosome appears uniformly stained in a certain color.

The DNA probes used for this analysis consist of a mixture of solid staining probes for the full set of human chromosomes, labeled with a combination of several fluorochromes, the ratio of which is selected individually for each pair of chromosomes. The use of appropriate registration methods and computer programs for image analysis, which evaluate the intensity of the luminescence of all fluorochromes for each point of the image, makes it possible to carry out karyotyping, in which each pair of chromosomes has its own unique "pseudocolor".

M-FISH (multitarget multifluor multicolor or multiplex. FISH) is the generic name for traditional multicolor FISH using fluorochrome-specific filter sets. The M-FISH principle consists in separate digital recording of the signal of all used fluorochromes, which is achieved by sequentially changing filter sets. All images are recorded in separate files, which makes it possible to carry out their efficient processing associated with signal and background separation, as well as signal quantification. Processing of all recorded information with the help of special software translates information about the level of fluorochrome signals at each point of the image into pseudocolors. One of the main limitations of the number of DNA probes used is the number of available fluorochromes, with non-overlapping excitation and emission spectra, and the availability of appropriate filter sets.

24-color FISH The most widely used M-FISH is 24-color FISH for the simultaneous identification of material from all human chromosomes. It is highly efficient in detecting chromosomal translocations, but is not designed to detect deletions and inversions. However, this problem can be partially solved by simultaneous staining of chromosomes with DAPI, which makes it possible to analyze the differential striation of chromosomes whose chromosome material has already been identified. Unfortunately, it should be noted that the quality of chromosome DAPI banding after M-FISH is significantly inferior to GTG differential staining and even DAPI banding after conventional in situ hybridization.

Rx. FISH The M-FISH principle has been used to generate a multicolor bar code for human chromosomes and a multicolor chromosome banding method based on interspecies in situ hybridization. In contrast to 24 color FISH, this method makes it possible to directly detect part of the intrachromosomal rearrangements. DNA probes used in Rx. FISH labeled with a combination of 3 fluorochromes, which provides 7 pseudo colors. They specifically stain individual regions of chromosomes, creating their color striation. This feature of DNA samples for Rx. FISH is determined by the way they are obtained. They are chromosome-specific DNA libraries of two gibbon species: Hylobates concolor and Hylobates syndactylus.

As a result of the intense chromosomal rearrangements that took place during the formation of modern species of gibbons, the material of their chromosomes turned out to be highly shuffled in comparison with the organization of chromosomes in humans, whose chromosomes are known for their conservatism. The ratio of human chromosome 1 to gibbon chromosomes is shown in Figure 1,

Figure 2 shows a general view of human chromosomes obtained as a result of Rx. FISH. a) Metaphase plate b) Arrangement of chromosomes.

However, RXFISH has serious limitations - chromosome rearrangements that have taken place within one color RX band cannot be detected using this method unless they lead to significant and easily visible changes in the size of this band. - probes stain several chromosomal regions of different chromosomes in one pseudocolor. However, as the number of fluorochromes used increases, the RXFISH method will undoubtedly be more informative than the routine 24-color FISH.

The advantages of RXFISH at present include the following points: The method allows the analysis of the entire human genome in a single multicolor FISH experiment. Commercially available sets of labeled DNA probes and the necessary detection systems are available. The method allows for the rapid identification of a significant part of intra- and interchromosomal rearrangements. Automatic identification of metaphase chromosomes of “color banding” is possible. Each human chromosome has a unique color bar code. RXFISH is integrated into the Cyto workstation. Vision and standard fluorescence microscopy systems.

Spectral karyotyping (SKY-spectralkaryotyping) The basic principles of microscopic image analysis in spectral karyotyping are practically the same as those used in M-FISH. The differences are related to the way the image is registered. SKY technology makes it possible to obtain spectral curves for all points of the image during one measurement, regardless of whether it is associated with epifluorescence or with traditional light microscopy. For spectral karyotyping of all human chromosomes, five fluorochromes are used, one in the green spectrum, two in red and two in infrared. Excitation and emission of all fluorochromes used in labeling DNA probes takes place with one set of filters, which makes it possible to avoid their sequential change, intermediate focusing, and, consequently, related problems, such as image spatial shift, determination of threshold values and segmentation masks . Based on the analysis of the spectral curves, the presence or absence of specific fluorochromes at a given point is determined.

The next step is the classification procedure, which allows you to directly and unambiguously determine the chromosomal affiliation of the analyzed material. This ensures reliable identification (up to a chromosome) of marker chromosome material, as well as chromosome derivatives resulting from various rearrangements. The great advantage of SKY is that DAPI coloring is registered parallel to the spectral image. Software improvement of DAPI banding allows achieving differential striation in quality close to GTG banding. The possibility of parallel analysis of the spectral image and qualitative differential staining of chromosomes greatly simplifies the interpretation of SKY results and allows more accurate determination of the points of chromosome breaks. The undoubted advantages of SKY include the possibility of using fluorochromes with overlapping excitation and emission spectra, which significantly expands the list of usable fluorochromes, and also increases the number of fluorochromes that can be used simultaneously. The listing of a new fluorochrome does not require the purchase of a new set of filters, since one filter unit for spectral FISH and one filter unit for DAPI is sufficient for SKY.

The disadvantages of SKY include: - long exposure time, necessary for recording microscopic images. - SKY is somewhat less efficient than M-FISH when it comes to research with relatively small DNA probes.

Color changing karyotyping (CCKs Color changing karyotyping) This method is based on the analysis of the difference in signal length between DNA samples hybridized with DNA probes associated with fluorochromes and DNA probes carrying antibodies. The method is feasible with only 3 filters and does not require special cameras and software. To identify chromosomes, one hybridization is carried out and two images are obtained. The method is based on the difference in the length of the fluorescent signal, since the exposure time of DNA samples associated with antibodies is 80 - 90% less than that of samples associated with fluorochromes. After the hybridization reaction, the swabs are exposed to the appropriate primary antibodies and then visualized to obtain the first image. The slides are then exposed to secondary antibodies and avidin bound to the same fluorochromes. The strokes can be counterstained using DAPI.

In the future, images of the preparations are again obtained using 3 filters in turn. These images are compared using special software and a second image is obtained, in which only the chromosomes associated with certain antibodies will be visible. Thus, some chromosomes will only fluoresce on the first or second scan, while others will change color on different scans.

Comparative genomic hybridization (CGH) The method was created to identify quantitative disorders in the genome. It is based on carrying out an in situ hybridization reaction using the entire genome as a DNA probe. The isolated and normal donor DNA is labeled with fluorochromes of different colors, thus turning them into DNA probes. Equivalent amounts of these probes are mixed and used in hybridization with a control cytogenetic preparation. After FISH, metaphases are analyzed on a fluorescent microscope, using a specialized computer image analysis program to determine the fluorescence intensity of two fluorochromes along the entire length of each chromosome.

In the absence of quantitative changes in the karyotype of the test sample, a certain ratio of the luminescence intensity of the two fluorochromes will be observed. In the case of gene amplification, the intensity of the signal of the corresponding fluorochrome will increase, and in the case of loss of part of the genetic material, on the contrary, it will weaken. Thus, CGH makes it possible to detect genomic imbalance, but this method cannot be used to detect balanced translocations and inversions, and trisomies and deletions can only be detected if the size of the unbalanced region is at least 10 million base pairs.

Chromosomal imbalance in the test sample is estimated from the difference in fluorescence intensity of two different fluorochromes by calculating the fluorescent ratio (RF).

The CGH method has a number of advantages over other methods for analyzing genome changes: First, it does not depend on the source of the test material, and can be successfully performed with a small amount of DNA tested, including archival material. Secondly, it allows obtaining detailed information about the loss or increase in the number of copies of genetic material throughout the genome in a single experiment. Thirdly, the CGH method does not require the preparation of metaphase chromosome preparations from the tissue under study, that is, it does not depend on the cell culture process and related artifacts.

Genomic situ hybridization (genomicin situ hybridization in, GISH) is a variant of the situ hybridization method, which consists in the fact that for hybridization with fixed samples, the total genomic DNA of one species of organism is used as a fluorescently labeled probe, with which the total genomic DNA of another species of organism competes; used to determine interspecies and intraspecies differences in genomes, chromosomal rearrangements, deletions and substitutions.

FISH variations 1) Q-FISH – quantitative FISH: Developed by Lansdorp et al: U. M. Martens, J. M. Zijlmans, S. S. Poon, W. Dragowska, J. Yui, E. A. Chavez, R. K. Ward, and P. M. Lansdorp. 1998. Short telomeres on human chromosome 17 p. Nat. Genet. 18:76-80. quantitative method. Designed to work with flow cytometry. Originally used to measure chromosome length (resolution: 200 bp) by counting the number of telomeric repeats. PNA-conjugated probes are used. Initially, metaphase chromosomes were studied (QFISH proper), now this method is also applicable to interphase chromosomes (IQ-FISH). Q-FISH is carried out on cell culture, tissue sections (both on glasses). At the moment, Q-FISH is an important tool in the study of the role of telomeres in the processes of aging and cancer formation.

PNA-FISH - Peptide nucleic acids FISH: Peptide nucleic acids (PNAs) are synthetic analogues of DNA in which the sugar backbone of deoxyribose phosphate, which supports the nitrogenous base, is replaced by an uncharged peptide backbone. As a result of this structure: when the PNA-oligomeric probe hybridizes with complementary DNA/RNA, no electrostatic repulsion occurs. PNA-DNA (PNA-RNA) duplexes are much more stable than natural homo/heteroduplexes. High specificity of PNA-DNA binding: PNA-DNA hybridization is much more sensitive to base pair mismatch than DNA-DNA hybridization. Thus, using a PNA probe, two centromeric repeats can be distinguished, differing in only one base pair. PNA has a relative hydrophobicity (compared to DNA), as a result of which PNA diffuses better through cell walls => wide application in microbiology. Based on high binding specificity: It is believed that PNA technology will soon become the basis for the creation of allele-specific probes for in situ hybridization. It is used in genetics, cytogenetics, epigenetics, microbiology, etc.

3) Flow-FISH - FISH for flow cytometry: Proposed in 1998 by: Rufer, N. , Dragowska, W. , Thornbury, G. , Roosnek, E. & Lansdorp, PM Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. nature biotechnol. 16, 743–747 (1998). A combination of Q-FISH and flow cytometry that allows signal analysis (measurement) and sorting. For flow-FISH, a cell suspension is used (for measuring the length of chromosome telomeres), chromosome spreads and isolated chromosomes (for further mapping). As in Q-FISH, PNA-tagged telomeric probes are used (for example, to visualize and measure the length of telomeric repeats). The main advantage is a faster method (analysis/sorting of a large number of cells/chromosomes). It is widely used to study various problems: aging, telomere preservation, the study of hematopoietic stem cell suspensions ex vivo, the study of bacteria.

Multiparametric analysis allows for differentiation of cell types within one sample, allowing for internal control, and analysis of leukocyte subtypes.

Benefits of flow-FISH: Easily reproducible results Ability to analyze subgroups of cells in a population using physical immunofluorescence and markers Better performance than Q-FISH Ability to quantify the fluorescence of thousands of cells.

INVESTIGATION OF ISOLATED CHROMOSOMES BY FLOW CYTOMETRY 1. Chromosome sorting by flow cytometry - Karyotyping by flow cytometry is used for further gene mapping and building chromosome libraries. - Identification and localization of genes on sorted chromosomes is carried out with the subsequent use of FISH methods / PRINS method (primed in situ labeling) or its variations and chromosome-specific PCR. - By 2011, only the chromosomes of 17 species of cultivated plants have been successfully sorted so far. - Sorting of metaphase or pachytenic chromosomes with a high division index.

Fluorescent label: - Chromosomes are labeled with nucleic acid specific fluorochromes. - Fluorochromes are selected in accordance with 1) specificity to nitrogenous bases, 2) experimental conditions (including taking into account the wavelengths of available lasers). 1. For monovariant analysis, use dyes that are not specific to AT or GC vapors: propidium iodide (excitation peak: 535 nm, emissions: 617 nm, laser required: 488 nm argon laser or lamp + long-pass filter) ethidium bromide (excitation peaks: : 300 and 520 nm, emissions: 600 nm, required laser: 488 nm argon laser or lamp + long-pass filter) These labels stain DNA regardless of the content of A, G, T or nitrogenous bases. 2. For bivariate analysis use: chromomycin (specific for G-C base pairs), peak A 3 excitation: 458 nm, emission 580 nm. Laser: , 458 nm minimum 400 m. V power. Hoechst 33258 (A-T bp specific), excitation: 351-364 nm, emission: 470 nm. Laser: 351 -364 nm (powerful). The lasers must be separated in time and space due to the partial overlap of the fluorochrome spectra.

2. Study of chromosomes/nucleotide sequences after sorting (for building physical and genetic maps, etc.) FISH BAC-FISH PRINS C-PRINS Study of large genomes with long chromosomes. Mapping of sequences with a large number of repeats (ex: telomeric and centromeric regions). Use of short probes. Due to the large number of repetitions, the signal is strong. Standard probe size: 15 - 30 nucleotides. FISH

FISH problems: It is difficult to localize single-locus DNA sequences (i.e., non-repetitive unique DNA sequences) because the signal will be very weak when using standard short probes. Increasing the length of the probe to several kilobases to enhance the signal will result in decreased sensitivity and => non-specific binding. Solution: BAC-FISH -BAC-FISH is a combination of the FISH method and the use of genomic DNA clones embedded in bacterial artificial chromosomes (BACs), allowing the insertion of large DNA sequences. - An efficient method for identifying and mapping individual chromosomes of organisms with small genomes. BAC probes, overgos (overlapping oligonucleotides) are used as a probe.

Alternative to FISH: PRINS PRINS (primed in situ labeling) is a method of labeling chromosomes by annealing an oligonucleotide DNA primer with a homologous sequence of denatured chromosomal DNA and subsequent enzymatic extension of the primer in situ with labeled nucleotides. First described by Koch et al. in 1989 (Koch, JE , Kølvraa, S. , Petersen, KB , Gregersen, N. , and Bolund, I. (1989) Oligonucleotide-priming methods for the chromosome-specific labeling of alpha satellite DNA in situ. Chromosoma 98 , 259–265). PRINS is an alternative to FISH. It is used for localization of nucleotide sequences, recognition and counting of metaphase or interphase chromosomes or chromosome pairs (including chromosomal aneuploidy). annealing of the unlabeled primer (primer-primer) with the DNA of interest; elongation of the primer with the help of thermostable DNA polymerase and labeled nucleotides; termination of the reaction (attachment of a blocking molecule to the 3'-end) Primer: PCR restriction fragment; - indirect (biotin/ dig fluorochrome-conjugated avidin/ anti-dig) - Only one pair of homologous chromosomes (one chromosome) can be identified in the results of each PRINS reaction. The next PRINS reaction on the same slide can only be carried out after blocking the previous one. - Used for DNA sequences with a large number of repeats.

Advantages of PRINS: 1. Requires minimal sequence information required for the synthesis of an oligonucleotide primer. 2. The fastest and simplest method for detecting a sequence of interest on a chromosome (compared to using heavy FISH probes that hybridize over a very long period of time). 3. Exclusion of the probe labeling step. 4. The ability to elongate the primer with labeled nucleotides to enhance the c-PRINS signal when detecting short unique sequences. To identify low-copy repeats or short unique sequences, a more sensitive method is used - cycling PRINS (c-PRINS). C-PRINS was proposed by Gosden et al. in 1991, an improved widely used protocol by Kubaláková et al. , 2001 (Kubaláková M, Vrána J, Cíhalíková J, Lysák MA, Dole J (2001). Localization of DNA sequences on plant chromosomes using PRINS and C-PRINS. Methods in Cell Science 23: 71-82). C-PRINS includes a series of thermal cycles similar to PCR.

Sheath fluid for FISH: -BD Bioscience Standard (GM Baerlocher, I Vulto, G de Jong, PM Lansdorp. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). 2006. Nature Protocols 1, - 2365 – 2376) -40 m. M KCl + 10 m. M Na. Cl (Vrána J, Kubaláková M, Simková H, Cíhalíková J, Lysák MA, Dolezel J. Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics. 2000; 156(4): 2033-41) -Mg . So 4 buffer without dithiothreitol (Lj. Li, L. Ma, K. Arumuganathan, YC Song. Flow-sorted chromosomes: a fine material for plant gene physical mapping. Caryologia. 2006, vol. 59, no. 2: 99 -103) -autoclaved 0.1% (wt/vol) Na. Cl-50m. M Na. Cl (M Kubaláková, P Kovářová, P Suchánková, J Číhalíková, J Bartoš, S Lucretti, N Watanabe, SF Kianian, J Doležel. Chromosome Sorting in Tetraploid Wheat and Its Potential for Genome Analysis. Genetics. 2005, 170(2): 823–829) -Chromosome-stabilizing polyamine buffer (protein-containing sheath fluid) (Darzynkiewics Z, Robinson JP, Crissman H. Flow Cytometry, 2nd Ed. Part B. San Diego, CA. Academic Press, Inc. 1994)

Standard microscopes do not make it possible to examine cells at the molecular level. A powerful increase alone is not enough here. Digital imaging, additional reagents and other materials and instruments are required. For the analysis of DNA and RNA, a method is currently used, which is called In situ hybridization. Because it involves staining samples followed by radiation analysis, it is also called fluorescence hybridization or FISH.

Fluorescent In situ hybridization is widely used in genetic research, cancer diagnostics, pregnancy management, and many other areas of science. The method, as the name implies, is based on the property of DNA and RNA molecules to form stable bonds with probes, that is, to form hybrid molecules. The words "In situ" mean that all observations are made "in situ", that is, directly without the use of an additional medium.

DNA probes (probes) are complementary to the molecules in the test sample. They include nucleosides, which are labeled with fluorophores (substances that give the molecule the property of fluorescence). This method is called direct labeling; if hybrid conjugate molecules are used as markers, indirect labeling is obtained. With direct labeling, hybridization can be observed under a fluorescence microscope immediately after its completion. For indirect labeling, another staining procedure is performed. It separates the conjugate molecules from the studied samples by color.

The hybridization method with indirect labeling requires more time and reagents, but it allows you to achieve more reliable results. The signal level in this case will be higher, and in addition, its stepwise amplification is also possible. Cloned DNA sequences (PCR products, genomic DNA, labeled oligonucleotides, and others) are used as labeling probes. Probe labeling is performed in several ways. Common methods are nick translation and polymerase chain reaction (PCR) with labeled nucleotides.

The order of the procedure

The In situ method begins with a preparatory stage - designing the probes. The dimensions of the probes should not be large enough to interfere with the research process. Probes that are too small are also undesirable, as they do not guarantee reliable results. Therefore, probes up to 1 thousand bp in size are taken for research. If the probe is double-stranded DNA, then the acid is denatured before hybridization. When the desired result is obtained (certain regions of chromosomes or all chromosomes are stained), further hybridization of DNA probes with repeating sequences is blocked. To do this, unlabeled DNA repeat molecules are added to the hybridization mixture.

The next stage of research is the preparation of preparations of interphase nuclei or metaphase chromosomes. The cells are fixed in the substrate on the glass, after which the DNA is denatured. To reduce the temperature of denaturation and preserve the morphology of nuclei and chromosomes, denaturation is carried out with the addition of formadide. After that, probes are added to the material and hybridization is performed for several hours. Upon its completion, a multi-stage washing is carried out to remove probes that have not connected with the sample molecules.

A modern method of cytogenetic analysis, which allows to determine the qualitative and quantitative changes in chromosomes (including translocations and microdeletions) and is used for the differential diagnosis of malignant blood diseases and solid tumors.

Russian synonyms

Fluorescent in situ hybridization

FISH analysis

English synonyms

Fluorescence in-situ hybridization

Research method

Fluorescent in situ hybridization.

What biomaterial can be used for research?

Tissue sample, tissue sample in paraffin block.

How to properly prepare for research?

No preparation required.

General information about the study

Fluorescent in situ hybridization (FISH) in- situ hybridization) is one of the most modern methods for diagnosing chromosomal abnormalities. It is based on the use of DNA probes labeled with a fluorescent label. DNA probes are specially synthesized DNA fragments, the sequence of which is complementary to the DNA sequence of the studied aberrant chromosomes. Thus, DNA samples differ in composition: different, specific DNA samples are used to determine different chromosomal abnormalities. DNA probes also vary in size: some may be directed to an entire chromosome, others to a specific locus.

During the hybridization process, if there are aberrant chromosomes in the test sample, they bind to the DNA probe, which, when examined with a fluorescent microscope, is determined as a fluorescent signal (a positive result of the FISH test). In the absence of aberrant chromosomes, unbound DNA samples are “washed out” during the reaction, which, when examined using a fluorescent microscope, is defined as the absence of a fluorescent signal (negative FISH test result). The method makes it possible to assess not only the presence of a fluorescent signal, but also its intensity and localization. Thus, the FISH test is not only a qualitative but also a quantitative method.

The FISH test has a number of advantages over other cytogenetic methods. First of all, the FISH study can be applied to both metaphase and interphase nuclei, that is, to non-dividing cells. This is the main advantage of FISH over classical karyotyping methods (eg, Romanowsky-Giemsa staining of chromosomes), which are applied only to metaphase nuclei. This makes FISH a more accurate method for detecting chromosomal abnormalities in tissues with low proliferative activity, including solid tumors.

Since the FISH test uses stable DNA of interphase nuclei, a wide variety of biomaterials can be used for research - fine-angle aspiration biopsy aspirates, smears, bone marrow aspirates, biopsy specimens, and, importantly, preserved tissue fragments, such as histological blocks. Thus, for example, a FISH test can be successfully performed on repeated preparations obtained from a histological block of a breast biopsy specimen when confirming the diagnosis of "breast adenocarcinoma" and the need to determine the HER2/neu status of the tumor. It should be emphasized that at the moment the FISH study is recommended as a confirmatory test when receiving an indeterminate result of the immunohistochemical study of the tumor for the tumor marker HER2 / neu (IHC 2+).

Another advantage of FISH is its ability to detect microdeletions that are not detected by classical karyotyping or PCR. This is of particular importance in cases of suspected DiGeorge syndrome and velocardiofacial syndrome.

The FISH test is widely used in the differential diagnosis of malignant diseases, primarily in oncohematology. Chromosomal abnormalities in combination with the clinical picture and immunohistochemical data are the basis for the classification, determination of treatment tactics and prognosis of lympho- and myeloproliferative diseases. Classical examples are chronic myeloid leukemia - t (9; 22), acute promyelocytic leukemia - t (15; 17), chronic lymphocytic leukemia - trisomy 12 and others. As for solid tumors, the FISH study is most often used in the diagnosis of cancers of the breast, bladder, colon, neuroblastoma, retinoblastoma, and others.

The FISH study can also be used in prenatal and preimplantation diagnosis.

The FISH test is often carried out in combination with other methods of molecular and cytogenetic diagnostics. The result of this study is evaluated in conjunction with the results of additional laboratory and instrumental data.

What is research used for?

For the differential diagnosis of malignant diseases (blood and solid organs).

When is the study scheduled?

If you suspect the presence of a malignant blood disease or solid tumors, the tactics of treatment and the prognosis of which depend on the chromosomal composition of the tumor clone.

What do the results mean?

Positive result:

The presence of aberrant chromosomes in the test sample.

Negative result:

The absence of aberrant chromosomes in the test sample.

What can influence the result?

Number of aberrant chromosomes.

Immunohistochemical study of clinical material (using 1 antibody)
Immunohistochemical study of clinical material (using 4 or more antibodies)
Determination of HER2 tumor status by FISH
Determination of HER2 tumor status by CISH method

Who orders the study?

Oncologist, pediatrician, obstetrician-gynecologist, geneticist.

Literature

Wan TS, Ma ES. Molecular cytogenetics: an indispensable tool for cancer diagnosis. Anticancer Res. 2005 Jul-Aug;25(4):2979-83.
Kolialexi A, Tsangaris GT, Kitsiou S, Kanavakis E, Mavrou A. Impact of cytogenetic and molecular cytogenetic studies on hematologic malignancies. Chang Gung Med J. 2012 Mar-Apr;35(2):96-110.
Mühlmann M. Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions. Genet Mol Res. 2002 Jun 30;1(2):117-27.