Fluorescent in situ hybridization. Fluorescence in situ hybridization (FISH)

In some cases, a cytogenetic study is not enough to issue a conclusion about the karyotype; in these cases, molecular cytogenetic methods are used, in particular, fluorescence in situ hybridization (FISH).

The emergence of new technologies of molecular cytogenetics, based mainly on in situ hybridization of nucleic acids, has significantly expanded the possibilities of chromosomal diagnostics. The in situ hybridization method was developed to localize specific DNA sequences directly on cytological preparations. There was a transition in the identification of chromosomes and chromosomal regions from the analysis of the cytological organization of the chromosome to the analysis of the DNA sequences that make up them. Comparison of the effectiveness of classical cytological methods for the detection and analysis of chromosomal rearrangements, such as differential chromosome staining, with modern molecular cytogenetic technologies has shown that in hematological disorders, cytological analysis of chromosomes detects and correctly identifies only about a third of chromosomal rearrangements detected using spectral karyotyping (SKY) ... About a third of the rearrangements are incorrectly identified by cytological methods, and a third remains completely unnoticed. Classical methods of cytogenetic analysis can reveal only about 15% of chromosomal rearrangements identified using SKY.

The FISH method uses fluorescent molecules to stain genes or chromosomes in vivo. The method is used to map genes and identify chromosomal aberrations.

The technique begins with the preparation of short DNA sequences, called probes, that are complementary to the DNA sequences of the object of study. The probes hybridize (bind) to complementary regions of DNA and, due to the fact that they are labeled with a fluorescent label, allow you to see the localization of genes of interest within DNA or chromosomes. Unlike other methods of studying chromosomes that require active cell division, FISH can be performed on non-dividing cells, which makes the method flexible.

FISH can be used for a variety of purposes using three different probe types:

* locus-specific probes that bind to specific regions of chromosomes. These probes are used to identify the available short sequence of isolated DNA, which is used to prepare a labeled probe and its subsequent hybridization with a set of chromosomes;
* alphoid or centromeric repeat probes are repeating sequences of centromeric regions of chromosomes. With their help, each chromosome can be colored in a different color, which allows you to quickly determine the number of chromosomes and deviations from their normal number;
* probes for the entire chromosome are a set of small probes that are complementary to individual regions of the chromosome, but generally cover its entire length. Using a library of such probes, it is possible to "color" the entire chromosome and obtain the differential spectral karyotype of an individual. This type of analysis is used to analyze chromosomal aberrations, for example, translocations, when a piece of one chromosome is transferred to the shoulder of another.

Fluorescent in situ hybridization (FISH)

The material for research is blood, bone marrow, tumor biopsy, placenta, embryonic tissue or amniotic fluid. Samples for research must be delivered to the laboratory fresh. Slides (slides) are prepared directly from tissue samples or after culture. Both metaphase and interphase cell preparations can be used. Fluorescently labeled specific DNA probes hybridize to chromosomal DNA, and multiple probes can be used simultaneously at different loci.

FISH is a useful and sensitive method of cytogenetic analysis for the detection of quantitative and qualitative chromosomal aberrations, such as deletions (including microdeletions), translocations, doubling and aneuploidy. FISH on interphase chromosomes is a rapid method for prenatal diagnosis of trisomies 21, 18 or 13 chromosomes or sex chromosome aberrations. In oncology, FISH can detect a number of translocations (bcr / abl, MLL, PML / RARA, TEL / AML1) associated with hematological malignant neoplasms. The method can also be used to monitor the residual effects of cancer after chemotherapy and bone marrow transplantation and to identify enhanced oncogenes (c-myc / n-myc) associated with a poor prognosis for some tumors. FISH is also used to monitor the survival rate of a bone marrow allograft obtained from an individual of the opposite sex.

FISH is a sensitive method for identifying chromosomal aberrations and for one-step rapid analysis of large (> 500) cell numbers. The method is highly accurate in identifying the nature of chromosomes and unknown fragments of chromosomal DNA.

Traditional cytogenetics when studying the karyotype, it was always limited by the band resolution level. Even with the use of high-resolution chromosome differential staining methods, we only detected more bands on the chromosome, but were not sure if we were getting to the molecular level of resolution. Recent advances in DNA technology and cytogenetics have made it possible to use FISH methods to analyze changes in chromosomal DNA at the molecular level. Molecular cytogenetics has provided a revolutionary breakthrough in cytogenetics, allowing:

Analyze the structure of DNA chromosomes in the range of 10-100 kilobases;
to carry out diagnostics of non-dividing interphase cells, which had a huge impact on prenatal diagnostics and preimplantation genetic diagnostics (PGD).

FISH technology uses a DNA probe that binds or anneals specific DNA sequences within a chromosome. The denatured probe is incubated with the cell's native DNA, also denatured to a single-stranded state. The probe replaces biotin-deoxyuridine triphosphate or digoxigenin-uridine triphosphate with thymidine. After renaturation of the native DNA probe with a probe, the probe-DNA complex can be detected by the addition of fluorochrome-labeled avidin, which binds to biotin, or fluorochrome-labeled anti-digoxigenin. Additional amplification of the signal can be obtained by adding antiavidin and studying the resulting complex using fluorescence microscopy. By marking different DNA probes with several different fluorochromes, it is possible to simultaneously visualize several chromosomes or chromosomal segments within one cell in the form of multi-colored signals.

Possibility of determination specific gene segments, present or absent on chromosomes, made it possible to diagnose gene sequence syndromes at the DNA level, as well as translocations in interphase nuclei, often in individual cells.

Material for FISH can be either metaphase chromosomes obtained from dividing cells, or interphase nuclei from cells that are not in the stage of division. Sections are pretreated with RNase and proteinase to remove RNA, which can cross-hybridize with the probe and chromatin. They are then heated in formamide to denature the DNA and fixed with ice-cold alcohol. The probe is then prepared for hybridization by heating. Thereafter, the probe and chromosome preparation are mixed and sealed with a coverslip at 37 ° C for hybridization. By varying the incubation temperature or salt composition of the hybridization solution, binding specificity and background labeling can be reduced.

Application of fluorescent in situ hybridization - FISH technology

FISH technology efficiency was first demonstrated in the localization of genes on. With the introduction of the fluorescent labeling method, in situ hybridization has proven to be indispensable for the diagnosis of chromosomal abnormalities that are not detected by traditional banding methods. FISH also played a key role in one of the most extraordinary discoveries in modern genetics - genomic imprinting.

Its development technology FISH received in three forms. Centromeric, or alpha-satellite, probes are characterized by relative chromosomal specificity; they are most often used in the genetics of interphase cells. These probes generate somewhat diffuse signals of adequate strength in the centromere region, but do not cross-hybridize with chromosomes having similar centromeric sequences. Currently, single-copy probes have been developed that give a discrete signal from a specific band of the chromosome and make it possible to avoid the phenomenon of cross-hybridization. These probes can also be used to determine the copy number and specific regions of the chromosome, presumably associated with a particular syndrome. Single-copy and centromeric probes designed for chromosomes 13, 18, 21, X and Y are used for prenatal diagnosis.

It is also possible to "stain" whole chromosomes with FISH... Thanks to spectral karyotyping technology, which uses a mixture of different fluorochromes, it is now possible to create a unique fluorescent pattern for each individual chromosome with 24 separate colors. This technology makes it possible to determine complex chromosomal rearrangements that are not visible when using traditional cytogenetic techniques.

Method FISH in prenatal diagnosis. For women of older reproductive age, pregnancy can be a cause not so much for joy as for anxiety. With the age of a woman, the risk of developing fetal chromosomal abnormalities is associated. Amniocentesis performed at the 16th week of pregnancy, followed by karyotype analysis, takes 10-14 days. The use of FISH in the preliminary examination can speed up the diagnosis and reduce the waiting time. Most geneticists and laboratories are of the opinion that the FISH method should not be used in isolation to make decisions about the future management of pregnancy. The FISH method must be complemented by a karyotypic analysis, and its results should at least correlate with the pathological picture of ultrasound examination (US) or biochemical screening based on the mother's blood.

Gene syndromes sequences also known as microdeletion syndromes, or segmental aneusomy. These are deletions of adjacent chromosome fragments, usually involving many genes. Gene sequence syndromes were first described in 1986 using classical cytogenetic techniques. Now, thanks to FISH, it is possible to identify submicroscopic deletions at the DNA level, which made it possible to identify the smallest deleted region associated with the development of a particular syndrome, called the critical region. Once the critical region for a syndrome has been identified, it is often possible to identify specific genes, the absence of which is recognized as associated with the syndrome. In a recently published manual on gene sequence syndromes, 18 deletion and microdeletion syndromes associated with 14 chromosomes are reported. Some of the most common gene sequence syndromes and their clinical manifestations are shown in Table. 5-2.

Telomeres- formations that cover the ends of the long and short arms of chromosomes. They consist of repetitive TTAGGG sequences and prevent fusion of the ends of the chromosomes with each other. Telomeric probes play an important role in recognizing complex translocations that cannot be detected by traditional cytogenetic methods. In addition, one of the discoveries of the Human Genome Project was the fact that regions of chromosomes adjacent to telomeres are rich in genes. It has now been shown that submicroscopic subtelomeric deletions are responsible for the occurrence of many genetically determined diseases.

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 their complementary sequences in targets - analyzed DNA or RNA molecules. To identify areas of hybridization, a DNA sample (DNA probe) is labeled with a reporter group: ü a radioactive isotope, ü a fluorochrome, ü an enzyme that gives a color or luminescent product, ü a hapten to 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 to each other - and their exact location on the chromosomes. The method of molecular hybridization has high sensitivity (it is possible to detect a small amount of a labeled probe (10 -15 and 10 -19 M) and, accordingly, a complementary sequence in the target) and rapidity of analysis, which makes it possible to use this method both for research activities and for the diagnosis of hereditary and infectious diseases in medicine, veterinary medicine and plant growing.

The emergence of new technologies of 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. 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 to detect and localize specific DNA sequences on chromosomes, m. RNA, etc. The method is based on the hybridization of a fluorescently labeled DNA / RNA probe with a complementary DNA / RNA sequence. Identification of the label occurs using a fluorescence microscope. The FISH method was introduced over 30 years ago. It has become widely accepted as a method for physically mapping genes on chromosomes. Later, it began to be applied in other areas of research (in the fields of clinical genetics, reproductive medicine, toxicology, evolutionary biology, comparative and cell genomics, and chromosomal biology). At the moment, FISH is mainly used to build physical and genetic maps of chromosomes, to identify structural rearrangements, translocations, microdeletions, gene amplifications in interphase and metaphase chromosomes. As a result of the development of 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 (improved 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 staging FISH can be presented as follows: 1. Preparation of a histological or cytological specimen. The preparation of the histological specimen is carried out according to the standard scheme: cutting, marking, wiring, filling, microtomy, placing the section on a glass slide and dewaxing. When preparing a cytological preparation, special precipitating solutions and centrifugation are used, which makes it possible to obtain a concentrated suspension of cells. 2. Pre-processing (if necessary). The drug is treated with proteases to eliminate the presence of proteins that hinder hybridization. 3. Application of 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 preparation is cooled to a certain temperature (370 C in the case of clinical studies) and incubated in a humid chamber for several hours (the incubation duration is indicated in each specific protocol). Currently, automatic hybridizers are used for denaturation and hybridization. 5. Flushing. After hybridization is complete, it is necessary to wash off unbound probes, which, otherwise, will create a background that makes it difficult to evaluate the results of the FISH analysis. For rinsing, a solution containing citrate and sodium chloride (SSC) is usually used. 6. Counter-staining. Using fluorescent dyes (DAPI - 4, 6-diamidine-2 phenylindole; propidium iodide), all nuclear DNA is stained. 7. Analysis of the results using a fluorescent microscope. Routine operations (dewaxing, pre-treatment, washing) can be automated.

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

Advantages of the FISH method: 1. the ability to study genetic material in interphase nuclei; 2. obtaining objective results according to the principle "yes / no" is a quantitative method; 3. relatively simple interpretation of the results; high resolution. Disadvantages of the FISH method: 1. fluorescent dyes quickly "fade"; 2. high-quality fluorescent dyes are required to analyze the results. microscope.

The FISH method is based on a hybridization reaction between a DNA probe created using special technologies, which is a nucleotide sequence of limited size, and a complementary portion of the nuclear DNA of the cytogenetic preparation under study. DNA-probe - the main element for FISH staging carries a "tag", ie, contains nucleotides directly associated with a fluorochrome or hapten for further visualization with antibodies carrying a fluorochrome. Unbound labeled DNA is washed off, and then the hybridized DNA probe is detected using a fluorescence microscope.

DNA labeling is divided into direct and indirect. Direct labeling introduction into DNA of reporter elements - - fluorochromes (rhodamine, diethylaminocoumarin, Texas red, etc.). Indirect labeling method - the DNA sample is pre-conjugated with intermediate ligands (biotin, dioxygenin, 2,4-dinitrophenol), the presence of which on the cytological preparation is then detected using fluorochromes. In this case, the sensitivity of the method increases significantly, since the ligand can contain several sites for interaction with the fluorochrome. For the first time in situ hybridization with a 32 P-labeled probe was described in 1969. The development of non-radioactive systems for labeling and detecting DNA probes has made the in situ hybridization method safe, simple to implement, 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 amplify 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 Visualization of chromosomes: using DAPI (2 mg / ml). 4 ', 6-diamidino-2-phenylindole; strong binding to A-T rich DNA regions; excitation by UV light; emission 461 nm (blue).

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

Characteristics of various types of probes 1.Locus-specific probes (LSI - locus - specific identifiers, which 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 a 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, interphase nuclei of amniocytes.

2. DNA - probes for telomeric regions of chromosomes (TEL telomericprobe) are designed to detect deletions and rearrangements affecting the terminal regions of the arms of chromosomes. Such probes are specific for the p- or q-arms of chromosomes and are complementary to a region about 300 kb in length. 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 identify both telomeric regions of the chromosome on one cytogenetic preparation.

3. centromere probes-repeats, alphoid 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 mainly in centromeric or pericentromeric heterochromatin regions of chromosomes. With their help, each chromosome can be stained in a different color, which allows you to quickly determine the number of chromosomes and deviations from their normal number, 4. probes for the whole chromosome, whole chromosome probe (WCP - Whole-Chromosome-Painting are a set) of small probes, complementary to individual parts of the chromosome, but generally covering its entire length. Using a library of such probes, it is possible to "paint" the entire chromosome and obtain the 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.

Fields of application FISH is widely used to solve the following clinical diagnostic tasks: 1. Perimplantation prenatal and diagnosis of chromosomal abnormalities. It is used in in vitro fertilization (IVF) clinics and perinatal centers. Allows you to timely (before embryo implantation in the case of IVF or in the early stages of fetal development) 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 to diagnose them. 3. Diagnosis of solid tumors. Currently, more and more causal relationships are established between the development of specific oncological diseases and specific changes in the genome. Therefore, using appropriate DNA probes, oncological FISH diagnostics can be performed.

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 test, unstained smears of blood, bone marrow, or other tissue preparations are used. Stage 1 - the 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. To detect monoclonal antibodies and DNA probes, fluorophores that differ in color are used. Study of the preparation under a fluorescence microscope in the presence of the necessary set of filters allows simultaneous analysis of the immunophenotype of hybridization and signals in interphase nuclei.

Chromosomaldeletionof TNFAIP 3 in c. HL detectedby interphasecytogenetics. FICTION analyzes of. representative c. HL cases combining. 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 not only a combination of fluorophores into pseudocolors, but also their ratio, intensities

Multicolor chromosome staining (Muli. Color Banding - MCB) This method is not intended for a complete analysis of all chromosomes, but for a detailed analysis of an individual 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. The overlapping of the intensity profiles of the signals from the DNA probes provides variations in the ratios of the fluorescence intensities of various fluorophores along the chromosome. The intensity ratios can be translated into pseudocolors, and, thus, each point of the image, and, therefore, each of the chromosomal loci, will have its own pseudocolor. This version of the multicolor FISH method proved to be highly effective in the analysis of not only interchromosomal, but also intrachromosomal rearrangements in cancer. However, for its successful application, it is necessary to first determine the chromosome that will be examined. This method of molecular cytogenetics is suitable for the analysis of karyotype abnormalities associated with certain chromosomes.

To date, sets of DNA probes have been created that provide MSV for all human chromosomes Multicolor banding of the fifth human chromosome (illustration Meta. Systems Gmb. H) (in situ hybridization of region-specific DNA libraries of chromosome 5; signal profiles of region-specific DNA libraries on chromosome 5; multicolor banding of chromosome 5; idiogram of chromosome 5; fluorochromes used for MCV).

Metaphase cytogenetics, which historically emerged earlier than interphase, allows one to determine a wide range of chromosomal abnormalities, but for this it is necessary that the cells under study are at the stage of meiotic metaphase.

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

DNA probes used for this analysis consist of a mixture of solid staining probes for a complete set of human chromosomes, labeled using 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, evaluating 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 kits. The principle of M-FISH consists in separate digital registration of the signal of all used fluorochromes, which is achieved by successive replacement of filter sets. All images are recorded in separate files, which allows their efficient processing associated with separation of the signal and background, as well as quantitative assessment of the signal. Processing of all recorded information using special software translates information about the level of fluorochrome signals at each point of the image into pseudo colors. One of the main limitations of the number of used DNA probes is the number of available fluorochromes, with non-overlapping excitation and emission spectra, and the availability of appropriate filter sets.

24-color FISH M-FISH is most widely used as a 24-color FISH for the simultaneous identification of material from all human chromosomes. It is highly effective in detecting chromosomal translocations, but it is not designed to detect deletions and inversions. However, this problem can be partially solved by the simultaneous staining of chromosomes with DAPI, which makes it possible to analyze the differential striation of chromosomes, the chromosomal affiliation of the material of which has already been identified. Unfortunately, it should be noted that the quality of DAPI banding of chromosomes 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 the multicolor bar code of human chromosomes and the multicolor chromosome banding method based on interspecies in situ hybridization. In contrast to 24 color FISH, this method allows one to directly reveal some of the intrachromosomal rearrangements. DNA probes used in Rx. FISH are labeled with a combination of 3 fluorochromes, resulting in 7 pseudo colors. They specifically stain individual regions of chromosomes, creating their color striation. This is a feature of DNA probes 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 intensive chromosomal rearrangements that took place during the formation of modern species of gibbons, the material of their chromosomes turned out to be strongly 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) Layout of chromosomes.

However, RXFISH has rather serious limitations - chromosome rearrangements that took place within one color RX-band cannot be detected using this method, if they do not lead to significant and easily visible changes in the size of this band. - the probes stain several chromosomal regions of different chromosomes in one pseudo color. 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 currently include the following points: The method allows the analysis of the entire human genome in one multicolor FISH experiment. Commercially available kits of labeled DNA probes and the required detection systems are available. The method allows quick identification of a significant part of intra- and interchromosomal rearrangements. Automatic identification of “color banding” metaphase chromosomes is possible. There is a unique color bar code for each human chromosome. RXFISH is integrated into the Cyto workstation. Vision and standard fluorescence microscopy systems.

Spectral karyotyping (SKY-spectralkaryotyping) The basic principles of the analysis of microscopic images in spectral karyotyping practically do not differ from those used in M-FISH. The differences are related to the way the image is registered. SKY technology allows one measurement to obtain spectral curves for all image points, regardless of whether it is related to epifluorescence or traditional light microscopy. For spectral karyotyping of all human chromosomes, five fluorochromes are used, one in the green spectrum, two in the red and two in the infrared. Excitation and emission of all fluorochromes used for labeling DNA probes takes place with one set of filters, which avoids their sequential change, intermediate focusing, and, consequently, associated problems, such as spatial image 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 identity of the analyzed material. This ensures reliable identification (up to chromosome accuracy) of the material of marker chromosomes, as well as chromosome derivatives resulting from various rearrangements. The great advantage of SKY is that the DAPI color is recorded parallel to the spectral image. DAPI banding software improvement allows achieving differential striation in quality close to GTG banding. The possibility of parallel analysis of the spectral image and qualitative differential chromosome staining greatly simplifies the interpretation of SKY results and makes it possible to more accurately determine the points of chromosomal breaks. The undoubted advantages of SKY include the possibility of using fluorochromes with overlapping excitation and emission spectra, which significantly expands the list of suitable 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 filter set, since one filter block for spectral FISH and one filter block for DAPI is sufficient for SKY.

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

Color changing karyotyping This method is based on the analysis of the difference in signal length between DNA samples hybridized with DNA probes bound to fluorochromes and with 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 performed 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 bound to antibodies is 80 - 90% shorter than that of samples bound to fluorochromes. After the hybridization reaction, the smears are exposed to the corresponding primary antibodies, and then visualized, obtaining the first image. Then the preparations are exposed to secondary antibodies and avidin bound to the same fluorochromes. Strokes can be counter-stained 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 in the first or second image, while others will change their color in different images.

Comparative Genomic Hybridization (CGH) The method was developed to detect quantitative abnormalities in the genome. It is based on an in situ hybridization reaction using the entire genome as a DNA probe. Isolated and normal donor DNA is labeled with fluorochromes of different colors, thus converting them into DNA probes. Equivalent amounts of these probes are mixed and used in hybridization with a control cytogenetic preparation. After FISH, the metaphases are analyzed on a fluorescence microscope, and the fluorescence intensity of two fluorochromes along the entire length of each chromosome is determined using a specialized computer image analysis program.

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 a part of the genetic material, on the contrary, it will weaken. Thus, CGH allows detecting genomic imbalances, but this method cannot be used to detect balanced translocations and inversions, and trisomies and deletions can be determined only if the size of the unbalanced region is at least 10 million base pairs.

Chromosomal imbalance in a test sample is assessed by the difference in fluorescence intensity of two different fluorochromes by calculating the fluorescence ratio (FR).

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 material under study, and can be successfully carried out with a small amount of tested DNA, including archival material. Secondly, it allows you to obtain detailed information about the loss or increase in the number of copies of genetic material throughout the genome in one experiment. Thirdly, the CGH method does not require the preparation of preparations of metaphase chromosomes from the tissue under study, that is, it does not depend on the process of cell cultivation and related artifacts.

Genomic 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 intraspecific 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 for flow cytometry applications. It was originally used to measure the length of chromosomes (resolution: 200 bp) by counting the number of telomeric repeats. PNA-conjugated probes are used. Initially, we studied metaphase chromosomes (QFISH itself), now this method is applicable to interphase chromosomes (IQ-FISH). Q-FISH is performed on cell culture, tissue sections (both on slides). Currently, Q-FISH is an important tool in studying the role of telomeres in aging and cancer.

PNA-FISH - Peptide nucleic acids FISH: Peptide nucleic acids (PNAs) are synthetic analogs of DNA in which the sugar backbone of deoxyribose phosphate supporting the nitrogenous base is replaced with an uncharged peptide backbone. As a result of this structure: when the PNA oligomeric probe hybridizes with complementary DNA / RNA, electrostatic repulsion does not occur. 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 for base pair mismatch than DNA-DNA hybridization. Thus, using a PNA probe, it is possible to distinguish between two centromeric repeats that differ in only one base pair. PNA is relatively hydrophobic (compared to DNA), which means that PNA diffuses better through cell walls => widespread use in microbiology. Based on the high specificity of binding: 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: 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). Combination of Q-FISH and flow cytometry to analyze (measure) signal and sort. For flow-FISH, a cell suspension (to measure the length of chromosome telomeres), chromosomal spreads and isolated chromosomes (for further mapping) are used. As in Q-FISH, PNA-labeled telomeric probes are used (for example, for visualization and measurement of telomeric repeat length). The main advantage is the faster method (analysis / sorting of a large number of cells / chromosomes). It is widely used to study various problems: aging, preservation of telomeres, study of suspensions of hemotopoietic stem cells ex vivo, 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 result Ability to analyze subgroups of cells in a population using physical immunofluorescent and markers Better performance than Q-FISH Ability to quantify fluorescence of thousands of cells.

STUDY OF DETACHED CHROMOSOMES using flow cytometry methods 1. Sorting chromosomes using flow cytometry - Karyotyping using flow cytometry is used to further map genes and build chromosome libraries. - Identification and localization of genes on sorted chromosomes is carried out with the subsequent application of FISH / PRINS (primed in situ labeling) methods 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 pachytene chromosomes with a high division index.

Fluorescent label: - Chromosomes are labeled with fluorochromes specific for nucleic acids. - Fluorochromes are selected in accordance with 1) specificity for 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, emission: 617 nm, required laser: 488 nm-argon laser or lamp + long-pass filter) ethidiumbromide (excitation peaks : 300 and 520 nm, emission: 600 nm, required laser: 488 nm-argon laser or lamp + long-pass filter) These tags color the DNA regardless of the content of A, G, T or Nitrogen 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 (specific for A-T base pairs), excitation: 351 -364 nm, emission: 470 nm. Laser: 351 -364nm (powerful). Lasers must be separated in time and space due to partial overlap of fluorochrome spectra.

2. Study of chromosomes / nucleotide sequences after sorting (to build 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 to 30 nucleotides. FISH

FISH problems: It is difficult to localize single-locus DNA sequences (ie, non-repeating unique DNA sequences) as the signal will be very weak when using standard short probes. Increasing the length of the probe to several kilobases to amplify the signal will lead to a decrease in sensitivity and => to nonspecific binding. Solution: BAC-FISH -BAC-FISH - a combination of the FISH method and the use of clones of genomic DNA embedded in bacterial artificial chromosomes (BAC), allowing the insertion of large DNA sequences. -Effective 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 for 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 the localization of nucleotide sequences, recognition and counting of metaphase or interphase chromosomes or chromosomal pairs (including chromosomal aneuploidy). annealing of an unlabeled primer (primer - primer) with the DNA under study elongation of the primer using thermostable DNA polymerase and labeled nucleotides stopping the reaction (attachment of a blocking molecule to the 3 'end) Primer: restriction fragment PCR product oligonucleotide Labeling of nucleotides: direct - 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 glass can only be carried out after blocking the previous one. - Suitable for DNA sequences with a large number of repetitions.

Benefits of PRINS: 1. Minimal sequence information required to synthesize an oligonucleotide primer is required. 2. The fastest and simplest method for detecting the sequence of interest on the chromosome (in comparison with the use of heavy probes for FISH, hybridizing for a very long period of time). 3. Elimination of the probe tagging stage. 4. Ability to elongate the primer with labeled nucleotides to enhance the c-PRINS signal when detecting short unique sequences. To detect low-copy repeats or short unique sequences, a more sensitive method is used - cycling PRINS (c -PRINS). C-PRINS proposed by Gosden et al. in 1991, an improved protocol widely used 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 -50 m. 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)

Fluorescence in situ hybridization

Fluorescent hybridization in situ , or the FISH method (eng. 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 the spatiotemporal features of gene expression in cells and tissues.

Probes

With fluorescent hybridization in situ use DNA probes (DNA probes) that bind to complementary targets in the sample. DNA probes contain nucleosides labeled with fluorophores (direct labeling) or conjugates such as biotin or digoxigenin (indirect labeling). With direct labeling, the target-bound DNA probe can be observed with a fluorescence microscope immediately after hybridization is complete. In the case of indirect labeling, an additional staining procedure is required, during which biotin is detected using fluorescently labeled avidin or steptavidin, and digoxigenin using fluorescently labeled antibodies. Although the indirect version of labeling DNA probes requires additional reagents and time expenditures, this method usually allows a higher signal level to be achieved due to the presence of 3-4 fluorochrome molecules on the antibody or avidin molecule. In addition, in the case of indirect labeling, cascade amplification of the signal is possible.

To create DNA probes, cloned DNA sequences, genomic DNA, PCR reaction products, labeled oligonucleotides, and DNA obtained by microdissection are used.

Probe labeling can be carried out in different ways, for example, by nick translation or by PCR with labeled nucleotides.

Hybridization procedure

Fluorescent hybridization experiment scheme in situ to localize the position of the gene in the nucleus

At the first stage, the design of the probes takes place. The probe size should be large enough for hybridization to occur at a specific site, but not too large (no more than 1000 bp) so as not to interfere with the hybridization process. When specific loci are identified or when whole chromosomes are stained, it is necessary to block the hybridization of DNA probes with non-unique repeated DNA sequences by adding unlabeled DNA repeats (for example, Cot-1 DNA) to the hybridization mixture. If the DNA probe is double-stranded DNA, it must be denatured before hybridization.

At the next stage, preparations of interphase nuclei or metaphase chromosomes are prepared. The cells are fixed on a substrate, usually on a glass slide, and then DNA is denatured. To preserve the morphology of chromosomes or nuclei, denaturation is carried out in the presence of formamide, which allows the denaturation temperature to be reduced to 70 °.

Bound DNA probes are visualized using a fluorescence microscope. The intensity of the fluorescent signal depends on many factors - the efficiency of labeling with a probe, the type of probe, and the type of fluorescent dye.

Literature

Rubtsov N.B. Methods for working with mammalian chromosomes: Textbook. manual / Novosib. state un-t. Novosibirsk, 2006.152 p.

Rubtsov N.B. Nucleic acid hybridization in situ in the analysis of chromosomal abnormalities. Chapter in the book "Introduction to molecular diagnostics" T. 2. "Molecular genetic methods in the diagnosis of hereditary and oncological diseases" / Ed. M.A. Paltseva, D.V. Zaletaeva. Educational literature for students of medical universities. M .: Medicine, 2011. T. 2. S. 100-136.

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See what "Fluorescence in situ hybridization" is in other dictionaries:

This term has other meanings, see hybridization. DNA hybridization, nucleic acid hybridization in vitro combination of complementary single-stranded nucleic acids into one molecule. With complete complementarity ... ... Wikipedia

Method of determination fluorescent in situ hybridization.

Study material See description

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The study is used to select individual adjuvant chemotherapy for breast cancer or stomach cancer.

Breast cancer (BC) ranks first among oncological diseases in women. Breast tumor cells can contain different types of receptors that are sensitive to certain substances (hormones or other biologically active molecules). Depending on the presence of hormone receptors (estrogens and progesterone) or human epidermal growth factor receptor type 2 (Human Epidermal Growth Factor Receptor 2, HER2) in tumor cells, hormone receptor-positive, HER2-positive and triple negative breast cancer are isolated. It is important to take this into account in the individual selection of therapy and to predict the success of treatment.

HER2 is a receptor that is present in tissues and normally, participating in the regulation of cell division and differentiation. Its excess on the surface of tumor cells (overexpression) predetermines the rapid uncontrolled growth of the neoplasm, a high risk of metastasis, and low efficiency of some types of treatment. Overexpression of HER2 in some subtypes of breast cancer leads to increased proliferation and angiogenesis, as well as dysregulation of apoptosis (genetically programmed cell self-destruction). Currently, there are drugs that target the HER2 receptor. These are, in particular, Herceptin (trastuzumab), which is a monoclonal antibody against HER2 / neu receptors.

Amplification and overexpression of the HER2 oncogene (ErbB-2) are relatively specific events for breast carcinoma and practically do not occur in tumors of other localizations. Stomach cancer (GC) is one of the few exceptions: activation of HER2 is observed in about 10-15% of cases of malignant neoplasms of this organ and correlates with the aggressive course of the disease. In HER2-positive breast cancer, there may be an excess of HER2 receptors on the surface of the tumor cells (referred to as HER2-positive or Hercept-positive). This phenomenon is observed in 15-20% of women with breast cancer. Assessment of HER2 status is important for determining treatment tactics.

The main standardized methods for detecting HER2 / neu overexpression and / or amplification of the HER2 / neu gene are immunohistochemical (IHC) method and fluorescence in situ hybridization (FISH). Both studies are carried out on histological preparations (sections of tumor material embedded in paraffin) using polyclonal antibodies (IHC), fluorescent probes (FISH) and various imaging systems.

When evaluating the results of the IHC reaction, expression only in the invasive component of the tumor is taken into account. The assessment of the reaction results is carried out using a point scale: 0, 1+, 2+, 3+, developed by the test manufacturer and received expert approval. Hercept status, assessed as 0 and 1+, should be considered negative - there is no overexpression of the protein, which correlates with the lack of amplification of its gene. Hercept status, assessed as 3+, is positive, i.e. protein overexpression is present, which is consistent with the presence of gene amplification. Hercept status 2+ is considered to be uncertain, i.e., from the expression of the protein determined on the basis of an immunohistochemical reaction, it is impossible to confidently judge the amplification of the gene, therefore, a study is required that directly reveals the presence or absence of amplification. The FISH method is used on sections from the same sample (block) on which the immunohistochemical study was performed. In FISH hybridization, the assessment of the presence of amplification of the HER2 gene is carried out by calculating the ratio of red fluorescent (corresponding to the labeled HER2 genes) and green fluorescent signals, which mark the centromeric region of the 17th chromosome. A ratio greater than 2 indicates the presence of HER2 amplification. FISH is more sensitive than IHC, as it allows direct assessment of the presence or absence of amplification.

Material for research: paraffin block with tumor biopsy.

Attention! NECESSARILY:

HER2 / neu IHC stained glass slide
a referral from a doctor or a discharge with the results of histological and IHC studies with antibodies to Her-2 / neu

Literature

Zavalishina L.E., Frank G.A. Morphological study of HER2 status. Methodology and atlas. - M .: Ed. "Media Medica". 2006: 98.
Malignant diseases in Russia in 2011 (morbidity and mortality). Ed. IN AND. Chissova, V.V. Starinsky, G.V. Petrova. - M .: FGBU "MNIOI im. P.A. Herzen »of the Ministry of Health of Russia. 2013: 289.
Oncology. Clinical guidelines. Association of Oncologists of Russia. Ed. IN AND. Chissova, S.L. Daryalova. - M .: Ed. GEOTAR-Media. 2008: 702.
Bang Y. J., Van Cutsem E., Feyereislova A., Chung H. C., Shen L., Sawaki A. et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomized controlled trial. Lancet. 2010; 376: 687-697.
Dabbs D.J. Diagnostic Immunohistochemistry: Theranostic and Genomic Applications. Elsevier, 4th Edition. 2013: 960.
Ferlay J., Shin H.R., Bray F., Forman D., Mathers C., Parkin D.M. GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 10. Lyon, France: International Agency for Research on Cancer; 2010. Available from: http://globocan.iarc.fr.
Goldhirsch A., Glick J.H., Gelber R.D., Coates A.S., Thurlimann B., Senn H.J. Meeting Highlights: International Expert Consensus on the Primary Therapy of Early Breast Cancer 2005. Annals of Oncology. 2005; 16 (10): 1569-1583.
Kurman R.J., Carcangiu M.L., Herrington C.S., Young R.H. WHO Classification of Tumors of the Female Reproductive Organs. WHO Press, 4th Edition. 2014; 4: 316.
NordiQC. http://www.nordiqc.org.
Park D.I., Yun J.W., Park J.H. et al. Her2 / neu amplification is an independent prognostic factor in gastric cancer. Digestive Diseases and Sciences. 2006; 51 (8): 1371-1379.
Slamon D. et al. Adjuvant Trastuzumab in HER2-Positive Breast Cancer. The New England Journal of Medicine. 2011; 365: 1273-1283.