KHARKIV NATIONAL MEDICAL UNIVERSITY FACULTY OF POSTGRADUATE EDUCATION DEPARTMENT OF MEDICAL GENETICS

METHODICAL RECOMMENDATIONS OF PRACTICAL CLASS FOR THE STUDENTS OF THE 4TH MEDICAL FACULTY, OF THE 3RD YEAR OF EDUCATION (ELECTIVE COURSE) ON THE THEME: CYTOGENETIC METHODS OF RESEARCH IN CLINIC

Established at the Departments meeting _________2007

Head of the Department, MD, professor _________________ E.Ya. Grechanina

Kharkiv 2007

Theme Cytogenetic methods of research in clinic (the third course) Human cytogenetics has its origin since a discovery of Tjio and Levan in 1956 and then an independent discovery of Ford Hamerton. Adding water to suspension of mytotically dividing human cells before their fixing on the glass, they could separate chromosomes from each other and count their number that was 46. Since that moment a research of qualitative disorders of caryotype, such as monosomias, trysomias and polyploidia, which are the most common cause of early abortions and severe inherited diseases, became possible. Next years the cytogenetic investigations were added by achievements of technological progress in molecular biology and chemistry and extended knowledge about human genome. Difficult and minimal changes of chromosomes became accessible to cytogeneticists. Methodics for estimation of caryotype in cells that are not dividing became available, receiving of isolated chromosomes for molecular analysis became possible and predicted the detailed investigation of human genome, mapping of genes and indification of different mutations. General aim meeting with cytogenetic methods and their use in medical practice. Skills Specific tasks Aims of final level 1. to know an essence, types, and 1. to choose the patients for providing possibilities of cytogenetic method cytogenetic research 2. to know the region of using 2. to interpret the results of cytogeneic cytogenetic method research Place of providing classes a classroom at the Department and cytogenetic laboratory of the Kharkiv Specialized Medical Genetic Center Duration of the class 45 minutes Plan of the class Introduction. 2 min. 1. Essence and types of cytogenetic method 8 min. 2. Methods of coloring chromosomes and the essence of methods 5 min. 3. Readings and the region of using cytogenetic method 10 min. 4. Molecular and cytogenetic methods of diagnostics 10min. 5. Educational control and correction of level of knowledge 10 min. Equipment of the class computer system METASYSTEMS, slides, photos, pictures, and genetic cards.

Tasks for independent preparing and independent correction of final level of knowledge. For determination if the final level of knowledge is corresponding to necessary you should fulfill the following tasks and compare results with answers. Test tasks. . Basic enzyme, providing enzymatic synthesis of gene (DNA): 1. Cytochromoxydase 2. Revertase 3. Endonuclease 4. RNA-polymerase 5. Superoxidase B. Light stripes on chromosomes in their differential coloring is: 1. Heterochromatine 2. Euchromatine 3. Coloring mistake 4. Chiasms C. Unit of genetic code: 1. Dynucleotide 2. Triplet 3. Pyrymidine base 4. Intron D. For studying of role of genetic and environmental factors a mehod is used: 1. Clinical-genealogical 2. Direct NA-probing 3. Microbiological 4. Cytological 5. Twin E. Preparation that allowed to determinate the correct number of chromosomes (46) in human caryotype in 1956: 1. Kolchycine 2. Cytoarsein 3. Phytohemaglutinine 4. Fluorescent dyes F. Preparation of kolchycine stops the cells dividing on the stage: 1. Anaphase 2. Prophase 3. Metaphase 4. Thelophase G. Material for providing polymerase chain reaction can be: 1. chorion cells 2. microorganisms

3. biological fluids (sperm, saliva) 4. old blood spots 5. venous blood 6. embryo on the pre-implantation stage H. Providng indirect method f molecular diagnostics is possible if: 1. original gene is mapped 2. mutation is not identified 3. gene is not sequened 4. proband is absent 5. nucleotide sequences planking gene are known and there are DNA-zonds and oligoprimers for them Information that is necessary for reinforcement of knowledge level, its possible to find in the following origins: 1. . -. , . .. , . .. , . .. . -V . 2007. 534 . . 2. .. .- .: , 2002 3. .., .., .., .. . . - .: , 1982 4. .., ., .., .. - . . - .:,1989. 5. .., .., .. .-.:,19836. 6. .., .., - .. - ., , 19903. 7. : / . .. .: ,1980 8. .. 9. .. . .- .: .. 1991 10. ., . ..1, , 1989 Study the following material after adoption of basic data: 1. .., .., .., .. . - // . - 2002. - 7. .., .., .., .. .

2.

 in situ // . - 2001. -7 3. .., .., .., .. . - // . - 2000. - 8. .., .. // . - 2004. - . 3, 4. : / . . .. - , .. . - ., 2003. .., .., .., .. . // . - 2000. - 8. Chudoba I., Plesch A., Loerch T. et al. High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes // Cytogenet. Cell Genet. - 1999. - V. 84. Henegariu O., Neerema N., Bray - Ward P., Ward D. Color - changing karyotyping: an alternative to M - FISH / SKY // Nature Genetics. - 1999. - V. 23., N. 3. Rubtsov N., Karamysheva T., Babochkina T., Zhdanova N. et al. A new simple version of chromosome microdissection tested by probe generation for 24 - multi color FISH, multi - color banding (MCB), ZOO - FISH and in clinical diagnostics // Medgen. - 2000. - V. 12. Speel E., Hopman A., Komminoth P. Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S) // The Journal of Histochemistry and Cytochemistry. - 1999. - V.47, N. 3. Subero M., Ignacio J., Viardot A., Siebert R. et al. Secondary aberration in fillicle center lymphoma: evaluatio of the diagnostic power of comparative genomic hybridization (CGH) and standard chromosome analysis (CA) // Medgen - 2000. V. 12.

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Technological map of providing the class Stage Time, Educational materials min. Means Equipment Tasks Place of providing the class Classroom

1. Determination of 15 original level of preparing on the questions of cytogenetic diagnostics 2. Thematic review 55

3. Results

20

Genetic cards, Electronic Classroom, caryograms, photos, microscope, cytogenetic and pictures computer laboratory system MetaSystem Tasks Classroom

General theoretical questions of the theme. Introduction. Determination of cytogenetic method of diagnostics. Significance of cytogenetic method in clinical practice: diagnostics of chromosomal diseases; diagnostics of range of mendeling diseases connected with chromosomal instability; diagnostics of oncological diseases; mutagenic influence of chemical matters etc. on stability of genome. Types of cytogenetic methods. Methods of collecting samples for providing cytogenetic research. Methodical bases for receiving preparations of chromosomes, and investigating tissues. Methods of coloring chromosomes (routine and differential). Readings for using cytogenetic method with the aim of diagnostics of chromosomal diseases. Promethaphase analysis of chromosomes. Autoradiographic research. Method of molecular cytogenetic diagnostics. Essence of the method of fluorescent hybridization and readings to using.

Graph of logical structure of the theme Cytogenetic methods of research in clinic. Cytogenetic diagnostics

Readings

Limits

G painting

Q painting

C painting

Metaphase cytogenetics

Whole painti ng chrom osome s

Comp arativ e geno mic hybrid izatio n

Multicolored caryotyping

Caryo typing with changi ng color

Multicolore d banding chromosom es Fluorescent in situ hybridization

Combinatio n FISH with other methods

R painting

Molecular genetic researches

Classification of cytogenetic methods of research Methods of painting chromosomes Routine painting Differential painting

Region of using

Interphase cytogenetics

Adition 1. Methods of painting chromosomes. The central task in providing cytogenetic analysis is determination of caryotype. Caryotyping is the start point of whole research, determining a possibility of using all other methodical approaches. Investigation of chromosomal set can be provided using different methodics of routine coloring when all the chromosomes have the same even color (Fig. 1). Such method allows determining the qualitative disorders of caryotype and detecting some marker chromosomes without confirmation of their origin.

Fig. 1. Dycentric chromosome found with using method of routine painting As such approach is informative a little, the main method of analysis of caryotype is technique of G differential coloring chromosomes (G - bending), that Torbjorn Caspersson was elaborated in the end of 1960-s. The base of the method is the acquisition by chromosomes the pattern from light and dark stripes in processing them by trypsine and then painting by Hymza dye. The character of draught is specific for each pair of chromosomes. Dark stripes (G - stripes, G - bends) are in the places of localization of intersticial heterochromatine. Such sites dont contain the same DNA practically, and they are full of - - pairs. In prometaphase when the chromosomes are on the early stage of condensation its possible to count about 2000 stripes, in other cases 400-800 stripes (Fig. 2). This method of painting allows identifying the chromosomes, detecting the deletions, inversions, insertions, translocations, fragile sites and more completed rebuilding. Pre-processing of cytogenetic preparations precedes to painting by the G - method. In connection with methodic it can be an incubation in buffers without Ca2+ Mg2+, in t <= 37 or t >= 60; incubation in solutions of proteolytic enzymes (trypsine, pepsine and others); incubation with deproteinized matters (urea, 2-merkaptoetanol); incubation in standard salt solution (SSC) under action of alkaline and high temperature. There are different methodics of G differential coloring chromosomes, but as a rule GTG (pre-processing by trypsine, coloring by Hymza dye) is used. Reaction of trypsyne provides transition of regular spiral structure to irregular segmentation that creates obstacles to perception of Hymza de in G negative sites and increases it in G positive segments of chromosomes.

Fig. 2. Segment of metaphase plate (G differential painting chromosomes of leukocytes of peripheral blood) R - method allows getting diametrical draught of chromosomes, thats converse the same in G painting (light R stripes are corresponding to dark G - stripes and vice versa). Preparations, received in R coloring, are analyzed by phase contrast microscopy. In getting of R draught of chromosomes for pre-processing the balanced salt solution of Arl is used in t = 78 - 96, and also in specific indexes of and time of exposition, which are various in different methodics. Preparing of preparations is more completed than in G differential coloring, because the pre-processing by ()2 in 60 or N and formaldehyde are often used. If the painting on R method provides in lower indexes of solutions and with longer exposition, telomeric regions will be colored only ( - method).Q differential coloring chromosomes is received with using of fluorochromes, exciting by blue-violet light, imitate red, yellow and orange lighting. Among the dyes usually acrihin is used, but preparations are more qualitative in painting by acrihin - iprit or acrihin - propyl; a bit worse result in using of derivative bibenzymidazolum Hoechst 33258. Much fluorescenting chromosomal sites are corresponding to dark G - discs; difference is that the secondary pulling of chromosomes 1 and 16 are colored by G method only, but intensity of painting segments of chromosomes 3, 4, 13 - 15, 21 - 22 and Y is weaker in using Q method. If we put a cytogenetic preparation in alkaline and then stay it in SSC solution in t = 60 - 65 for a long time, the structural chromatine of pericentromeric regions will be colored only. This approach that received the name of - method is used for detecting pericentric inversions. Appearance of new technologies of molecular cytogenetics that are based mainly on in situ hybridization of nucleic acids, increased possibilities of chromosomal diagnostics significantly.

Addition 2. Molecular cytogenetic methods of investigation

Interphase cytogenetics
1. Multiple banding of chromosomes ()

2. Fluorescent in situ hybridization (FISH) 3. Combination FISH with other methods

o o o

Cytology + FISH Hystology + FISH Immunophenotyping + FISH (FICTION)

Metaphase cytogenetics 1. Whole coloring chromosomes (Whole painting) 2. Comparative genome hybridization (CGH)
3. Caryotyping with changing color ()

4. Multicolored caryotyping
o o

Spectral caryotyping (SKY) Multicolored FISH (M - FISH, M - BAND)

Interphase cytogenetics allows providing chromosomal analysis on the stage of interphase, when they are in despringed condition, because hybridization in situ is independent on the phase of cellar cycle. Such research is impossible using methods of classical cytogenetics. This new approach to studying caryotype gives a possibility to make an objective estimation of dimensions of cellar clone, carrying chromosomal abberation, because the cellar populations become available for investigation. Method of fluorescent in situ of hybridization (FISH) was elaborated for detecting of specific sequences of DNA of cytological preparations. It allows pass to analysis of DNA sequences of chromosomes. The base of the FISH method is reaction of hybridization between DNA-probes, created on special technologies and presented the nucleotide sequence of limited size, and complementary site of nuclear DNA of investigating cytogenetical preparation. DNA-probe carries a mark- contains nucleotides connected with fluorochrome directly or with hapten for further visualization by antibodies carrying fluorochrome. Not connected marked DNA is washed and then detection of hybridized probe is provided with using fluorescent microscope. Firstly in situ

hybridization with 32P-marked probe was described in 1969. Improving methods of marking DNA-probes, providing hybridization and detection of marked DNA on cytological preparations allow passing to localization of unique sequences. Elaboration of non-radioactive systems of marking and detecting DNA-probes has done the method of in situ hybridization safe, simple for providing and processing results. Development of technologies of digital record of microscopic pictures and their computer elaboration resulted in creation of new approaches to visualization and identification of chromosomal material. At present time there are some basic approaches using by modern molecular cytogenetics: 1. Identification of material of long chromosomal regions and whole chromosomes. 2. Detection of specific DNA sequence in interesting region. 3. Analysis of disorder of balance of single chromosomal regions. For identification of material of chromosomal regions and whole chromosomes chromosome-specific and region-specific probes are used (painting probes), coloring the whole chromosomes or chromosomal regions in using FISH methodics. Parallel development of methods of in situ hybridization, creation and detection of DNA-probes and improving epyfluorescent microscopy, digital record and computer processing of micro pictures resulted in appearance of new technologies allowing using some dozens DNA-probes in one experiment. DNA-probes are central and leading link in FISH providing because determination of chromosomal anomaly is possible if corresponding DNA-probe is available. DNA-probes for centromeric chromosomal sites (CEP - centromeric probe) work for identification of centromeric regions of chromosomes and detection of quantitative disorders of caryotype both in interphase and metaphase. They contain short nucleotide sequences that are complementary to multiple chromosomespecific satellite DNA - repeats, localized at centromeric chromosomal site. Pan centromeric probes allow detecting the centromeric regions of all chromosomes and they are used for detection of aneuploidia, polyploidia, dycentric chromosomes and other complex aberrations. Its possible to use individual centromeric probes to some chromosomes if investigating pathology is connected with quantitative disorders of the chromosomes (monosomias, trysomias). To exclude cells with haploid and polyploid chromosomal set in analysis the control centromeric DNA-probe for any chromosome without diagnostic meaning is used. DNA-probes to telomeric sites of chromosomes (TEL - telomeric probe) are intended for detection of deletions and rebuildings affected the ending sites of chromosomal arms. Such probes are specific for - or q- arms of chromosomes and complementary to the site that is 300 ... from the end of chromosome. As a rule, DNA-probes for short and long chromosomal arms are connected with different fluorochromes that allow detecting both telomeric chromosomal sites on one cytogenetic preparation. To specify differential diagnostics of deletions and monosomias its proposed to use the DNA-probe to centromeric site of investigating chromosome connected with fluorochrome that is differ from

fluorochromes of telomeric DNA-probes. Locus-specific DNA-probes (LSI - locus - specific identifiers) hybridize with unique sequences of DNA. They are intended for detecting significant chromosomal aberrations in different pathological conditions. On the base of interphase FISH analysis the integration of molecular cytogenetics with other methods occurs. The material can be not only cytogenetic preparations but also standard colored blood and medullar smears, or traces of lymphonodes and hystological material stored in the form of paraffin blocks. Combination FISH with morphological methods of investigation allows comparing cytogenetic and morphological peculiarities of cells directly. Unfortunately, such method isnt enough standardized for wide using in clinical diagnostic researches. In 1992 Weber-Matthiesen et al. had proposed using the combination of methods of immunophenotyping and for investigation of turnoral cells called FICTION (Fluorescence Immunophenotyping and Interfase Cytogenetics as a Tool for Investigation of Neoplasms). For such analysis uncoloured blood and medullar smears or preparations of other tissues are used. On the first stage the preparations incubate with specific monoclonal antibodies and then conjugate with fluorochromes for further visualization of antigen-monoclonal body complex. Then they provide hybridization in situ with DNA-probes. Different colored fluorochromes are used for detection of monoclonal antibodies and DNA-probes. Investigation of preparation by fluorescent microscope with necessary set of filters allows providing analysis of immunophenotype and hybridization signals in the interphaze nuclei. Digital record of micro images has opened a possibility to transfer both combination of fluorochromes and correlation of their intensity into pseudo colors. The method was productive for elaboration of the method of multicolored chromosomal banding (MultiColor Banding - ). It provides not the whole chromosomal analysis but the detail analysis of a single chromosome. Regionspecific DNA tests are marked by different fluorochromes of combination of fluorochromes. The level of signal of each DNA-probes varies on intensity achieving maximum in the centre and falling practically to zero on its limits. Recovering of profiles of intensity of the signals of DNA-tests provides variations of correlations of intensity of fluorescence of different fluorochromes along the chromosome. Relations of intensities can be transferred to pseudo colors and each point of imaging. Thus, each chromosomal region has its own pseudo color. Such variant of multicolored FISH has high effectiveness in analysis both interchromosomal and intrachromosomal rebuilding in turnoral diseases. However, for its successful providing its necessary to determine a chromosome previously for investigation. Thus, the method of molecular cytogenetics is suitable for analysis of disorders of caryotype associated with specific chromosomes. At present time there are sets of DNA-probes for providing MCB of all human chromosomes (Fig. 3). Due to saving of aerial chromosomal organization on all

stages of cellar cycle, MCB allows providing analysis of chromosomal reorganization both in interphase nuclei and metaphase nuclei.

Fig. 3. Multicolored banding of human chromosome 5 (in situ hybridization of region-specific of DNA-libraries on chromosome 5; profiles of signals of region-specific of DNA-libraries on chromosome 5; multicolored banding of chromosome 5; ideogram of chromosome 5; fluorochromes, used for ). Metaphase cytogenetics that historically appeared earlier than interphase, allows determining a wide spectrum of chromosomal disorders, but its necessary that the investigating cells are at the metaphase of meiosis. Method of comparative genome hybridization (Comparative Genome Hybridization - CGH) was created for detection of quantitative disorders in genome. It is based on the providing reaction of hybridization in situ with using whole genome like DNA-probe. Extracted and normal donor DNA is marked by different colour fluorochromes converting them into DNA-probes. Equivalent numbers of these probes are mixed and used in hybridization with control cytogenetic preparation. After providing FISH metaphases are analyzed by fluorescent microscope determinating with the help of specialized program of computer analysis of image the intensity of fluorescence of two fluorochromes along each chromosome. In absence of quantitative changes in caryotype of investigating sample a correlation of intensity of lighting of two fluorochromes is watched. In the case of amplification of genes the intensity of the signal of corresponding fluorochrome is increasing, but in the case of partial loosing genetic material it is decreasing. Thus, CGH allows detecting a genome dysbalance. But this method cant be used for finding balanced translocations and inversions, and trysomias and deletions can be determined in the case if the size of imbalanced site is not less than 10 mln. pairs of nucleotides. Multicolor caryotyping has found a wide using for determination of origin of different marker chromosomes in turnoral haematological diseases. Such analysis is provided in using DNA-probes of whole painting to arms or whole chromosomes (probes WCP - whole chromosomal painting). Such probes are hybridized with multiple short DNA sequences situated along the chromosome. Thus, in studying by fluorescent microscope a chromosome looks like whole

colored in specific color. Technique of whole chromosomal painting became a reason for new type of caryotype research multicolour caryotyping. DNA-probes using for the analysis consist of mix of probes of whole painting for whole human chromosomal set marked with the help of combination of some fluorochromes, correlation of which is chosen for each chromosomal pair. Using corresponding methods of registration and computer programs of analysis of image estimating the intensity of lighting of all fluorochromes for each point of image allows providing caryotyping in which each chromosomal pair has its unique pseudo color. Simultaneous using n number of fluorochromes allows providing simultaneous analysis of 2n-1 DNA-probes. Combination using of direct- and hapten-marked DNA-probes allows decreasing number of fluorochromes necessary for visualization in metaphase of material of all human chromosomes to three. But the version of multicolour FISH is more complicated. There are different variants of multicolour FISH for decision of different tasks in cytogenetic diagnostics. At present time there are two basic types of providing pseudo multicolor images. In the simplest variant the sets of specific combinations of exciting and closing filters and apparatus for black and white digital registration of signal are used. Modern technique is able to provide registration both signal intensity and its spectral characteristics. Such technical equipment is the base of spectral caryotyping (SKY). M-FISH (multitarget, multifluor, multicolor or multiplex FISH) is general name of traditional multicolour FISH with using sets of fluorochrome-specific filters. Principe of M-FISH is separated digital registration of signal of all information and using fluorochromes in change of filter sets. Processing of whole recorded information connected with separating signal and background and also quantitative estimation of signal is conducted with the help of special program providing that transfers the information about level of signals of fluorochromes in each point of image to pseudo colors. One of the main limits of the number of using DNAprobes is the number of available fluorochromes with uncovering spectra of exciting and emission and corresponding filter sets. 24-color FISH (variant of M-FISH) has the widest spreading. It is intended for simultaneous identification of material of all human chromosomes. It has high efficiency in detection of chromosomal translocations because each chromosomal pair is colored into its pseudo color, but it doesnt allow detecting deletions and inversions. Such problem can be partially solved by simultaneous painting chromosomes DAPI that gives a possibility of providing analysis of differential draught of chromosomes. Unfortunately, the quality of DAPI-banding of chromosomes after M-FISH is worse than GTG-differential painting and DAPI-

banding

after

usual

in

situ

hybridization.

Method of multicolor chromosomal banding (RxFISH) is based on the intertype in situ hybridization. It allows detecting a part of interchromosomal rebuilding providing analysis of whole human genome in one experiment. DNA-probes, using in RxFISH, are marked by combination of three fluorochromes that provides 7 pseudocolors. They paint specifically single regions of chromosomes, creating their coloured draught. Such peculiarity of DNA-tests for RxFISH depends on type of their getting. These are chromospecific DNA libraries of two types of hylobates: Hylobates concolor and Hylobates syndactylus (Fig. 4). As a result after intensive chromosomal rebuilding in formation of modern types of hylobates the material of their chromosomes was mixed a lot compared with organization of human chromosomes. In hybridization the DNA sites of different chromosomes of hylobates connected with different fluorochromes connect with complementary DNA sites on the same human chromosome creating its colored draught in investigation with using fluorescent microscopy (Fig. 5). Unfortunately, providing RxFISH has enough serious limits of chromosomal rebuilding. Those which had place inside of one colored Rx-band cant be detected by this method if they dont lead to significant and visible changes of its size. The probes paint some chromosomal regions of different chromosomes into one pseudo color. Also there is a lack of RxFISH such as a big size of many colored bands, but each of human chromosomes 15, 18, 19, 21, 22, x and Y presents one colored band. Using large number of fluorochromes and new DNA-tests in future can significantly increase abilities of the method. But in increasing number of using fluorochromes RxFISH will be more informative than routine 24-colored FISH.

Fig. 4. Colored draught of human chromosome 1 in RxFISH.

Fig. 5. Colored draught of human chromosome in RxFISH. ). Metaphase plate. b). Chromosomal distribution The main principles of analysis of microscopic images in spectral karyotyping (spectral karyotyping - SKY) dont differ from using in M-FISH. The differences are connected with the type of registration of image requiring correct describing spectral characteristic of object. In one measure there are all spectral curves for all points of image. For spectral karyotyping of all human chromosomes 5 fluorochromes are used: one is in green spectrum, two are in red spectrum and two are in infrared spectrum. Exciting and emission of all fluorochromes using in marking DNA-probes is provided in one filter set that allows avoiding their sequent changing, intermediate focusing and problems connected with it, such as aerial moving of image, determination of cut-off values and segmentation masks. In one act of exposition for each point of image the whole spectral characteristic of light is recorded. On the base of analysis of spectral curves an availability and absence of specific fluorochromes in the point is determinated. The next step is a procedure of classification conducting with the help of special program providing that allows determining chromosomal belonging of analyzing material. The great advantage of SKY is that painting DAPI is registered parallel with spectral image. The possibility of parallel analysis of spectral image and qualitative differential chromosomal painting simplify significantly an interpretation of results of the SKY and allow determining correctly the points of chromosomal gaps. Classical methods of cytogenetic analysis allow detecting about 15% chromosomal rebuildings identifying with the help of SKY. This technology is highly effective for analysis of FISH results in interphase nuclei (spectral FISH). Next advantage of the SKY is possibility of using fluorochromes with covering spectra of exciting and emission that extends the list of fluorochromes that are suitable for using and increases the number of using fluorochromes. For providing SKY a block of filter for spectral FISH and a block of filters for DAPI are necessary. The lack of the SKY is long time for record of microscopic images. Also it can be waiting that SKY is a bit less effective than -FISH in providing investigations with small-size DNA-probes.

Color - changing karyotyping is proposed by range of authors like alternative method to M-FISH and SKY. This method is based on analysis of difference of signal length between samples of DNA hybridized with DNA-probes connected with fluorochromes and DNA-probes carrying antibodies. This method requires 3 filters only and doesnt require a special ward and program providing. For identification of chromosomes hybridization is provided once only and two images are received. The base of the method is difference of the length of fluorescent signal because the time of exposition of DNA-tests connected with antibodies is less on 80 - 90% than in tests connected with fluorochromes. After reaction of hybridization the smears are exhibited with corresponding primary antibodies and then visualization is provided receiving the first photo. Then the preparations are exhibited with secondary antibodies and avidine connected with the same fluorochromes. The smears can be contrpainted using DAPI. Then the photos of preparations are made with using three filters. These images are exhibited with using special program providing and the second photo with visible chromosomes connected with specific antibodies is made. Thus, some chromosomes will fluorescent on the first or the second photo only, but other ones will change their color on different photos. From written above the conclusion can be done that the fullest analysis of karyotype (determination of disorders of chromosomal set, diagnostics of translocations, deletions, duplications, inversions and insertions) can be provided by different methods of differential painting (G- and R-banding), and also RxFISH and SKY in combination with DAPI, that allow to get finally the diametrical chromosomal draught. Methodic FISH with using locus - specific DNA probes is suitable perfectly for detection of specific chromosomal aberrations that are important for the diagnostics and prognosis. For qualitative changes of genetic information the CGH method can be used, however it doesnt allow to determinate localization of the sites with amplified and deleted genes. As a result, the type of methodic depends on the tasks of investigators. Addition 3 Readings for providing cytogenetic investigations Readings for cytogenetic investigations are enough wide, especially in obstetricalgynecological practice. Below there is a list of conditions in which its necessary to provide a cytogenetic investigation of a patient (proband) or, if its necessary, of his relatives with diagnostic aims. 1. Suspicion on chromosomal disease on clinical symptomatic (for confirmation of diagnosis). 2. Multiple inborn developmental defects in a child (not genic syndrome); + the childs parents have multiple developmental defects.

3. Repeated (more than two) spontaneous abortions, mortinatality or natality of children with inborn developmental defects. 4. Disorder of reproductive function of unclear genesis in females and males (primary amenorrhea, sterility.); anomalies of sexual development in males and females. 5. Essential mental and developmental retardation in a child. 6. Prenatal diagnostics (late reproductive age, translocation in parents, previous child with chromosomal disease). 7. Suspicion on syndromes characterized by chromosomal instability (register of chromosomal aberrations) 8. Leucosis (for differential diagnostics, estimation of treatment efficiency and prognosis). 9. Estimation of mutagenic influences (radiation, chemical affections). Addition 4 Karyotyping in sterility is read in following cases: 1. Non-obstructive azoospermia
2.

Severe oligozoospermia (<5 mln/ml)

3. Delay of sexual development 4. Primary amenorrhea

5. 6.

Secondary amenorrhea (premature menopause)

Habitual abortion of early term of pregnancy (two or more spontaneous abortions at the first trimester of the pregnancy) both parents are investigated. Families with gravitated obstetrical anamnesis (habitual abortion, especially of early terms, mortinatality, multiple inborn developmental defects (MIDD) chromosomal disorders - 5 - 15% cases. 7. Investigation of donors of sperm and ovums. Addition 5 Sister chromatid exchane (SCE) Genotoxic action of chemical matters in vivo is often accompanied by destabilization of genome and its rebuildings. If the contact with mutagens leads to formation of the DNA lacerations, in the process of their reparation the exchanges of homological sites of sister chromatids in the interphase nuclei are watched. Their frequency is the measure of mutagenic interaction for cells. There

were elaborated the methods, which allow finding SCE in somatic cells of animals and plants. Cells (lymphocytes of peripheral blood usually) incubates in vitro in accompanying 5-bromdezoxiuridine thats included into DNA instead of timidine for two cellar cycles. One of the chromatids includes the analogue of nucleoside into both DNA chains and another one includes it into one DNA chain only. Fluorescent dye has different interaction with such homological chromatids that can be detected on intensity of painting with the help of microscope one homological chromatid looks lighter than another one. In SCE light and dark sites in chromatids of individual chromosomes are sequenced and the number of such sites is the measure of SCE frequency. The higher SCE the more intensive was predicative mutagenic interaction on the somatic cell. Determination of SCE can be used for retrospective estimation of mutagenic interaction because SCE are prolonged for some time after initiating action of mutagen in its absence, for example, for removal of origin of ionizing radiation of whole detoxication of xenobiotic.