WO2000032810A1 - $i(in situ) hybridization method using probes containing dendrimer structures - Google Patents

Abstract

The invention relates to an in situ hybridization method which is characterized in that at least one nucleic acid probe is used for the in situ hybridization which probe comprises at least one dendrimer that is labeled with one or more labeling molecules.

Description

 IN SITU HYBRIDIZATION PROCESSES WITH PROBES CONTAINING DENDRIMER STRUCTURES

The invention relates to an in situ hybridization method and especially in situ hybridization methods using nucleic acid probes, in which the label is bound to the nucleic acid sequence via at least one dendrimeric structure.

The in si tu hybridization (ISH) or, synonymously, in si tu cytohybridization represents a special application of nucleic acid hybridization, in which the hybridization reaction in tissue sections, tissue culture cells, chromosome preparations and cell nuclei, each by means of appropriate pretreatment on a solid support , e.g. a slide that has been fixed.

For this purpose, DNA or RNA probes are currently used which have been labeled with hapten (e.g. biotin or digoxigenin), fluorescence-labeled nucleotides (dNTPs) or also radioactively labeled nucleotides. The probes are usually labeled using "nick translation", "random priming" or the polymerase chain reaction (PCR). In the case of labeling with haptens, such as biotin or digoxigenin, the hybridized probes are detected by means of fluorescence-coupled "reporter molecules" (e.g. avidin or antibodies against digoxigenin). If fluorescence-labeled dUTPs are used, the detection is carried out directly in the microscope, for radioactively labeled probes, for example, by means of X-ray film emulsions with which the slides are coated after the hybridization reaction has taken place.

For example, fluorescence in situ hybridization (FISH) on chromosomes and tissues is currently used in particular in various fields of basic biological research and medical diagnostics. The in situ hybridization method not only allows the molecular homology of a probe to be determined for a specific genome section, but also with the help of the probe to localize the homologous DNA or RNA sequence spatially in the cell cluster, in a cell or within a set of chromosomes . This is the most important difference to classic molecular biological hybridizations (eg blot hybridizations, such as "Southern blot" or "Northern blot", among others).

Compared to the classic molecular biological hybridization experiments that are carried out on isolated DNA, the detection of complementary DNA sequences within a cell or chromosome cluster is more complex and complex. For example, sophisticated and complicated techniques are required for the appropriate sample preparation. Another important point is that the actual hybridization reaction takes place in a much more complex, since biological or essentially biological system. Even after denaturation for the dissociation of the DNA double strands, the DNA is essentially in its biological environment, that is to say surrounded by histone proteins, cell membranes and other cellular components. These cell components present in the vicinity of the DNA interfere with the hybridization reaction, for example by being able to prevent or restrict the access of the nucleic acid probes to the DNA.

In recent years, fluorescence-labeled DNA probes of different complexity have been developed, which are able to "stain" the smallest chromosome regions, larger chromosome sections, or entire chromosomes. These DNA probes are also known as "chromosome paints". The possibility of detecting a DNA probe depends directly on the intensity of the fluorescence signal and the sensitivity of the microscopic procedure. The more fluorescent molecules the probe contains, the stronger the signal. The sensitivity of the recording of the signals can be increased by highly sensitive camera systems, eg cooled "charge coupled device" (CCD) cameras, such as these be used for astronomy, and increased by support by means of digital image processing.

The use of differently labeled fluorescent probes in the same experiment allows FISH signals to be displayed in different colors. Different combinations of haptens and / or directly labeled probes can be used ("combinatorial labeling"). In another approach, the differently labeled probes can produce differently colored chromosomes not only through different combinations but also through different concentrations of the fluorescent labeling molecules ("ratio labeling"). With both methods it is in principle conceivable to display all 24 different chromosomes of the human chromosome set in different colors. So far, however, this has only been shown for "combinatorial labeling" (Speicher et al., Nature Genet 12: 368-375, 1996; Schröck et al., Science 273: 494-491, 1996; see also US Pat. No. 8, 640 , 657).

In comparison to "combinatorial labeling", in which a separate fluorescence filter is required for each color, "ratio labeling" can be used with less complex microscopes in order to arrive at the same number of different colors. The generation of precise and reproducible ratios of the respective marking intensities with classic marking techniques is difficult or impossible. To date, there have been no published experiments in which ratio labeling has been successfully demonstrated for all 24 different human chromosomes.

In the above-mentioned common labeling systems, labeled dUTPs are added to the reaction for the synthesis of the nucleotide probes. To ensure the incorporation of these molecules, they are offered in very high concentrations, but only a fraction is actually incorporated. Nucleotides that are not installed are discarded. The labeled dUTPs, however, account for more than 90% of the material costs in the manufacture of labeled DNA probes. The low installation rate is therefore an essential factor for the extreme cost of FISH experiments. A significant reduction in production costs could be achieved if the insertion of labeled nucleotides can be drastically improved.

The previously common methods for labeling the probes have the further disadvantage that the extent of the labeling can only be controlled inadequately, e.g. if two different haptens are to be installed in the same probe. A precise, reproducible "ratio labeling" is not possible within the scope of such markings.

The chemistry of polyfunctional organic "starburst" polymers (cascades, silvans, arboranes, dendrimers) has developed into an important branch of polymer science since the late 1970s. What these polymers have in common is that they have a plurality of organic molecular chains which extend outward from a common center. Two basic strategies have been proposed for their synthesis: a divergent synthesis, in which the structure is built from the center towards the periphery, and a convergent synthesis with growth of the molecule from the periphery to the center.

The structural concept underlying these "starbursf polymers" was also taken up in the field of biotechnology, in connection with the possibility offered by these structures to provide a plurality of marking molecules on a single molecular basic structure and thus to achieve an amplification of a marking signal.

For example, oligonucleotides that have been labeled using dendrimers were used to amplify signals in molecular-biological hybridization experiments on isolated nucleic acids. Urdea et al. (Gene 61: 3, 253-264, 1987) used this system to increase the sensitivity of the detection of hepatitis B viruses in human serum. Shchepinov et al. (Nucl. Acid Res. 25:22, 4447-4454, 1997) synthesized oligonucleotides and radioactively labeled using dendrimers and achieved signal amplification when using these oligonucleotides as probes in hybridization assays on "oligonucleotide arrays". In this work it could also be shown that Ddrimer-bearing oligonucleotides can be used as primers in PCR reactions.

To the knowledge of the applicants, hybridizations using nucleic acid probes in which a plurality of labeling molecules are bound to the nucleic acid sequence available for hybridization via a dendrimeric structure were thus only carried out on isolated nucleic acid molecules, that is to say in purely molecular-biological hybridization methods.

As already explained above, in comparison to purely molecular-biological hybridization methods in in-situ hybridization methods there is a much more complex reaction system in which, in particular, macromolecular cell components, such as histone proteins and cellular membranes, are present in addition to the DNA to be analyzed. These macromolecular cell components, some of which are even after denaturation to separate the DNA duplexes are in close association with the DNA to be analyzed, interfere with nucleic acid probes and, if the probes are unsuitable, can lead to hybridization with the cellular DNA to be analyzed not taking place despite complementary sequences. Reasons for the unsuitability of a nucleic acid probe for hybridization in situ can be, for example, steric and / or electrostatic properties of the probe molecule. However, the entire prerequisites that must be present for a nucleic acid probe in order to make it suitable for in-situ hybridization have not yet been researched to such an extent that it would be possible to predict an appropriate suitability if the molecular structure of the nucleic acid probe were known.

A simple conclusion from the suitability of a nucleic acid probe for purely molecular biological hybridization experiments on "isolated" DNA for a corresponding suitability for in situ hybridization methods is therefore not possible.

Compared to the relevant state of the art in connection with in situ hybridization methods, the object of the invention is to provide an alternative in si u hybridization method which has a number of advantages compared to the known methods: when using equivalent amounts signal amplification of nucleic acid probe (s) is made possible; the incorporation of marker molecules into the nucleic acid probe can be controlled more precisely by incorporation into the dendrimeric structure and the amount of signal amplification can thus be varied according to individual needs; due to the specifically possible incorporation of labeling molecules, there is also the possibility of labeling a nucleic acid probe with more than one type of labeling molecule in labeling ratios which can be varied according to individual requirements, which enables a more reproducible and more widely applicable ratio labeling; Due to the different synthetic strategy for the production of the nucleic acid probes, the immense costs due to the low incorporation rate of the labeled dNTPs into the nucleic acid probes for the Si u hybridization methods of the prior art are significantly reduced.

A method is described for the first time in which nucleotide probes, to which dendrimers labeled with labeling molecules such as haptens, fluorescent dyes and radioactive compounds are attached, are used in in-situ hybridization and in particular in molecular cytogenetics and histology. The invention relates to the in situ hybridization methods and kits for use in these methods as defined in the claims.

Due to the complex composition of the hybridization sample with macromolecular cell components that are closely associated with the DNA to be analyzed and in view of the extremely high steric space requirement of the nucleic acid probes used according to the invention due to the protruding dendrimeric structures, it was completely surprising to determine the suitability of these nucleic acid probes for in situ hybridization. As explained, this suitability could not be expected on the basis of the hybridization experiments in solution known from the prior art or of oligonucleotides on oligonucleotide arrays, since in these experiments the DNA to be analyzed was always isolated from other cellular components.

In the procedure proposed here

Complex probes (e.g. using "degenerate oligonucleotide primed PCR, Telenius et al., 1992, Genes, Chromosomes & Cancer 4: 257-263) containing dendrimers are prepared and

• These probes are hybridized to complex biological structures such as cells, cell nuclei and chromosomes.

The general structure of the nucleotide probes used according to the invention comprises three basic components:

a polymer chain which is able to recognize DNA sequences as complementary,

one or more polyfunctional dendrimeric structures, each of which is based on a central branching structural unit and is composed of a plurality of monomeric branching units and which are also referred to below as dendrimers or "trees", and

- One or more labeling molecules, for example hapten molecules and hapten-labeled compounds, fluorescent marker molecules or radioactively labeled compounds, which are attached to the outer ends of the dendrimers.

The polymer chain The polymer chains have the function of specifically recognizing certain polynucleic acid chains (RNA or DNA) on the basis of their nucleic acid sequence. Although the polymer chain will often be built up from the four monomeric building blocks of DNA or RNA or hybrids from the DNA and RNA building blocks, there is the possibility that the polymer's chemical composition differs significantly from these classic nucleic acid building blocks. Possibilities of variation of the polynucleic acid chains lie on the one hand in the modification of the purine or pyrimidine bases, for example by generating the following building blocks: 5-methylcytosine, 5-bromocytosine, 5-ethynyluridine, 5- (1-propynyl) -2'-deoxyuridine , 2, 6-diaminopurine, 2-deoxyuridine, 2-deoxy-inosine, 2-deoxynebularine, 3-nitropyrrole, 5-nitroindole; in the modification of the ribose ring, for example to produce 2′-0-methyl and 3′-deoxy derivatives, and also in the modification of the covalent bond between the sugar residues, the backbone of the polymer strand. In these backbone modifications, the phosphorus atom can be preserved and the oxygen atoms can be partially replaced, for example, by sulfur atoms, methylene, dialkylamino, methoxy, ethoxy groups. An example of this are phosphothioate oligonucleotides. The entire phosphorus diester bridge can also be replaced by other functional units, such as carbamates, urea, guanidines, amines, amides, ethers, thioethers, ketones, acetals, hydroxylamines or sulfamates.

A completely alternative concept is the construction of the polymer from so-called PNAs or peptide nucleic acids. These are achiral, uncharged polyamide nucleic acids, which act as oligonucleotide analogs and which consist of a polyamide backbone made from repeating aminoethylglycine units. The purine or pyrimidine bases are covalently bound to the backbone via an acetyl spacer.

The polymer chain can likewise be constructed as a hybrid of the monomeric building blocks, for example DNA, RNA, PNA and O-methyl-RNA, and optionally have one or more different ones of the further modifications explained above by way of example. In connection with the present invention it is essential that the resulting polymeric chain has the ability to specifically recognize a particular sequence section of RNA or DNA, ie to hybridize with it. This is the case when the polymeric chain can spatially align the optionally modified purine and pyrimidine bases so that they can base pair with complementary purine and pyrimidine bases of the DNA or RNA strand to be analyzed.

The dendrimers

The dendrimers can be covalently attached to the 5 'end of the polymer chain, internally via a modified base or via the backbone. The degree of branching of the dendrimers and ultimately also the number of functional groups that are available for the attachment of marker molecules is determined by the structure of the mono units from which the tree structure is built. The simplest monomer is a phosphoramidite based on glycerin. Two hydroxyl groups are protected with a dimethoxytrityl group and a further hydroxyl group is activated as cyanoethyl phosphoramidite. The number of reactive 5'-hydroxyl groups doubles in each coupling cycle, whereby 2 ⁿ functional hydroxyl groups are obtained with n condensations. Synthones based on pentaerythritol triple the number of reactive hydroxyl functions.

A large number of monomeric units and their combinations are available for the construction of the dendrimers. The degree of branching can be increased by a larger number of hydroxyl groups (eg sorbitol; saccharides) or the hydroxyl group can be replaced by other nucleophilic groups, such as amino or thiol functions. Methylene groups can be substituted by N-alkyl derivatives, P-alkyl derivatives or the alkyl spacers by polyoxyethylene chains of various lengths. By selecting suitable orthogonal protection group technology and strategy, these tree structures can be built up selectively.

With regard to examples of possible dendrimers which can be used according to the invention and their production processes, reference is made to the following examples and the publications by Urdea et al. (1987, op. Cit.) And Shchepinov et al. (1997, op. Cit.), The disclosure of which is incorporated by reference in this application.

As already mentioned, the tree structures can be built up selectively by choosing a suitable orthogonal protection group technique and strategy. For this purpose, for example, a monomer comprising several hydroxyl groups can be used, in which the individual hydroxyl groups are protected by different types of protective groups. Due to a suitable selection of the protective groups, probes are thus accessible which contain a different number of markings and possibly also types of markings in a desired ratio. For example, a glycerin-based monomer can be used in which one hydroxyl function is protected with a dimethoxytrityl group and the other with a levulinyl group.

Nucleic acid probes currently particularly preferred in the context of this invention are based on the dendrimer structure concept described in the examples below and which can also be taken from FIG. 1 by way of example.

The marker molecules

All marking molecules known in the prior art for use with nucleic acid probes or primers, such as hapten molecules, fluorescent marker molecules (fluorochromes), chromophores, chemiluminescent molecules, radioactively marked molecules or enzymatically active molecules or fragments, can be used as marking molecules. Labeling molecules, such as haptens or fluorescent dyes, which are available as phosphoramidite, can be coupled directly to the free hydroxyl functions which form the outer ends of the dendrimers. Labels that are present as active esters must be attached via amino-functionalized dendrimer ends.

Digoxigenin, biotin, dinitrophenol, estradiol, benzopyran or also fluorescein isothiocyanate (FITC) can be used as haptens. After the hybridization reaction, haptens can be detected using an anti-hapten labeled, preferably using a fluorescent molecule. For example, avidin (e.g. avidin-Cy5, avidin-FITC, avidin-Cy3) is used to detect biotin-labeled probes. Digoxigenin is raised using an anti-digoxigenin antibody, e.g. of a FITC-conjugated anti-digoxigenin antibody or rhodamine-anti-digoxigenin antibody. FITC labeling molecules can, for example, be detected using a primary unlabeled rabbit anti-FITC antibody and a secondary FITC-conjugated goat anti-rabbit antibody in succession.

The fluorescent dyes which can be used as a labeling molecule in the present context are numerous. Fluorescein isothiocyanate (FITC), cyanine dyes such as e.g. Cyanin2, Cyanin3, Cyanin3.5, Cyanin5, Cyanin5.5 and Cyanin7, Rhodamin, Rhoda in 110, Lissamin, Phycoerythrin, Fern, FluorX, Hex (Hexachlorfluorescein), Tet (Tetrachlorofluorescein), Texas Red, Fluorescein, Rox , Tamra, Joe, the BODIPY dyes, Oregon Green 488, Oregon Green 500, Oregon Green 514, Spectrum Green, Spectrum Orange, the Alexa dyes, pyrene, eosin or erythrosine.

Donor-acceptor pairs (energy transfer pairs) that are covalently bound to the dendrimers are also conceivable here.

Since fluorescence quenching can occur if the density of fluorescent dyes is too high, the provision of spacer structural units, such as long-chain Al- kyl or polyoxyethylene chains of different lengths, which serve as spacers, at the outer dendrimer ends are a tried and tested means of suppressing this phenomenon. The preparative measures to be taken for this are well known to the person skilled in the art in this field.

The incorporation of the respective marking or reporter molecules at any desired position and in any number, preferably in an automated synthesis, is achieved in particular through the use of "multi-reporter phosphoramidites", e.g. Multi-biotin phosphoramidite.

The nucleic acid probes which can be used according to the invention can be produced with their entire nucleic acid sequence by, in particular, automated synthesis, as is explained, for example, in Example 1. However, they are preferred starting from the oligonucleotide primers containing the dendrimers, which e.g. are prepared as described in Example 1. In this case, the nucleic acid probe is produced, for example, by a PCR reaction to extend the oligonucleotide portion to the desired length. Chromosome-specific DNA serves as the PCR template. Various methods are known to the person skilled in the art for the production of chromosome preparations suitable for this purpose. With this method it is possible to incorporate 100% of the label of the primer into the synthesized probe. Alternatively, the nucleic acid probes are labeled by means of "random priming" using the oligonucleotide primers containing the dendrimer (s).

The in situ hybridization method according to the invention is characterized in that at least one nucleic acid probe is used for the si u hybridization, which comprises at least one dendrimer that is labeled with one or more labeling molecules. According to preferred embodiments of the invention, more than one label molecule and / or more than one label molecule type can be bound to a dendrimer structure his. In addition, the polymer chain can also carry more than one dendrimer structure labeled with labeling molecule (s). For example, a nucleotide probe that can be used in accordance with the invention can carry a plurality of dendrimers, each of which has different labeling molecules bound to it, here also being considered as a possibility of providing several different types of labeling molecules on one and the same dendrimer. In a particular embodiment, the nucleic acid probe comprises different types of marker molecules in a certain, precisely defined relationship to one another.

In the method according to the invention, the nucleic acid probe explained above can be used, for example, as a “chromosome paint”, as a centromer-specific probe or as a probe for identifying a gene. The in situ hybridization method according to the invention can be carried out on a large number of biological or clinical samples from humans, animals or plants, on cells in mitosis and meiosis or on interphase cells, for example on chromosomes, cell nuclei, cells obtained from biological samples, such as lymphocytes from peri pure blood, cells from cell culture, tissue sections or spread DNA.

The hybridization method according to the invention using dendrimer-labeled nucleic acid probes is otherwise carried out by standard in si u hybridization techniques (see, for example, Gall and Pardue, 1981, Meth. Enzym. 21: 470-480; Henderson, 1982, Int. Review of Cytology 76: 1-46). Any necessary adaptation of the hybridization conditions can easily be carried out by the person skilled in the art on the basis of the numerous known in si u hybridization protocols.

In general, in situ hybridization involves the following major steps:

(1) fixation of the biological sample to be analyzed;

(2) treatment of the biological sample to increase the accessibility of the target DNA (eg denaturation by means of heat or alkali); (3) if necessary, a pre-hybridization treatment to reduce non-specific binding (for example by blocking the hybridization capacity of repetitive sequences);

(4) hybridization of the nucleic acid probe (s) with the nucleic acids in the biological sample;

(5) washing steps to remove nucleic acid probe molecules not bound during the hybridization reaction;

(6) detection of the nucleic acid probes hybridized to the target DNA;

(7) If necessary, counterstaining of the chromosms.

According to a special embodiment of the method according to the invention, the in situ hybridization reaction can also take place by means of the “pri ed in situ” (PRINS) method. A mixture of primer, nucleotides and DNA polymerase is placed directly on previously denatured chromosomes on the slide. After hybridization of the primer or primers to the target sequence on the chromosome, an elongation takes place by means of the polymerase, which uses the chromosomal DNA as a template. A nucleotide of the freely added nucleotides is usually labeled, be it with a hapten or a fluorescent dye. In the method described here, the marking is introduced via the marking of the primer as in the FISH experiments described above. This can contain, for example, a dendrimer with eight biotin or FITC molecules or other dye molecules.

The hybridization process is otherwise carried out by standard reactions (see, for example, Hindkj-er et al., In "In Situ Hybridization Protocols", vol. 33, editor KH Andy Choo, Humana Press). In general, primed in situ hybridization comprises the following Steps:

(1) Fixation of the biological sample to be analyzed

(2) Treatment of the biological sample to increase the accessibility of the target DNA (eg denaturation using heat or alkali) (3) Hybridization of the primer (s) to the chromosome and elongation by the polymerase

(4) Stop the reaction by incubating in stop buffer

(5) Washing steps to remove unbound primers

(6) In the case of indirect labeling, detection using fluorescent-labeled antibodies or avidin

(7) If necessary, counterstaining of the chromosomes

The detection reaction for the hybridization signal corresponds to that which is described below for the "chromosome painting" experiment.

The invention also relates to a kit for use in the in situ hybridization method according to the invention. As an essential component, the kit comprises one or more of the nucleic acid probes explained above, which can be provided in the kit both individually and in the form of mixtures. To provide the nucleic acid probes in the kit, they are formulated in a manner customary in the prior art and, if appropriate, mixed with customary auxiliaries. The kit can also contain other reagents required for the si u hybridization, such as formamide and dextran sulfate, and, if necessary, additional reagents required for the detection of the labeling molecules, such as e.g. one or more antibodies. These additional reagents can be provided separately or, if possible, in the form of mixtures of certain components. The preparations of the reagents can likewise comprise auxiliaries customary in the prior art, which, however, should not have a negative effect on the hybridization reaction and the subsequent detection.

In addition, the kit may include instructions for use, which may also include drawings and reference pictures. The new method should help to drastically reduce the effort (material and labor costs) when labeling the probes and will be essential for the economic success of DNA / RNA probes that are labeled at low cost.

Significant signal amplification is achieved by coupling a plurality of marker molecules to the nucleic acid probe via a single dendrimer or alternatively also via several dendrimers. Depending on the special structure of the dendrimer, a more than additive effect can be observed, i.e. the signal undergoes a greater amplification than would have been expected due to the number of labeling molecules. It is believed that this is due to some influence of the dendrimer structure.

Another advantage of this marking is that for the first time a precisely defined "ratio labeling" of DNA probes can be carried out. By carefully selecting the protective group combinations, probes are accessible that contain a different number of markings in a desired ratio. E.g. Labeled probes with PCR primers containing dendrimers labeled with FITC or cyanine 3, the ratios of the labels can be determined precisely on the basis of the number of individual fluorochrome-labeled dendrimers. A nucleotide probe can e.g. contain three FITC dendrimers, the other FITC and cyanine 3 in a ratio of 1: 2, etc. By selecting a suitable orthogonal protective group technique, these tree structures can, as explained, be built up selectively.

In a preferred embodiment, the monomer based on the glycerol structure is used for these purposes. One hydroxyl function is protected with a dimethoxytrityl group, the other with a levulinyl group.

Such a precise "ratio labeling" is not possible with conventional methods of PCR labeling or with "nick translation" and will allow the "multi-color" fluorescence microscopy can be drastically simplified. Compared to the classic "combinatorial labeling", fewer filters can be used to achieve the same number of different colors.

Another advantage of this type of marking is the possibility of simply performing "multi-color" PRINS. In a normal PRINS reaction with the addition of labeled molecules, this is only possible if the entire experiment is carried out two or more times, the labeled nucleotides of the previous reaction having to be washed out in each case. A method that could not prevail in practice due to the complexity.

The use of “multi-reporter phosphoramidites” to be coupled to one another multiple times enables the installation of the respective reporter molecules at any desired position and in any number, this synthesis preferably being automated.

To illustrate, the invention is described below using the example of three in situ hybridization methods according to the invention using dendrimer-labeled probe types, "chromosome paints" and satellite DNA as well as PRINS. In principle, however, any other dendri-armed DNA, RNA or PNA probe can be used for the method according to the invention, the polymer chain portion of which is or could be complementary to a sequence section of the DNA present in the sample to be examined.

EXAMPLES

A FISH experiments

1. Synthesis of nucleic acid probes

PCR primer synthesis

PCR primers were synthesized, each with a dendrimer containing eight biotin or eight fluorescein molecules. were. The synthesis of these multi-labeled primers was carried out on an ABI 392 DNA synthesizer using standard phosphoramidite chemistry (ABI). The synthesis cycle was carried out according to the conventional protocol described in ABI's User Manual for a 40 nmol synthesis. The concentration of the phosphoramidites (A, G, C, T) used was 0.05 mol / 1. The coupling yields were determined by measuring the conductivity of the DMT cation. The branching branches of the dendrimer were made with Cruachem Ltd.'s symmetrical "branching phosphor amidite" based on the molecular structure of glycerin. (((DMTO-CH ₂ ) ₂ CH-0-CEP); C ₅₄ N ₆ ιN ₂ 0 ₈ P); Order number 22-8424-17) synthesized. The branching phosphor amidite, which was dissolved with acetonitrile (max. 7 ppm water) to a concentration of 0.1 molar, was introduced into the synthesis cycle at a separate bottle position. The increase in the degree of branching could be followed via the conductivity measurements of the DMT cation (doubling of the measured values). After three cycles, eight free hydroxyl groups were obtained on the periphery of the dendrimer. The modification with biotin or fluorescein was carried out using the Amidite from Cruachem Ltd. (Fluorescein; order number 22-8409-35) and ABI (biotin; order number 401396). Both the structure of the dendrimer and the modification of the free hydroxyl groups with biotin or fluorescein to label the dendrimer were carried out according to the standard protocol mentioned above for a 40 nmol synthesis.

The CPG carrier (1000 A) was split off and the protective groups were split off with 33% ammonia solution (800 μl) at 55 ° C. for 6 hours. After the ammonia had been stripped off, the oligonucleotide was precipitated with 2 ml of ethanol after the addition of 60 μl of 3 M sodium acetate buffer, pH 5.2. After cooling for 20 min at -20 ° C., centrifuging at 13500 rpm, decanting the supernatant and dissolving the pellet in deionized or disinfected water, the amount of oligonucleotide was determined by UV measurement at 260 nm.

The structure is shown schematically in FIG. ture of the PCR primer oligonucleotide prepared above.

Preparation of the nucleic acid probes by means of PCR

For the synthesis of the nucleic acid probes by means of PCR, primers, each as explained in the previous section, were used with a dendrimer of eight. Biotin or eight fluorescein molecules were used. The primer 6-MW (5'-CCG ACT CGA GNN NNN NAT GTG G-3 '; N = A, C, G or T) (Telenius et al., 1992, op. Cit.) Was used for the PCR. The PCR buffer used consisted of 100 mM Tris-HCl, pH 9.0, 15 mM MgCl ₂ , 500 nM KC1, 1% Triton X-100, 0.1% (w / v) stabilizer.

Human, pre-amplified (“primary ^* ) chromosome-specific DNA served as the template for the PCR reaction.

A 50 μl reaction mixture for probe synthesis contained the following components:

1 μl DNA template (approx. 50 ng)

5 ul lOx PCR buffer

5 μl lOx dNTP mix (final concentration: 250 uM each)

5 ul 6 MW primer labeled with dendrimers

(about 20 uM)

2 ul 25mM MgCl ₂

0,. 5 μl Taq polymerase (5 U / μl; SuperTaq, HT Biotechnology Ltd.) 31.5 μl aqua dest.

The following PCR conditions were selected (Hybai TouchDown thermal cycler):

9 ° C 5 min. (Initial denaturation, lx)

94 ° C 1 min.

30 cycles 62 ° C 1 min.

72 ° C 1 min.

72 ° C 5 min. In this way, nucleic acid probes were obtained as a so-called DOP-PCR product, which were subsequently used in in situ hybridization experiments. The dendrimer and labeling section of the nucleic acid probes thus produced corresponds to the structure shown in FIG. 1; only the oligonucleotide section of the molecule shown there was extended by the PCR reaction.

2) FISH with "chromosome pain" and satellite DNA in si u hybridization

1 μl DOP-PCR product was used as a DNA probe in 14 μl hybridization buffer for hybridization on an area of 22 mm × 22 mm. The hybridization buffer consisted of 50% formamide, 20% dextran sulfate in 2x SSC. The DNA probe was denatured for 5 min at 70 ° C and then incubated for 60 min at 37 ° C so that repetitive DNA sequences could reassociate.

In the hybridization of satellite DNAs, the denatured probe was placed directly on the denatured chromosomes without waiting for the DNA to reassociate.

Chromosome preparations were produced according to standard protocols. An exemplary standard protocol comprises the following steps:

1) Colchicine synchronization of cells. After adding colchicine to a final concentration of 0.1 μg / ml, the culture was incubated at 37 ° C. for 1-2 hours.

2) Hypotonic treatment of the cells. After trypsinization and centrifugation of the cells, they were resuspended in 10 ml of 0.4% KCl solution and incubated at 37 ° C. for 12 min.

3) Fixation of the cells in fixative (glacial acetic acid / methanol in the ratio 3: 1). After adding about 1 ml of fixative, the cell suspension was centrifuged and the supernatant was removed. The pellet was resuspended in 10 ml fixative. Centrifugation and re- suspend were repeated twice. The last resuspension takes place in a smaller volume (approx. 5 ml).

The preparation was stored at -20 ° C until use.

The slides with the chromosome preparation applied to them were incubated for 50-100 seconds in a cuvette preheated to 68 ° C. with 70% formamide / 2x SSC and then dehydrated in an ascending ethanol series (70%, 90%, 100%).

The DNA probe was pipetted onto a previously selected area of the slide, provided with a 22 mm x 22 mm cover glass and sealed with Fixogum. The hybridization took place at 37 ° C ("chromosome paints") or 42 ° C (satellite DNAs) for 24 hours.

Detection reaction

After hybridization, the coverslips were removed and the slides 2 x 4 min in 1 x SSC / 50% formamide, 2 x 4 min in 2 x SSC (each at 45 ° C) and 1 x 4 min in 0.1 x SSC (45 ° C) washed.

Equilibration in 4x SSC / 0.2% Tween (room temperature) was followed by a 20 minute incubation in 4x SSC / 0.2% Tween / 3% bovine serum albumin (w / v) at 37 ° C.

A further washing step in 4x SSC / 0.2% Tween (room temperature) was followed by the detection reaction of the DNA probes labeled with biotin using fluorescein-coupled avidin. In this case, the detection reaction of the DNA probes labeled with biotin was carried out using fluorescein-coupled avidin and fluorescein-coupled anti-avidin antibody. The former was diluted 1: 250 in 4x SSC / 1% Tween, 15% human AB serum, the antibody 1: 125 in the same solution. Both were combined in a 1: 1 ratio. 100 μl of this mixture were used per slide. There was an incubation at 37 ° C for 20 min. The mixture was then washed three times at 42 ° C. with 4x SSC / 1% Tween. If PCR primers were used that were exclusively labeled with directly fluorochrome-coupled dendrimer, a chromosomal counterstaining with DAPI followed instead.

a) Microscopy

The microscopic analysis was carried out on a Zeiss fluorescence microscope (Axioplan II), which was equipped with fluorescence filters from Chroma Techn., Bratlbourgh, USA.

In the manner described, a human metaphase was clearly stained with the "paint" specific for chromosome 4 using the method according to the invention illustrated in this example. The hybridization signals were visible even under low magnification (25x objective) and showed stable fluorescence. Chromosomes stained by indirect detection using biotin / fluorescein-coupled avidin showed a somewhat stronger fluorescence than those in experiments in which fluorescein was directly linked to the dendrimers.

B PRINS experiment

The primer synthesis was carried out as described under "FISH experiments" point 1. Primers were chosen that bind specifically to alpha-satellite monomers of chromosome X: XI (5 '-ATT TCT TTG GAA TCG GGA ATA TTT CC - 3') and X2 (5 '- CTC TCG TCT TTC TGT GAA GAT AAA G - 3 ') acc. To Waye JS and Willard H.F., 1985.

PRINS hybridization

A 50 μl reaction mixture contained the following components:

5 ul 10 x PCR buffer

1 μl 50 x dNTP mix (final concentration 200 μM each)

1 μl primer XI (approx. 50 ng / μl)

1 μl primer X2 (approx. 50 ng / μl)

0.2 μl Taq polymerase (5u / μl)

41.8 μl aqua dest. 10 x PCR buffer and Taq polymerase were the same as previously described in "FISH experiments".

This reaction mixture was applied to denatured chromosome preparations (preparation according to standard protocol) and incubated in a moist chamber at 65 ° C. for 2 min.

The reaction was then stopped by incubating the slides in a cuvette preheated to 65 ° C. and containing stop buffer (50 mM NaCL / 50 mM EDTA) for 5 min. The preparations were finally washed in 4 x SSC / 0.2% Tween for 5 min at room temperature. If the primers were labeled with biotin, a detection reaction followed, as already described. If primers were used which had only been labeled directly, only counterstaining with DAPI (4 ', 6'-diamidino-2-phenylindole) followed. The microscopy was carried out as already described.

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Claims

EXPECTATIONS

in situ hybridization method, characterized in that at least one nucleic acid probe is used for the in situ hybridization, which comprises at least one dendrimer, which is labeled with one or more labeling molecules.

A method according to claim 1, characterized in that the nucleic acid probe is a DNA, RNA or PNA sequence or a hybrid of DNA, RNA and / or PNA.

A method according to claim 1 or 2, characterized in that more than one labeling molecule is bound to the dendrimer.

A method according to claim 3, characterized in that two or more different types of marker molecules are bound to the dendrimer.

Method according to one of claims 1 to 4, characterized in that the marker molecule (s) is or are selected from hapten molecules, fluorescent marker molecules, chro ophores, chemiluminescent molecules, radioactively labeled molecules or enzymatically active molecules or fragments .

A method according to claim 5, characterized in that the marker molecule (s) is or are selected from biotin, digoxigenin, dinitrophenol, fluorescein isothiocyanate (FITC), cyanine dyes such as cyanine2, cyanine3, cyanine3.5, cyanine5, Cyanine5.5 and cyanine7, rhodamine, rhodamine 110, lissamin and phycoerythrin.

7. The method according to claim 6, characterized in that the marking molecule (s) is or are selected from biotin and fluorescein isothiocyanate (FITC).

8. The method according to claim 4, characterized in that the nucleic acid probe comprises a dendrimer which is labeled with one or more hapten molecules and one or more fluorescent marker molecules.

9. The method according to claim 7, characterized in that the nucleic acid probe comprises a dendrimer which is labeled with one or more biotin molecule (s) and one or more FITC molecule (s).

10. The method according to any one of claims 1 to 8, characterized in that the nucleic acid probe comprises two or more dendrimers.

11. The method according to claim 9, characterized in that at least one of the dendrimers is labeled with more than one labeling molecule.

12. The method according to claim 9, characterized in that at least one of the dendrimers is labeled with two or more different types of labeling molecules.

13. The method according to any one of claims 9 to 11, characterized in that the nucleic acid probe contains two or more dendrimers, each labeled with haptens or fluorescence, ark molecules or combinations of haptens and / or fluorescence marker molecules, in different ratios.

14. The method according to claim 13, characterized in that the nucleic acid probe contains dendrimers, which are each labeled with FITC or biotin, in different ratios.

15. The method according to any one of the preceding claims, characterized in that the nucleic acid probe is a "chromosome paint".

16. Ver a ren nac e nem he nsprüc e 1 to 14, characterized in that the nucleic acid is a centromer-specific probe.

17. The method according to any one of claims 1 to 14, characterized in that the nucleic acid probe is a probe for identifying a gene.

18. The method according to any one of the preceding claims, characterized in that the hybridization reaction takes place on chromosomes.

19. The method according to any one of claims 1 to 17, characterized in that the hybridization reaction takes place on cell nuclei.

20. The method according to any one of claims 1 to 17, characterized in that the hybridization reaction takes place on spread DNA.

21. The method according to any one of the preceding claims, characterized in that the hybridization reaction takes place in a tissue section.

22. The method according to any one of the preceding claims, characterized in that the in situ hybridization reaction takes place by means of the "primed in situ (PRINS) -" method.

23. The method according to claim 22, characterized in that the PRINS reaction is carried out by means of nucleic acid probes which contain differently labeled dendrimers, or by means of different nucleic acid probes which each contain differently labeled dendrimers.

24. The method according to any one of the preceding claims, characterized in that the nucleic acid probes containing dendrimer are prepared using oligonucleotide primers containing the dendrimer (s).

25. The method according to claim 24, characterized in that the nucleic acid probes are produced by polymerase chain reaction (PCR reaction) to extend the oligonucleotide primer containing the or the dendrimers.

26. The method according to claim 24, characterized in that the labeling of the nucleic acid probes with the dendri structures is carried out by means of "rando priming" using oligonucleotide primers containing the dendrimers.

27. Kit for use in an in situ hybridization method according to one of claims 1 to 26, characterized in that it comprises at least one nucleic acid probe which comprises at least one dendrimer which is labeled with one or more labeling molecules, optionally further reagents for cytogenetics and possibly includes instructions for use.

28. Kit according to claim 27, characterized in that it comprises, as further reagents for the cytogenetics, formamide, dextran sulfate and / or optionally additionally required reagents for the detection of the marking molecules, such as one or more antibodies.

29. Kit according to one of claims 27 or 28, characterized in that it comprises several different DNA, RNA and / or PNA probes.

30. Kit according to claim 29, characterized in that it comprises several different "chromosome paints".

31. Kit according to claim 30, characterized in that it comprises several different "chromosome paints" which are each marked differently.

32. Kit according to claim 30 or 31, characterized in that it comprises one or more different "chromosome paints" as probes for the identification of one or more genes and / or other DNA sequences.

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