COMPOSITION AND METHOD FOR HYBRIDIZATION

Description

[0001] The present invention relates to the technical field of detection methods for nucleic acids, in particular DNA and / or RNA.

[0002] In particular, the present invention relates to a composition for use in hybridization, in particular for the detection and / or detection of nucleic acids, as well as its use according to the invention. Furthermore, the present invention relates to a method for detecting nucleic acids or chromosomal aberrations. Finally, the present invention relates to a kit for detecting nucleic acids or chromosomal aberrations.

[0003] Many tumor diseases are caused by structural and numerical chromosomal mutations, such as translocations, inversions, segmental duplications, deletions, insertions, duplications, aneuplodies, and amplifications. Detection of these alterations as predictive, prognostic, or differential diagnostic markers is usually achieved by in situ hybridization (ISH).

[0004] In situ hybridization is based on the hybridization or pairing of complementary bases of single nucleic acid strands, especially single DNA strands, so that specific nucleic acid sequences in a sample, especially in a tissue or cell preparation, can be detected. For this purpose, directly or indirectly labeled, synthetically produced probes are hybridized with single nucleic acid strands in the sample and subsequently detected.

[0005] For detection purposes, fluorescently labeled nucleic acid fragments or fluorescently labeled hybridization probes (fluorescent ISH (FISH)) can be used. Antigen-labeled probes, especially hapten-labeled probes, can also be used, which are then visualized using antibodies through color reactions, allowing light microscopic analysis (brightfield ISH (BrISH), chromogenic ISH (CISH), silver ISH (SISH)).

[0006] To perform in situ hybridization, a biological sample to be examined, in particular a preparation preferably based on tissue sections or cytological specimens, is usually first prepared. The samples are fixed on slides and dehydrated in preparation for in situ hybridization. To ensure that the labeled probes can hybridize with the nucleic acids, in particular RNA or DNA, in the cells or cell nuclei, a denaturation step is carried out first, so that the nucleic acid or nucleic acid segment to be detected, as well as the hybridization probes used, are present in the sample in single-stranded form. It is possible to denature the sample and hybridization probe separately or together (co-denaturation). The hybridization probes used are then hybridized with the nucleic acids contained in the sample.The hybridization probes are usually dissolved or stabilized in hybridization solutions, which are applied to the samples to be examined for hybridization. The hybridized probes, or those bound to the target nucleic acids, can then be detected as described above.

[0007] As described in detail below, the hybridization conditions, which influence the binding specificity of the hybridization probes as well as the stringency of the hybridization, are crucial for obtaining well-evaluable results: For the hybridization and the success of the hybridization, the denaturation of the sample and hybridization probes and the subsequent renaturation of the nucleic acids, in particular the DNA, i.e. the hybridization of single nucleic acid strands in the sample with single nucleic acid strands of the hybridization probes, are of central importance.

[0008] The separation of double-stranded nucleic acid molecules into single-stranded nucleic acids, or denaturation, can be carried out particularly at high temperatures of approximately 90 to 100 °C. However, such high temperatures are detrimental to the morphology of the biological sample, especially the tissue sections or cells that are preferably used.

[0009] To preserve sample morphology and improve hybridization stringency, in situ hybridization is typically performed using formamide-containing solutions to denature the double-stranded nucleic acids in biological samples. The use of formamide in the solutions used for hybridization reduces the melting temperature of double-stranded nucleic acids, particularly DNA-DNA and DNA-RNA duplexes, which generally ranges from 90 to 100 °C, to 65 to 80 °C.

[0010] However, the hybridization solutions known from the state of the art are sometimes associated with certain disadvantages, as described below: Although the use of formamide-containing hybridization solutions can improve the morphology of the samples used and thus also the hybridization result due to the reduced melting point of the DNA, formamide-containing hybridization solutions are usually associated with very long reaction times. This is because formamide significantly slows down renaturation, i.e. the attachment or binding of the hybridization probes on the one hand and the nucleic acids to be detected in the samples to be examined, on the other. In the state of the art, hybridization or renaturation times of 10 to 24 hours are usually envisaged. In individual cases, a hybridization or renaturation time of up to 72 hours may even be necessary.

[0011] To overcome the problem of long hybridization times, there are approaches in the prior art that involve replacing formamide in hybridization solutions with other solvents: For example, WO 91 / 02088 A1 uses lactam-based solvents in hybridization solutions. WO 00 / 69899 A1 describes compositions for stabilizing nucleic acids or nucleic acid analogues for biotechnological applications, which contain solvents other than formamide for dissolving the nucleic acids. However, these formamide-free solutions often do not produce satisfactory signal patterns or signal patterns that are easy to evaluate. WO 2011 / 067678 A1 discloses compositions for in situ hybridization that do not require denaturation and use small amounts of formamide or do not contain formamide at all.

[0012] In addition, the hybridization solutions known to date in the state of the art, which contain the locus-specific hybridization probes required for in situ hybridizations, have further disadvantages, as described below: For example, with the known hybridization solutions, strong signals are often not obtained when carrying out in situ hybridizations as rapid tests, in which well-evaluable signal patterns are generated even with short hybridization times, although there is a need for this, e.g. for carrying out rapid tests in pre- or postnatal diagnostics or for tumor cytogenetics.

[0013] Furthermore, the hybridization solutions known from the state of the art are hardly suitable, or only to a very limited extent, for use in automated in situ hybridization methods. Compared to non-automated or manually performed in situ hybridization methods, larger amounts or volumes of hybridization solution must be applied to the samples in automated methods. With the hybridization solutions known from the state of the art, it has not yet been possible to dissolve or stabilize the amounts of hybridization probes required for in situ hybridization, even in high-volume solutions or in strong dilution, in such a way that good hybridization results can be achieved. However, since hybridization probes are very expensive, no approach or hybridization compositions for automated in situ hybridization are known in the state of the art whichwhich is also feasible taking economic aspects into account.

[0014] The present invention is therefore based on the object of providing compositions which are suitable for use in in situ hybridization, in particular for the detection of nucleic acids in a biological sample, and which at least largely avoid or at least mitigate the disadvantages of the prior art described above.

[0015] In particular, the present invention is based on the object of providing compositions or hybridization solutions for in situ hybridization that are also suitable for use in automated in situ hybridization methods or that allow for cost-effective implementation of automated in situ hybridization methods. Furthermore, the present invention is based on the object of providing compositions for in situ hybridization that lead to good results or readily analyzable signal patterns for both short and long hybridization or renaturation times. Description

[0016] To achieve the above-described object, the present invention proposes a composition according to claim 1; further advantageous embodiments are the subject of the dependent claims in this regard.

[0017] Furthermore, the present invention relates to the use of a composition according to the present invention according to the relevant independent claim.

[0018] Yet another subject of the present invention is a method for detecting nucleic acids or chromosomal aberrations according to the relevant independent claim.

[0019] Finally, the present invention relates to a kit or kit-of-parts or system for detecting nucleic acids or chromosomal aberrations according to the independent claim; further advantageous properties are the subject of the subclaim.

[0020] It goes without saying that particular configurations, embodiments or the like which are described only in connection with one aspect of the invention also apply accordingly to the other aspects of the invention without this requiring express mention.

[0021] Furthermore, with all relative or percentage quantities mentioned below, especially those based on weight or volume, it should be noted that, within the scope of the present invention, these must be selected by the person skilled in the art such that the sum of the respective ingredients, active ingredients, additives or auxiliaries, or the like, always results in 100% or 100% by weight. However, this will be understood by the person skilled in the art.

[0022] Furthermore, the person skilled in the art may deviate from the numerical, range or quantity specifications given below depending on the application or individual case, without departing from the scope of the present invention.

[0023] In addition, all parameter specifications or similar mentioned below can in principle be determined or ascertained using standardized or explicitly specified determination procedures or using determination methods that are familiar to the person skilled in the art.

[0024] The subject of the present invention is therefore - according to a first inventive aspect - a composition, in particular a composition for use in hybridization, in particular in situ hybridization, preferably automated in situ hybridization, in particular for the detection and / or proving of nucleic acids, preferably RNA and / or DNA, in a biological sample, preferably in one or more cells and / or in one or more cell nuclei, wherein the composition is defined in claim 1.

[0025] In other words, the invention thus provides for compositions for hybridization, synonymously also referred to as hybridization solutions, in which the hybridization probes are present in a stabilized or dissolved form based on the combination of at least one specifically selected polar protic or polar aprotic solvent on the one hand and at least one carboxylic acid amide or its salts, preferably formamide, in an amount of more than 10 wt.%, based on the composition.

[0026] Within the scope of the present invention, it was discovered, quite surprisingly, that even small amounts of hybridization probes, i.e., highly diluted hybridization probes, can be stabilized in high-volume hybridization solutions by the targeted combination of at least one specifically selected polar protic or polar aprotic solvent, on the one hand, and at least one carboxylic acid amide or its salts, in particular formamide or its salts, on the other hand, in defined amounts. These high-volume hybridization solutions according to the invention, containing the hybridization probes in high dilution, surprisingly yield excellent signal patterns even within the scope of automated in situ hybridization methods. This has not been achieved to date in the prior art.

[0027] Furthermore, it was completely surprising that the hybridization solutions according to the invention are not only suitable for use in automated hybridization procedures, but also lead to excellent results and signal patterns for both short and long hybridization or renaturation times. The hybridization solutions according to the invention are thus also suitable for use in rapid tests, such as Fast-ISH or Fast-FISH, or so-called "flexible" in situ hybridizations with variable hybridization times.

[0028] The present invention as a whole is associated with numerous advantages and special features, which are discussed below in a non-limiting manner and are to be regarded as an indication of the patentability of the present invention.

[0029] As previously described, it has surprisingly been possible within the scope of the present invention to stabilize even small amounts of hybridization probes in high-volume hybridization solutions or compositions, so that now automatic or automated in situ hybridization methods can be carried out while obtaining well-evaluable signal patterns with strong signals, without having to use larger amounts of probes compared to manual or established hybridization methods.

[0030] The compositions or hybridization solutions according to the invention are therefore particularly advantageous with regard to the economic aspects of hybridization processes, since automated hybridization processes can now be carried out cost-effectively. Furthermore, the overall efficiency of in situ hybridizations is increased, since automated processes involve a higher sample throughput.

[0031] Furthermore, the hybridization solutions according to the invention are versatile, as they are suitable for various in situ hybridization methods. In particular, the compositions according to the invention can be used equally for fluorescence in situ hybridization (FISH), brightfield in situ hybridization (BrISH), chromogenic in situ hybridization (CISH), and / or silver in situ hybridization (SISH).

[0032] Furthermore, the compositions are also suitable for in situ hybridizations with both long and short hybridization or renaturation times. The compositions according to the invention lead to excellent results, particularly in the context of rapid tests with short hybridization times, such as fast ISH methods, or flexible in situ hybridizations with variable hybridization times. This is particularly surprising given that the compositions according to the invention contain formamide in amounts of at least 10 wt. %, and formamide typically requires long hybridization times. The short hybridization times despite the use of formamide are only made possible by the targeted combination according to the invention of the at least one carboxylic acid amide, in particular formamide, with at least one polar protic or polar aprotic solvent in defined amounts.

[0033] The compositions according to the invention are characterized overall by their excellent stability, particularly of the hybridization probes contained therein. The stability of the compositions can be further increased if the compositions contain stabilizing or blocking agents, in particular based on nucleic acids and / or nucleic acid analogues, since these prevent premature degradation of the hybridization probes. Surprisingly, the inventive combination of at least one polar protic or polar aprotic solvent and formamide allows large amounts of stabilizing or blocking agents, in particular based on nucleic acids, to be dissolved in the compositions according to the invention.

[0034] Finally, the signal patterns of the in situ hybridizations obtained with the compositions according to the invention are characterized by their overall excellent quality. Distinct and easily interpretable signals are generated. Furthermore, nonspecific signals or nonspecific background staining can be at least substantially minimized, ie, the hybridization compositions according to the invention are characterized by their excellent stringency.

[0035] For a better understanding of the present invention, the key terms and designations of the composition according to the invention are defined below: The in situ hybridization used according to the invention is based on the hybridization or pairing of complementary bases of single nucleic acid strands, in particular single DNA strands, so that specific nucleic acid sequences, i.e. the chromosome or DNA regions to be detected, can be detected in a sample, such as a tissue or a cell preparation. During in situ hybridization, directly or indirectly labeled, synthetically produced, in particular locus-specific, hybridization probes based on nucleic acids are hybridized with single nucleic acid strands of the sample and subsequently detected.

[0036] In principle, in situ hybridization can take place or be performed at various stages of the cell cycle of the cells or nuclei being examined. It is generally performed in metaphase, when the chromosomes are in a condensed state, or in interphase, when the chromosomes are decondensed. Depending on the goal or purpose of in situ hybridization, it is not always possible to perform the test on condensed chromosomes in metaphase, particularly when examining solid tumor cells for chromosomal aberrations. In these cases, in situ hybridization is performed on cells in interphase.

[0037] The hybridization probes are preferably locus-specific. In the context of the present invention, locus-specific hybridization probes are understood to be probes that are specific for a chromosomal region or DNA region to be detected, or that are complementary to a chromosomal region or DNA region of the DNA material or genetic material to be detected in a sample to be examined. It can also be provided to detect the RNA of preferably individual genes, or to use locus-specific hybridization probes that are specific for the RNA of preferably individual genes. The hybridization probes used according to the invention are usually based on nucleic acids or nucleic acid fragments and / or nucleic acid analogues and are capable of specifically binding or hybridizing to the chromosomal region or DNA region to be detected. The nucleic acids and / or nucleic acid analogues can in particular be DNA, RNA, locked nucleic acids(LNA) or peptide nucleic acids (PNA). For detection purposes, the locus-specific hybridization probes are furthermore directly or indirectly labeled with detection labels. The chromosomal region or DNA region to be detected can have a variable length. In particular, it can be provided that a chromosomal region or DNA region or an RNA to be detected partially or completely comprises a single gene. Likewise, it can also be provided that a chromosomal region or DNA region to be detected partially or completely comprises several genes, preferably neighboring genes, preferably two genes. Furthermore, it can also be provided within the scope of the present invention that the hybridization probes used are Whole Chromosome Painting (WCP) hybridization probes or Partial Chromosome Painting(PCP) probes, which comprise a multitude of different locus-specific hybridization probes, each labeled with at least one detection label and allowing the detection of entire chromosomes and / or chromosome segments. The design of the hybridization probes as such is familiar to the person skilled in the art, so no further information is required in this regard.

[0038] Furthermore, with regard to the term "biological sample," this can refer, in particular, to tissues or tissue components to be examined, especially tissue sections. However, it may also be intended to use individual cells, cell aggregations, or isolated cell nuclei as biological samples.

[0039] In the context of the present invention, polar aprotic solvents are understood to mean, in particular, non-aqueous solvents that do not contain an ionizable proton in the molecule. In other words, aprotic solvents are based, in particular, on molecules that do not have functional groups from which hydrogen atoms can be released or dissociate in the form of protons. Furthermore, in the context of the present invention, aprotic polar solvents are preferably substituted with strongly polarizing functional groups, such as carbonyl groups, nitrile groups, or thiol groups, so that the molecules underlying the solvent exhibit a dipole moment. Polar aprotic solvents can be present in aliphatic, aromatic, or cyclic form. According to the invention, it has proven particularly advantageous to use polar aprotic solvents with a cyclic molecular structure.Furthermore, within the scope of the present invention, it is intended that the compositions according to the invention do not contain dimethyl sulfoxide (DMSO) or are free of DMSO. For further details on the term "polar aprotic solvent," reference can be made to RÖMPP Chemielexikon, 10th edition, Thieme Verlag, Stuttgart, New York, 1996, page 241, keyword: "Aprotic Solvents," as well as the literature cited therein, which is hereby incorporated in its entirety.

[0040] Polar protic solvents, on the other hand, are understood in the context of the present invention to mean, in particular, non-aqueous solvents that contain an ionizable proton in the molecule and / or are capable of releasing it and / or are capable of forming hydrogen bonds. In other words, in the context of the present invention, polar protic solvents are based in particular on molecules that have functional groups from which hydrogen atoms can be released or dissociate in the form of protons, or that are functionalized in such a way that hydrogen atoms can be released or dissociate in the form of protons. Polar protic solvents can also be referred to as polar amphiprotic solvents. For further details on the term "polar protic solvent," reference can be made to RÖMPP Chemielexikon, 10.Edition, Thieme Verlag, Stuttgart New York, 1996, page 3597, keyword: "Protic solvents" as well as the literature referred to therein, which is hereby fully included.

[0041] For the purposes of the present invention, carboxamides are understood to mean derivatives of ammonia and of primary and secondary amines in which one or more hydrogen atoms of the nitrogen are replaced by carboxylic acid residues or are substituted by carboxylic acid residues. Particularly good results with regard to in situ hybridizations are achieved when the compositions according to the invention contain formamide as the carboxamide, which is the amide of formic acid and thus the simplest carboxamide. In a particularly preferred embodiment, the carboxamide is therefore formamide. Furthermore, as regards the amount of carboxamide used, in particular formamide, this is more than 10% by volume according to the invention, ie an amount of exactly 10% by volume is excluded.

[0042] The composition according to the invention can be configured in a variety of ways. Preferred embodiments are described in detail below.

[0043] Within the scope of the present invention, it has surprisingly been possible to obtain well-evaluable signal patterns in the context of in situ hybridizations even with high-volume hybridization compositions which contain the hybridization probes only in low concentrations and / or amounts or small amounts of hybridization probes relative to the volume used.

[0044] Particularly good results are achieved according to the invention when the composition (a) contains the at least one, preferably locus-specific, hybridization probe in a concentration in the range from 0.1 ng / µl to 50 ng / µl, in particular 0.5 ng / µl to 50 ng / µl, preferably 0.7 ng / µl to 8 ng / µl, more preferably 1 ng / µl to 5 ng / µl, based on the composition. In the context of the present invention, it was completely surprising that the, preferably locus-specific, hybridization probes can be stabilized in the aforementioned concentrations in the hybridization solutions according to the invention and thus lead to excellent signal patterns even when used in automated hybridization methods. The provision of hybridization solutions which also allow an economical or cost-saving implementation of automated hybridization methods has not yet been achieved in the prior art.With regard to the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, in particular, it is preferably selected from the group of solvents with lactone, sulfone, nitrile, carbonate, and / or amide functionality, more preferably from the group of solvents with lactone, sulfone, carbonate, and / or amide functionality. The term "functionality" in this context describes the presence of functional groups in a molecule. In other words, the polar protic or polar aprotic solvent is preferably selected from the group consisting of solvents that have at least one lactone, sulfone, nitrile, carbonate, and / or amide functional group.

[0045] In particular, it can be provided that the at least one polar aprotic or polar protic solvent is selected from the group of ethylene carbonate, pyrrolidones, lactams, ethylene sulfite, γ-butyrolactone, ethylene trithiocarbonate, propylene carbonate and / or sulfolane, particularly preferably ethylene carbonate and / or pyrrolidones, more preferably ethylene carbonate.

[0046] It can also be provided within the scope of the present invention that the at least one solvent, preferably with a cyclic molecular structure, is a polar aprotic solvent, in particular selected from cyclic carbonates (cyclic carbonic acid esters), in particular cyclic carbonic acid esters of alkylene glycols, preferably ethylene carbonate (1,3-dioxolan-2-one) and propylene carbonate, particularly preferably ethylene carbonate, as well as cyclic mono-, di- and trithiocarbonates, in particular ethylene sulfite and ethylene trithiocarbonate.

[0047] Furthermore, good results are achieved according to the invention when the at least one solvent, preferably with a cyclic molecular structure, is a polar aprotic solvent, in particular selected from aprotic cyclic amides (lactams), in particular N-alkyl-substituted pyrrolidones, preferably N-methyl-2-pyrrolidone and / or N-ethyl-2-pyrrolidone.

[0048] It can also be provided according to the invention that the at least one solvent, preferably with a cyclic molecular structure, is a polar protic solvent, in particular selected from protic cyclic amides (lactams), in particular protic cyclic amides (lactams) with a hydrogen atom on the amide nitrogen (lactam nitrogen), preferably selected from the group of 2-pyrrolidone (γ-butyrolactam), 3-pyrrolidone, caprolactams and / or 2-piperidone (valerolactam), particularly preferably pyrrolidones, very particularly preferably 2-pyrrolidone (γ-butyrolactam).

[0049] The at least one polar protic or polar aprotic solvent preferably has a cyclic molecular structure. Preference is given to cyclic molecular structures with a ring system consisting of five or six ring atoms. The cyclic molecular structure can be a homocycle or heterocycle, preferably a heterocycle. A heterocycle is a compound with ring-forming atoms from at least two different elements. Preferably, in addition to ring-forming carbon atoms, the heterocycle also comprises one or more ring-forming nitrogen, sulfur, and / or oxygen atoms, preferably one or more nitrogen atoms (N-heterocycle) and / or one or more oxygen atoms. Furthermore, within the scope of the present invention, it can be provided that the at least one solvent is a polar protic solvent with a cyclic molecular structure, wherein the cyclic molecular structure comprises an N-heterocycle with a free hydrogen atom on the nitrogen atom.

[0050] Preferably, the cyclic molecular structure has lactone, sulfone, nitrile, carbonate, and / or amide functionality, more preferably lactone, sulfone, carbonate, and / or amide functionality. In a preferred embodiment, the invention thus relates to a composition comprising or consisting of: (a) at least one hybridization probe ("component (a)"); (b) at least one polar protic or polar aprotic solvent with a cyclic molecular structure, wherein the cyclic molecular structure has lactone, sulfone, carbonate, and / or amide functionality ("component (b)"); and at least one carboxylic acid amide and / or its salts ("component (c)") in an amount of more than 10% by volume based on the composition.

[0051] According to a particularly preferred embodiment of the present invention, the at least one polar protic or polar aprotic solvent can be selected from the group consisting of ethylene carbonate, 2-piperidone (valerolactam), 2-pyrrolidone (γ-butyrolactam), 3-sulfolene ("butdiene sulfones"), and / or γ-butyrolactone. The best results are achieved according to the invention when the polar protic or polar aprotic solvent is γ-butyrolactone, 2-pyrrolidone (γ-butyrolactam), and / or ethylene carbonate. In this context, reference is also made to the exemplary embodiments carried out by the applicant and described below, which demonstrate the superior properties of the aforementioned polar protic or polar aprotic solvents in the compositions according to the invention.

[0052] By using at least one polar protic or polar aprotic solvent in the composition according to the invention - in particular in combination with at least one carboxylic acid amide, preferably formamide - the disadvantages associated with the use of formamide in hybridization solutions, such as long hybridization times, can be compensated for. Furthermore - without wishing to be limited to this theory - the use of polar protic or aprotic solvents in the compositions according to the invention increases the solubility for nucleic acids overall, which in turn improves the stability of the hybridization compositions according to the invention. This is because the good solubility capacity for nucleic acids means that large amounts of non-hybridizing, non-specific nucleic acids, in particular stabilizing DNA, can be used as so-called blocking orStabilizing agents are incorporated into the compositions which, on the one hand, prevent degradation of the, preferably locus-specific, hybridization probes and, in addition, improve the overall signal pattern and prevent signals through non-specific binding or hybridization, ie also lead to an increase in stringency.

[0053] As far as the amount of the at least one polar protic or polar aprotic solvent used is concerned, this can vary within wide limits. According to the invention, it can be provided in particular that the composition (b) contains the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, in an amount which does not lead to the denaturation of nucleic acids. Within the scope of the present invention, it is particularly provided that the nucleic acids in the composition according to the invention are dissolved and stabilized by the at least one polar protic or polar aprotic solvent, whereby the solvent is used in an amount which on its own, i.e. without the additional use of at least one carboxylic acid amide and / or further denaturing agents, would not lead to the denaturation of the nucleic acids.

[0054] In particular, the composition according to the present invention may contain the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, in an amount in the range of 0.5 to 40 vol.% or wt.%, in particular 1 to 35 vol.% or wt.%, preferably 2 to 30 vol.% or wt.%, preferably 5 to 20 vol.% or wt.%, particularly preferably 7 to 15 vol.% or wt.%, based on the composition.

[0055] Furthermore, within the scope of the present invention, it is preferred if the composition (b) contains the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, in an amount of preferably at least 2 vol.% or wt.%, preferably at least 5 vol.% or wt.%, particularly preferably at least 7 vol.% or wt.%, very particularly preferably at least 10 vol.% or wt.%. Likewise, it can be provided that the composition (b) contains the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, in an amount of at most 50 vol.% or wt.%, in particular at most 40 vol.% or wt.%, preferably at most 30 vol.% or wt.%, preferably at most 20 vol.% or wt.%, particularly preferably at most 15 vol.% or wt.%, very particularly preferably at most 13 vol.% or wt.%, based on the composition.

[0056] The above percentages regarding the at least one polar protic or polar aprotic solvent are preferably percentages by weight. In other words, the composition according to the present invention contains the at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, preferably in an amount ranging from 0.5 to 40 wt.%, in particular 1 to 35 wt.%, preferably 2 to 30 wt.%, more preferably 5 to 20 wt.%, particularly preferably 7 to 15 wt.%, based on the composition.

[0057] Within the scope of the present invention, it has been shown overall that adherence to the aforementioned quantity ranges significantly improves the signal patterns obtained. The amount of the at least one carboxylic acid amide or its salts, in particular formamide or its salts, has also proven to be a critical factor in relation to the signal strengths and signal patterns. In connection with the amounts of the at least one polar protic or polar aprotic solvent on the one hand and of the at least one carboxylic acid amide, in particular formamide, on the other hand, reference is also made to the exemplary embodiments carried out by the applicant and described in detail below, which demonstrate that only the use of specially selected amounts of solvent and carboxylic acid amide leads to the excellent signal patterns when carrying out in situ hybridizations, in particular on an automated machine or as a rapid test with short orflexible hybridization times.

[0058] Within the scope of the present invention, it can be provided in particular that the composition (c) contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount which leads to the denaturation of nucleic acids. In a preferred embodiment, the composition (c) contains formamide and / or salts thereof in an amount which leads to the denaturation of nucleic acids or is suitable for this. In other words, it can therefore be provided within the scope of the present invention that the composition contains the at least one carboxylic acid amide, in particular formamide, in an amount which alone, ie without a polar protic or polar aprotic solvent, would already lead to the denaturation of the nucleic acids.

[0059] With regard to the amounts of the at least one carboxylic acid amide used, it has proven advantageous if the composition (c) contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount in the range from 10 to 60 vol.%, in particular 15 to 50 vol.%, preferably 17 to 40 vol.%, more preferably 20 to 30 vol.%, based on the composition. In a preferred embodiment, the composition according to the invention contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount in the range from 17 to 40 vol.%. In an even more preferred embodiment, the composition according to the invention contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount in the range from 20 to 30 vol.%.

[0060] Furthermore, it can be provided that the composition (c) contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount of at least 10 vol. %, in particular at least 15 vol. %, preferably at least 17 vol. %, more preferably at least 20 vol. %, based on the composition. Likewise, it can be provided that the composition (c) contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount of at most 60 vol. %, in particular at most 50 vol. %, preferably at most 40 vol. %, more preferably at most 30 vol. %, based on the composition. In a preferred embodiment, the composition according to the invention contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount of at least 17 vol. % based on the composition.In an even more preferred embodiment, the composition according to the invention contains the at least one carboxylic acid amide and / or its salts, in particular formamide and / or its salts, in an amount of at least 20% by volume based on the composition.

[0061] As mentioned above, amounts of the carboxylic acid amide and / or its salts of more than 10% have proven particularly effective. Thus, it is particularly preferred according to the invention for composition (c) to contain the at least one carboxylic acid amide and / or its salts, in particular formamide and / or its salts, in an amount between 10 and 60 vol.%, in particular between 15 and 50 vol.%, preferably between 17 and 40 vol.%, preferably between 20 and 30 vol.%, based on the composition. In a particularly preferred embodiment, composition (c) contains formamide and / or its salts in an amount between 17 and 40 vol.%.

[0062] Furthermore, it is particularly preferred that the composition (c) contains the at least one carboxylic acid amide and / or its salts, in particular formamide and / or its salts, in an amount of more than 10 vol.%, in particular more than 15 vol.%, preferably more than 17 vol.%, preferably more than 20 vol.%, based on the composition. In a particularly preferred embodiment, the composition (c) contains formamide and / or its salts in an amount of more than 17 vol.%.

[0063] In one embodiment, the composition according to the invention contains more than 10 vol. % of the carboxylic acid amide and / or its salts, preferably formamide and / or its salts. In a more preferred embodiment, the composition according to the invention contains more than 20 vol. % of the carboxylic acid amide and / or its salts, preferably formamide and / or its salts. In addition to the absolute amounts used, it has proven advantageous with regard to the signal patterns obtained with the composition according to the invention if the at least one polar protic or polar aprotic solvent on the one hand and the at least one carboxylic acid amide, in particular formamide, are used in a defined quantitative ratio to one another: Particularly good results are achieved in the context of in situ hybridizations if the composition contains component (b) and component (c) orwhich contains at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure, and at least one carboxylic acid amide and / or its salts, in particular formamide and / or its salts, in a volume-related ratio in the range particularly preferably 1:5 to 1:1, very particularly preferably 1:3 to 1:2.

[0064] Furthermore, it has proven advantageous if the composition contains at least one polysaccharide ("component (d)"), in particular biopolysaccharide, preferably neutral biopolysaccharide, preferably dextran and / or its derivatives or salts, particularly preferably dextran sulfate. Within the scope of the present invention, it has surprisingly been found that the hybridization results with the compositions according to the invention can be further improved if the compositions according to the invention contain at least one polysaccharide, in particular for stabilization. In this regard, reference is also made to the exemplary embodiments according to the invention, which demonstrate the effect of the polysaccharide in the compositions. Particularly good results are achieved when dextran sulfate is used as the polysaccharide.

[0065] With regard to the amount of the at least one polysaccharide in the compositions, it has proven particularly advantageous if the composition contains component (d) in an amount ranging from 0.1 to 50 wt.%, in particular from 1 to 40 wt.%, preferably from 5 to 30 wt.%, more preferably from 10 to 20 wt.%, based on the composition. In a preferred embodiment, the composition contains from 5 to 30 wt.% of the at least one polysaccharide, particularly preferably from 5 to 30 wt.% dextran sulfate.

[0066] Furthermore, within the scope of the present invention, the composition may contain at least one chemical buffer system, in particular in the form of buffer salt(s) ("component (e)"). In particular, the chemical buffer system serves to adjust or maintain the pH of the composition.

[0067] The amounts of the chemical buffer system used can vary widely. Within the scope of the present invention, it has proven advantageous if the composition (e) contains the chemical buffer system, based on the composition and calculated as the sum of all constituents of the chemical buffer system, in an amount in the range of 0.001 to 5 wt.%, in particular 0.01 to 4 wt.%, preferably 0.1 to 3 wt.%, more preferably 0.5 to 2 wt.%, particularly preferably 1 to 1.5 wt.%.

[0068] The selection of the buffer system is within the usual skill of the person skilled in the art. Particularly good results are achieved within the scope of the present invention when the chemical buffer system contains at least one salt, in particular at least one carboxylic acid salt, preferably a citrate, and / or at least one inorganic salt, in particular at least one alkali and / or alkaline earth salt, preferably at least one alkali and / or alkaline earth chloride, particularly preferably sodium chloride. The chemical buffer system is particularly preferably a citrate-based buffer system or a citrate-based buffer system based on trisodium citrate / sodium chloride.

[0069] According to a preferred embodiment, the chemical buffer system known to the person skilled in the art, e.g., an SSC (saline-sodium citrate) buffer system based on trisodium citrate (0.3 M at 20-fold concentrated SSC) and sodium chloride (3 M at 20-fold concentrated SSC) is used. Furthermore, it is also possible to use other buffer systems well known to the person skilled in the art, such as HEPES [2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid], SSPE [ s odium chloride / s odium p hosphate / E DTA], PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], TMAC [tetramethylammonium chloride], TRIS [tris(hydroxymethyl)aminomethane] or SET buffer.

[0070] Particularly good results within the scope of the present invention are also achieved when the composition has a pH in the range of 5.0 to 9.0, in particular in the range of 5.5 to 8.5, preferably in the range of 6.0 to 8.0, preferably in the range of 6.5 to 7.5. The chemical buffer system according to the invention is particularly suitable for adjusting this pH and maintaining it during storage and the hybridization reaction.

[0071] According to a preferred embodiment of the present invention, it can also be provided that the composition contains at least one blocking and / or stabilizing agent ("component (f)"), in particular wherein the blocking and / or stabilizing agent is based on nucleic acids and / or nucleic acid analogues, preferably on DNA and / or RNA. The nucleic acids and / or nucleic acid analogues as blocking and / or stabilizing agents can in particular also be locked nucleic acids (LNA) or peptide nucleic acids (PNA). The use of at least one blocking or stabilizing agent is advantageous in several respects: on the one hand, it stabilizes the hybridization probes used and prevents premature degradation of the probes. On the other hand, it minimizes nonspecific background signals in the signal pattern of in situ hybridization.

[0072] Due to the components used according to the invention, in particular due to the inventive combination of the at least one polar protic or polar aprotic solvent on the one hand and the at least one carboxylic acid amide, in particular formamide, on the other hand in each case defined amounts, it is possible to dissolve large amounts of blocking and / or stabilizing agent(s) in the composition according to the invention. Thus, it can be provided according to the invention that the composition (f) contains the at least one blocking and / or stabilizing agent in a concentration in the range from 0.001 µg / µl to 100 µg / µl, in particular 0.005 µg / µl to 80 µg / µl, preferably 0.01 µg / µl to 40 µg / µl, more preferably 0.05 µg / µl to 20 µg / µl, particularly preferably 0.1 µg / µl to 10 µg / µl, based on the composition.

[0073] Furthermore, with regard to the compositions according to the invention, it is advantageous if the composition contains at least one inorganic salt ("component (g)"), in particular alkali and / or alkaline earth salt, preferably alkali and / or alkaline earth chloride, particularly preferably sodium chloride.

[0074] The amounts of the at least one inorganic salt used can vary widely. Particularly good results are achieved with the composition according to the invention when composition (g) contains the at least one inorganic salt in an amount ranging from 0.01 to 15 wt.%, in particular 0.05 to 10 wt.%, preferably 0.1 to 10 wt.%, more preferably 0.5 to 5 wt.%, particularly preferably 1 to 3 wt.%, based on the composition.

[0075] Furthermore, it has surprisingly been found that the signal patterns obtained during in situ hybridization can be further improved if the compositions or hybridization solutions according to the invention contain at least one detergent and / or surfactant ("component (h)"). In the context of the present invention, a detergent or surfactant is understood to mean substances capable of reducing the surface tension of liquids or the interfacial tension between two phases. On this basis, the formation of dispersions or solutions is promoted or improved.

[0076] By using at least one detergent or surfactant in the compositions or hybridization solutions according to the invention, the specific hybridization of nucleic acids in the sample and hybridization probes is improved or enhanced, without wishing to be limited to this theory. On the other hand, the nuclear staining or staining of the nucleus typically performed during the evaluation or analysis of in situ hybridization samples or preparations is attenuated, which in turn leads to improved signal-to-background contrast and overall stronger signal intensities.

[0077] Particularly good results are achieved within the scope of the present invention when the detergent and / or surfactant is selected from nonionic surfactants, preferably polyalkylene glycol ethers, particularly preferably polyalkylene glycol ethers of lauryl alcohol and / or cetyl alcohol and / or cetylstearyl alcohol and / or oleyl alcohol, in particular lauryl alcohol. In this context, it is particularly preferred if the detergent and / or surfactant is selected from polyoxyethylene(4) lauryl ether, polyoxyethylene(9) lauryl ether and / or polyoxyethylene(23) lauryl ether, in particular polyoxyethylene(23) lauryl ether.

[0078] The amounts of detergent or surfactant used are variable. It has proven particularly advantageous if the composition contains the at least one detergent and / or surfactant in an amount ranging from 0.001 to 5 wt.%, in particular 0.01 to 3 wt.%, preferably 0.05 to 2 wt.%, more preferably 0.1 to 1.5 wt.%, particularly preferably 0.12 to 1 wt.%, based on the composition.

[0079] With regard to the advantageous properties of detergents or surfactants, reference is also made to the exemplary embodiments according to the invention, which demonstrate their positive effects on the hybridization results or signal strengths.

[0080] With regard to the design of the composition according to the invention, it can be provided, in particular, that it is an aqueous solution. In this context, it is advantageous if the composition contains water, in particular purified water, in an amount in the range of 10 to 99 wt.%, in particular in the range of 20 to 95 wt.%, preferably in the range of 30 to 90 wt.%, particularly preferably in the range of 40 to 85 wt.%, based on the composition.

[0081] Furthermore, within the scope of the present invention, it can be provided that the composition contains water in an amount such that the total, including all components, always results in 100% or 100% by weight, based on the composition.

[0082] Furthermore, the composition may also contain water as a carrier or excipient. Likewise, the composition may be aqueous.

[0083] The present invention - according to a particularly preferred embodiment - thus relates to a composition for use in hybridization, in particular in situ hybridization, preferably automated in situ hybridization, in particular for the detection and / or proving of nucleic acids, preferably RNA and / or DNA, in a biological sample, preferably in one or more cells and / or in one or more cell nuclei, in particular a composition as described above, wherein the composition contains: (a) at least one, preferably locus-specific, hybridization probe ("component (a)"), in particular in a concentration in the range of 0.1 ng / µl to 50 ng / µl, in particular 0.5 ng / µl to 50 ng / µl, preferably 0.7 ng / µl to 8 ng / µl, more preferably 1 ng / µl to 5 ng / µl, based on the composition; (b) at least one polar protic or polar aprotic solvent ("component (b)"), preferably with a cyclic molecular structure, in particular in an amount in the range of 1 to 35 vol.%, preferably 2 to 30 vol.%, preferably 5 to 20 vol.%, particularly preferably 7 to 15 vol.%, based on the composition; (c) at least one carboxylic acid amide and / or its salts ("component (c)"), in particular formamide and / or its salts, in particular in an amount in the range from 10 to 60% by volume, in particular 15 to 50% by volume, preferably 17 to 40% by volume, more preferably 20 to 30% by volume.-%, based on the composition; (d) optionally at least one polysaccharide ("component (d)"), preferably neutral biopolysaccharide, preferably dextran and / or its derivatives or salts, in particular in an amount in the range of 0.1 to 50 wt.%, in particular 1 to 40 wt.%, preferably 5 to 30 wt.%, preferably 10 to 20 wt.%, particularly preferably 13 to 18 wt.%, based on the composition; (e) optionally at least one chemical buffer system ("component (e)"), in particular in the form of buffer salt(s), in particular in an amount in the range of 0.001 to 5 wt.%, in particular 0.01 to 4 wt.%, preferably 0.1 to 3 wt.%, preferably 0.5 to 2 wt.%, particularly preferably 1 to 1.5 wt.-%, based on the composition and calculated as the sum of all components of the chemical buffer system; (f) optionally at least one blocking and / or stabilizing agent ("component (f)"), in particular in a concentration in the range from 0.001 µg / µl to 100 µg / µl, in particular 0.005 µg / µl to 80 µg / µl, preferably 0.01 µg / µl to 40 µg / µl, more preferably 0.05 µg / µl to 20 µg / µl, particularly preferably 0.1 µg / µl to 10 µg / µl, based on the composition; (g) optionally at least one inorganic salt ("component (g)"), in particular alkali and / or alkaline earth salt, preferably alkali and / or alkaline earth chloride, particularly preferably sodium chloride, in particular in an amount in the range from 0.01 to 15 wt.%, in particular 0.05 to 10 wt.%, preferably 0.1 to 10 wt.%, preferably 0.5 to 5 wt.%, particularly preferably 1 to 3 wt.-%, based on the composition; and (h) optionally at least one detergent and / or surfactant ("component (h)"), in particular in an amount in the range of 0.001 to 5 wt.%, in particular 0.01 to 3 wt.%, preferably 0.05 to 2 wt.%, more preferably 0.1 to 1.5 wt.%, particularly preferably 0.12 to 1 wt.%, based on the composition.

[0084] Yet another subject of the present invention according to a further particularly preferred embodiment is a composition for use in hybridization, in particular in situ hybridization, preferably automated in situ hybridization, in particular for the detection and / or proving of nucleic acids, preferably RNA and / or DNA, in a biological sample, preferably in one or more cells and / or in one or more cell nuclei, preferably a composition as described above, wherein the composition contains: (a) at least one preferably locus-specific hybridization probe ("component (a)"), in particular in a concentration in the range of 0.1 ng / µl to 50 ng / µl, in particular 0.5 ng / µl to 50 ng / µl, preferably 0.7 ng / µl to 8 ng / µl, more preferably 1 ng / µl to 5 ng / µl, based on the composition; (b) at least one polar protic or polar aprotic solvent ("component (b)") with a cyclic molecular structure, particularly preferably γ-butyrolactone, 2-pyrrolidone (γ-butyrolactam) and / or ethylene carbonate, particularly preferably ethylene carbonate, in particular in an amount in the range of 1 to 35 vol.%, preferably 2 to 30 vol.%, preferably 5 to 20 vol.%, particularly preferably 7 to 15 vol.%, based on the composition; (c) formamide and / or its salts ("component (c)"), in particular in an amount in the range of 15 to 50 vol.%, preferably 17 to 40 vol.%, preferably 20 to 30 vol.%.-%, based on the composition; (d) optionally dextran and / or its derivatives or salts ("component (d)"), in particular in an amount in the range of 0.1 to 50 wt.%, in particular 1 to 40 wt.%, preferably 5 to 30 wt.%, more preferably 10 to 20 wt.%, particularly preferably 13 to 18 wt.%, based on the composition; (e) optionally at least one chemical buffer system, in particular in the form of buffer salt(s), preferably citrate and / or sodium chloride, ("component (e)"), in particular in an amount in the range of 0.001 to 5 wt.%, in particular 0.01 to 4 wt.%, preferably 0.1 to 3 wt.%, preferably 0.5 to 2 wt.%, particularly preferably 1 to 1.5 wt.-%, based on the composition and calculated as the sum of all components of the chemical buffer system; (f) optionally at least one blocking and / or stabilizing agent ("component (f)"), in particular in a concentration in the range from 0.001 µg / µl to 100 µg / µl, in particular 0.005 µg / µl to 80 µg / µl, preferably 0.01 µg / µl to 40 µg / µl, more preferably 0.05 µg / µl to 20 µg / µl, particularly preferably 0.1 µg / µl to 10 µg / µl, based on the composition; (g) optionally at least one inorganic salt ("component (g)"), in particular alkali and / or alkaline earth salt, preferably alkali and / or alkaline earth chloride, particularly preferably sodium chloride, in particular in an amount in the range from 0.01 to 15 wt.%, in particular 0.05 to 10 wt.%, preferably 0.1 to 10 wt.%, preferably 0.5 to 5 wt.%, particularly preferably 1 to 3 wt.-%, based on the composition; and (h) optionally at least one polyalkylene glycol ether ("component (h)"), in particular in an amount in the range from 0.001 to 5 wt.%, in particular 0.01 to 3 wt.%, preferably 0.05 to 2 wt.%, more preferably 0.1 to 1.5 wt.%, particularly preferably 0.12 to 1 wt.%, based on the composition.

[0085] As can be seen from the above, based on the targeted combination according to the invention, even small amounts of hybridization probes or hybridization probes in low concentrations can be stabilized for the first time in compositions for use in in situ hybridization. The compositions according to the invention are suitable for use in both automated in situ hybridization methods and in in situ hybridization methods with short or flexible hybridization times, such as Fast ISH, and lead to easily analyzable signal patterns with strong signals. Compositions of this type are previously unknown in the prior art.

[0086] The compositions according to the invention can be used in particular for in situ hybridizations in connection with the diagnosis and / or prognosis of diseases, in particular malignancies, preferably carcinomas, sarcomas and / or leukemias.

[0087] The genes to be investigated in this context are preferably selected from the group of ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B, TGF, BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1, RUNX1T1, EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, FUS, PAX3, PAX7, PDGFB, SS18, TFE3, USP6, WT1, HER2 / ERBB2, FGFR1, ALK, CCND1, CDK4, CD274, PDCD1LG2, EGR1, EGFR, ESR1, ETV1, FGF3,4,19, FGFR2, FGFR3, FHIT (RCC), KRAS, MDM2, MDM4, MET, MYB, MYC, MYCN, PIK3CA, PTEN, SMARCB1, SOX2, TERT, TOP2A, TP53, TYMS and / or VHL.

[0088] Particularly good results are achieved within the scope of the present invention when the compositions according to the invention are used for the detection of chromosomal aberrations based on inversions and / or translocations: In this context, the compositions according to the invention can be used in particular for the detection of different translocations and / or inversions, in particular in lung tumors, wherein in particular the genes ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B and / or TGF are affected.

[0089] Furthermore, it can be provided to use the composition according to the invention for the detection of different translocations and / or inversions, in particular in lymphomas and leukemias, wherein in particular the genes BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1 and / or RUNX1T1 are affected.

[0090] According to a further preferred embodiment of the present invention, the composition according to the invention can be used to detect different translocations and / or inversions, in particular in sarcomas, wherein in particular the genes EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, FUS, PAX3, PAX7, PDGFB, SS18, TFE3, USP6 and / or WT1 are affected.

[0091] It can also be provided according to the invention to use the composition for the detection of inversions and / or translocations, in particular the genes ALK and ROS1 being affected.

[0092] Within the scope of a preferred embodiment of the present invention, the composition according to the invention can also be used to detect different translocations and / or inversions, in particular in lung tumors, in particular the genes ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B and / or TGF being affected.

[0093] Furthermore, it can be provided that the composition according to the invention is used for the detection of different translocations and / or inversions, in particular in lymphomas and leukemias, wherein in particular the genes BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1 and / or RUNX1T1 are affected.

[0094] According to a further preferred embodiment of the present invention, the composition according to the invention is used for the detection of different translocations and / or inversions, in particular in sarcomas, wherein in particular the genes EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, FUS, PAX3, PAX7, PDGFB, SS18, TFE3, USP6 and / or WT1 are affected.

[0095] It can also be provided according to the invention that the composition according to the invention is used to detect inversions and / or translocations, in particular the genes ALK and ROS1 being affected.

[0096] Another object of the present invention is also - according to a second Aspect of the present invention - the use of a composition as described above in hybridization, in particular in situ hybridization, preferably automated in situ hybridization, in particular for the detection and / or proving of nucleic acids, preferably RNA and / or DNA, in a biological sample, preferably in one or more cells and / or in one or more cell nuclei.

[0097] For further details on the use according to the invention, reference can be made to the preceding statements on the first aspect of the invention, relating to the composition according to the invention, which apply accordingly with regard to the use according to the invention.

[0098] Another object of the present invention - according to a third Aspect of the present invention is a method for detecting nucleic acids, preferably RNA and / or DNA, and / or chromosomal aberrations in a biological sample, preferably in one or more cells and / or in one or more cell nuclei by means of hybridization, in particular in situ hybridization, preferably automated in situ hybridization, using a composition as described above.

[0099] Within the scope of the present invention, it has surprisingly been found that the use of the compositions according to the invention in the inventive, particularly automated, hybridization method, in particular in situ hybridization method, leads to good signal patterns with strong and easily analyzable signals. Furthermore, the inventive method for detecting nucleic acids using the compositions according to the first aspect of the invention can surprisingly also be carried out with short hybridization times without a decrease in signal intensity or strength.

[0100] In particular, the subject of the present invention according to this aspect of the invention is a method for detecting nucleic acids, preferably RNA and / or DNA, and / or chromosomal aberrations in a biological sample, preferably in one or more cells and / or in one or more cell nuclei by means of hybridization, in particular in situ hybridization, preferably automated in situ hybridization, wherein the method comprises the following method steps: (a) Providing a biological sample, in particular based on one or more cells and / or one or more cell nuclei, preferably in the form of tissue, for in situ hybridization; (b) Providing a composition for use in hybridization, wherein the composition comprises at least one, preferably locus-specific, hybridization probe ("component (a)"), at least one polar protic or polar aprotic solvent, preferably with a cyclic molecular structure ("component (b)"), and at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount of more than 10 vol.-%, based on the composition ("component (c)"), in particular a composition as described above; (c) bringing the biological sample from process step (a) into contact with the composition from process step (b); (d) denaturing the biological sample from process step (a) and the composition from process step (b), wherein the biological sample and the composition are denatured separately from one another, in particular before carrying out process step (c), or together, in particular after carrying out process step (c); (e) subsequent hybridization of the at least one, preferably locus-specific, hybridization probe contained in the composition and the nucleic acids contained in the biological samples; (f) subsequent detection of the hybridized, preferably locus-specific, hybridization probes and / or the nucleic acids to be detected in the biological sample.

[0101] According to a particularly preferred embodiment of the method according to the invention, it can be carried out on or in a machine and / or automatically. On this basis, the efficiency and sample throughput of hybridization methods, in particular in situ hybridizations, can be significantly increased.

[0102] With regard to process step (a) or the provision of a biological sample for carrying out the method according to the invention, in particular in situ hybridization, this is carried out in a manner known per se to those skilled in the art, i.e. the biological samples are prepared for in situ hybridization in a manner known per se. In particular, process step (a) comprises the fixation and embedding of the samples on the basis of cells or tissues, the application of the samples to suitable supports, sample pretreatment, and drying of the samples. The provision or preparation of samples for in situ hybridization methods is generally known to those skilled in the art, so that no further explanation is required here.

[0103] In connection with process step (b) or the provision of the composition, reference is made to the above aspects of the invention, in particular the statements in connection with the composition according to the invention, in order to avoid unnecessary repetition.

[0104] Furthermore, with regard to process step (c), it has proven advantageous if the composition is used in an amount in the range of 0.1 to 5,000 µl, in particular 1 to 2,500 µl, preferably 2 to 1,500 µl, more preferably 5 to 1,000 µl, particularly preferably 10 to 500 µl, very particularly preferably 20 to 250 µl, and even more preferably 40 to 150 µl. The aforementioned amounts of the composition lead to high-quality and easily analyzable signal patterns both in automated or automatic in situ hybridizations and in in situ hybridizations with short hybridization times. The composition is used, in particular, in an amount sufficient to cover the sample area under investigation.

[0105] Process step (d) for denaturing the nucleic acids in the biological sample and in the composition is carried out in a manner generally known to those skilled in the art. Within the scope of the process according to the invention, the biological sample, on the one hand, and the composition, on the other hand, can be subjected to denaturation separately from one another before being brought into contact according to process step (e). Likewise, it can be provided that the biological sample and the composition are co-denatured together after carrying out process step (c). The setting of the denaturation conditions, in particular the temperature required for this purpose, is known per se to those skilled in the art and requires no further explanation. In particular, the temperature in process step (d) can be in the range from 60 to 90 °C, preferably in the range from 70 to 85 °C.

[0106] Process step (e), which serves to hybridize the preferably locus-specific hybridization probes contained in the hybridization compositions, on the one hand, and the DNA or chromosome regions to be detected contained in the biological samples, on the other hand, is also carried out in a manner generally known to those skilled in the art. In particular, with regard to setting a suitable hybridization temperature, no further explanation is necessary at this point.

[0107] In connection with process step (e), it has also been surprisingly found within the scope of the present invention that the hybridization, ie the attachment of the, preferably locus-specific, hybridization probes to the DNA or chromosome section to be detected, can be further improved if process step (e) is carried out under movement, in particular wave-like and / or continuous movement.

[0108] In the context of the present invention, it has also been surprisingly found that the method according to the invention using the compositions according to the invention leads to excellent results both with short hybridization times ("rapid ISH"), e.g., from 10 to 240 min, and with the usually used long hybridization times, e.g., 4 to 100 h.

[0109] Thus, within the scope of the present invention, it can be provided that, within the scope of the method according to the invention, the hybridization or method step (e) is carried out over a period in the range from 10 min to 240 min, in particular in the range from 30 min to 180 min, preferably in the range from 60 min to 150 min, more preferably in the range from 90 min to 130 min. Furthermore, it can also be provided that the hybridization or method step (e) is carried out over a period in the range from 1 h to 100 h, in particular 2 h to 80 h, preferably 3 h to 50 h, preferably 4 h to 30 h, particularly preferably 5 h to 25 h, even more preferably 8 h to 20 h.

[0110] The detection of the bound or hybridized, preferably locus-specific, hybridization probes in the biological sample is also carried out using methods known to those skilled in the art, depending on the labeling of the hybridization probes. No further explanation is required in this regard. Within the scope of the method according to the invention, preference is given to using fluorescently labeled hybridization probes (fluorescent ISH (FISH)), which are detected by fluorescence microscopy, or antigen-labeled probes, in particular hapten-labeled probes, which are visualized with the aid of antibodies through color reactions and detected by light microscopy (brightfield ISH (BrISH), chromogenic ISH (CISH), silver ISH (SISH)). How exactly the labeled hybridization probes used are to be detected within the scope of the method according to the invention is self-evident to those skilled in the art.

[0111] For further details on the method according to the invention, reference can be made to the preceding statements on the above aspects of the invention, which apply accordingly with regard to the method according to the invention.

[0112] Finally, the subject of the present invention is - according to a fourth Aspect of the present invention - a kit or kit-of-parts or system for the detection of nucleic acids, preferably RNA and / or DNA, and / or chromosomal aberrations in a biological sample, preferably in one or more cells and / or in one or more cell nuclei by means of hybridization, in particular in situ hybridization, preferably automated in situ hybridization, wherein the kit contains a composition as described above, wherein the kit is intended and / or used and / or suitable for carrying out the method described above.

[0113] According to a preferred embodiment, the kit according to the invention may contain the components of the composition as described above in a common storage vessel or application device or in spatially separate, mutually different storage vessels and / or application devices.

[0114] For further details on the kit according to the invention, reference can be made to the preceding statements on the above aspects of the invention, which apply accordingly with regard to the kit according to the invention. Short description of the characters

[0115] Further features, advantages and special features of the present invention will become apparent from the following description of preferred embodiments based on the Figures 1 to 4 .

[0116] They show: Fig. 1: A schematic representation of in situ hybridization results concerning the signal patterns, particularly the signal strength. Fig. 2: A schematic representation of the signal patterns and particularly the signal strengths when using an "Auto ALK Break Apart Probe" FISH probe in in situ hybridizations using automated staining machines. Fig. 3: A schematic representation of the signal patterns and particularly the signal strengths when using a "Flexible ROS1 Break Apart Probe" FISH probe in in situ hybridizations. Fig. 4: A schematic representation of the signal patterns and particularly the signal strengths when using a "Flexible HER2 / CEN17 Probe" FISH probe in in situ hybridizations. More detailed description of the characters

[0117] Due to the better representation in black and white, the figures shown here do not represent fluorescence images of the experimental results described below, but rather schemes that were created based, among other things, on the respective observed results.

[0118] Fig. 1 . shows a schematic representation of in situ hybridization results (exemplary for FISH, BrISH, and CISH), which allows conclusions to be drawn about the signal pattern obtained and, in particular, the signal intensities. Locus-specific hybridization probes can be used, in particular, probes specific for genomic regions of the human genes HER2, ALK, or ROS1.

[0119] In principle, the present invention assumes diploid cells, i.e., two signals are obtained for each detected genomic region, or a double signal in cells without aberrations. The obtained signal strengths are divided into the following five levels: a) very strong signals, b) strong signals, c) moderately strong signals, d) weak signals, e) no signals, cf. Fig. 1 ). Examples of signal patterns, in particular signal intensities, of in situ hybridizations with standard hybridization solutions on the one hand and hybridization solutions according to the invention on the other hand, or of in situ hybridizations according to the method according to the invention and according to standard methods, are shown below.

[0120] The inventive methods and compositions based on specific concentrations of formamide and polar protic or polar aprotic solvent with cyclic molecular structure lead to very strong or strong signals ( Fig. 1 a) and b These excellent signal patterns are observed both in flexible in situ hybridizations, i.e., in situ hybridizations with a two-hour hybridization time ("Fast-ISH") or with a 16-hour hybridization time, as well as in automated in situ hybridization procedures. The signal intensity is a result of both the brilliance and the contrast with the background (for example, a strong background leads to weaker contrast and thus less strong signals).

[0121] Compositions or hybridization solutions containing a polar protic or polar aprotic solvent with a cyclic molecular structure, but no formamide, do not lead to any hybridization signals ( Fig. 1 e) ). Compositions or hybridization solutions that contain formamide but no polar protic or polar aprotic solvent with a cyclic molecular structure also lead to no hybridization signals or at best to very weak or barely detectable hybridization signals ( Fig. 1 e) and f)).

[0122] Fig. 2shows a schematic representation of the signal patterns and signal intensities of in situ hybridizations performed using an FISH probe "Auto ALK Break Apart Probe" on an automated stainer. This locus-specific hybridization probe consists of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against sequences located proximal to the ALK breakpoint region in 2p23, and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against sequences located distal to the ALK breakpoint region in 2p23. The hybridization solution or composition is based on the following components: 18 wt% dextran sulfate, 600 mM NaCl, 22 vol% formamide, 1x concentrated SSC buffer, 12.5 wt%-% ethylene carbonate, blocking and stabilizing DNA at a concentration of 0.1 ug / ul and locus-specific hybridization probes at a concentration of 2 ng / µl, each based on the hybridization solution or composition.

[0123] After denaturation (20 minutes at 75 °C), the hybridization probe is hybridized with cell and tissue samples over a hybridization or renaturation period of 120 minutes at 45 °C in an automated system (Celerus Wave RPD System) with continuous wave movement of the slides.

[0124] When using appropriate filter sets, the hybridization signals for the non-rearranged ALK gene appear as green-orange fluorescence fusion signals. In the interphase of a normal cell (without ALK aberration), two green-orange double or fusion signals appear when using an appropriate green-orange dual bandpass filter set ( Fig. 2la). A 2p23 locus affected by an ALK translocation is indicated by a separate green signal and a separate orange signal ( Fig. 2 Ib).

[0125] The obtained signal intensities and thus the evaluability of the results depend crucially on the specific concentrations of the polar protic or polar aprotic solvents with a cyclic molecular structure (CPAL) and the formamide in the underlying hybridization solutions of the hybridization probes. At low concentrations of the polar protic or polar aprotic solvents with a cyclic molecular structure (CPAL), e.g., as in this case ethylene carbonate, between 5 wt.% and 13 wt.%, and formamide concentrations greater than 10 vol.%, very strong fluorescence signals appear with very low background (Fig. 2 l). Reference compositions that do not contain formamide do not lead to evaluable results, i.e., no gene-specific signals can be generated ( Fig. 2II). Reference compositions that do not contain a polar protic or polar aprotic solvent with a cyclic molecular structure, e.g. ethylene carbonate, lead to very weak and therefore difficult to evaluate signals ( Fig. 2 III).

[0126] Fig. 3shows a schematic representation of the signal patterns and signal intensities of in situ hybridizations performed using a FISH probe, the "Flexible ROS1 Break Apart Probe." This locus-specific hybridization probe consists of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against sequences located proximal to the ROS1 breakpoint region in 6q22, and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against sequences located distal to the ROS1 breakpoint region in 6q22. The hybridization solution or composition is based on the following components: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1x concentrated SSC buffer, 9.75 wt%-% ethylene carbonate, blocking and stabilizing DNA at a concentration of 2 µg / µl, and locus-specific hybridization probes at a concentration of 5 ng / µl, each based on the hybridization solution or composition. After denaturation (10 minutes at 75 °C), the hybridization probes are hybridized with cell and tissue samples for a hybridization or renaturation time of 10 minutes, either for 2 hours at 37 °C or for 16 hours at 37 °C.

[0127] When using appropriate filter sets, the hybridization signals for the non-rearranged ROS1 gene appear as green-orange fluorescence signals. In the interphase of a normal cell (without ROS1 aberration), two green-orange fusion or double signals appear when using an appropriate green-orange dual-bandpass filter set ( Fig. 3la). A 6q22 locus affected by a ROS1 translocation is indicated by a separate green signal and a separate orange signal ( Fig. 3 Ib).

[0128] The signal intensities obtained, and thus the evaluability of the results, depend crucially on the specific concentrations of the polar protic or polar aprotic solvents with a cyclic molecular structure (CPAL) and the formamide in the underlying hybridization solutions of the locus-specific hybridization probe. At low concentrations of the polar protic or polar aprotic solvents with a cyclic molecular structure (CPAL), e.g., ethylene carbonate in this case, in the range of 5 wt.% and 13 wt.%, as well as formamide concentrations of more than 10 wt.%, very strong fluorescence signals appear with very low background after both a hybridization time of two hours and 16 hours (Fig. 3 l). With regard to signal intensity, excellent results are achieved with the hybridization solutions according to the invention, both with short and long hybridization times.

[0129] Reference compositions containing a polar protic or polar aprotic solvent with a cyclic molecular structure, but no formamide, do not lead to any evaluable results after either two or 16 hours of hybridization, ie no gene-specific signals can be generated ( Fig. 3 II). Reference compositions containing formamide but no polar protic or polar aprotic solvent with a cyclic molecular structure (CPAL) lead to very weak signals ( Fig. 3 III).

[0130] Fig. 4shows a schematic representation of the signal patterns and signal intensities of in situ hybridizations performed using a flexible FISH probe, the "Flexible HER2 / CEN17 Probe." This probe consists of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against the 17q11.2-q12 region of the HER2 gene and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against the alpha satellite centromere region of chromosome 17 (D17Z1). The hybridization solution contains the following components: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate, blocking and stabilizing DNA at a concentration of 2 µg / µl and locus-specific hybridization probes at a concentration of 5 ng / µl probe DNA, each based on the composition.After denaturation (10 minutes at 75 °C), the hybridization probes are hybridized with cell and tissue samples either for two hours at 37 °C or for 16 hours at 37 °C.

[0131] When using appropriate filter sets, the hybridization signals for the non-rearranged HER2 gene appear as two green fluorescence signals and the hybridization signals for the non-rearranged centromere region of chromosome 17 appear as two orange fluorescence signals in the generated signal pattern. In the interphase of a normal cell without HER2 aberration and without aberrations of chromosome 17, two green and two orange signals appear when using an appropriate green-orange dual-bandpass filter set ( Fig. 4 la and Ila). A 17q11.2-q12 locus affected by HER2 amplification is indicated by additional green signals ( Fig. 4 Ib and IIb).

[0132] The signal intensities obtained, and thus the evaluability of the results, depend crucially on the presence of dextran sulfate in the underlying hybridization solutions or compositions. Hybridization solutions containing a polar protic or polar aprotic solvent with a cyclic molecular structure, e.g., ethylene carbonate, in an amount ranging from 5 wt% to 13 wt%, formamide in an amount of at least 10 wt%, and dextran sulfate in an amount of 15 wt%, produce very strong fluorescence signals with very low background in in situ hybridizations, both for short and long hybridization times of two or 16 hours, respectively ( Fig. 4 I and II). Hybridization solutions that do not contain dextran sulfate do not produce any analyzable results, ie, no gene-specific signals can be generated ( Fig. 4 III). Examples of implementation

[0133] A) Before individual, particularly preferred embodiments of the present invention are described, the underlying in situ hybridization methods as such are first described. Example A.1 ISH Procedure I (Flexible FISH with hybridization times of two hours or 16 hours)

[0134] The reagents used to perform the "flexible" rapid FISH came from the ZytoLight Flexible FISH-Tissue Implementation Kit (Z-2182-20, ZytoVision GmbH, Bremerhaven, Germany). The kit contains the necessary reagents (Heat Pretreatment Solution Citric, Pepsin Solution, 5x Flexible FISH Wash Buffer, and DAPI / DuraTect™ Solution) for performing FISH on formalin-fixed and paraffin-embedded tissue sections.

[0135] FISH was performed on 3- to 5-µm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissue from breast cancer, lung tissue, lymph node tissue, kidney tissue, prostate tissue, and placenta tissue. The tissue sections were mounted on coated slides and baked overnight at 58 °C.

[0136] To remove the paraffin, the specimens were incubated twice in 100% xylene for five minutes each at room temperature (RT). A descending ethanol series was then carried out for two minutes each at room temperature (twice each with 96%, 90%, and 70% denatured ethanol). After two incubation steps of two minutes each in ultrapure water at room temperature, heat pretreatment was carried out for 20 minutes at 98°C in Heat Pretreatment Solution Citric, followed by two further incubation steps of two minutes each in ultrapure water at RT. The proteolytic pretreatment was performed by dropwise application of Pepsin Solution (RTU) to the specimens and subsequent incubation in a humid chamber at 37°C for a period of 5 to 30 minutes. The digestion was followed by two incubations for two minutes each at RT in ultrapure water and an ascending ethanol series (70%, 90%, 96%) for one minute each at RT.

[0137] After air-drying the specimens, 10 µl of a hybridization solution containing the fluorescently labeled hybridization probes was applied directly to the sections using a pipette. After applying a suitable coverslip, the specimens were sealed with Fixogum and stored on a hotplate at 75 °C for ten minutes for co-denaturation. For hybridization, the specimens were transferred to a preheated humid chamber and incubated at 37 °C for either two hours or overnight (approximately 16 hours).

[0138] Before stringency washing, the Fixogum was removed, and the specimens were incubated in 1x Flexible Wash Buffer for approximately two minutes at RT. The coverslip was then removed, and the actual washing was performed by incubating in 1x Flexible Wash Buffer for ten minutes at 72°C, followed by a further three minutes at RT. This was followed by an ascending ethanol series (70%, 90%, 96%) for one minute each at RT before the specimens were air-dried in the absence of light. Finally, DAPI DuraTect Solution was applied, and the specimen was coverslipped.

[0139] The evaluation was carried out using a fluorescence microscope (Axio Scope.A1 with illumination unit HXP 120V, Carl Zeiss Microscopy GmbH) and appropriate filter sets for the respective underlying absorption and emission ranges. Example A.2 ISH Procedure II (Automated FISH on the Celerus Wave RPD System)

[0140] The reagents used for automated FISH (on the Celerus Wave RPD System from Celerus Diagnostics, California, USA) were contained in the LRM bin (Celerus Diagnostics, California, USA). The LRM (Linear Reagent Magazine) contained all necessary reagents for heat pretreatment, proteolysis, and the wash buffer for the necessary washing steps. DAPI / DuraTect™ (ZytoVision GmbH, Bremerhaven, Germany) was used for coverslipping the specimens and staining the nuclei. The automated probe was located in the PAC bin, which was inserted into the LRM bin.

[0141] Automated FISH was performed on 3- to 5-µm-thick sections of formalin-fixed, paraffin-embedded (FFPE) human tissue from breast cancer, lung, lymph nodes, kidneys, prostate, and / or placenta, which were mounted on coated slides and baked overnight at 58°C. To remove the paraffin, the specimens were treated according to the predefined program of the Celerus Wave®< RPD System. This was followed by heat pretreatment for 15 minutes at 95°C, followed by two incubation steps in wash buffer for six minutes at 40°C. After drying for 10 minutes at 45°C, proteolytic pretreatment followed. This was performed with pepsin by incubating the specimens for a period ranging from 5 to 40 minutes at 60°C. The digestion was followed by two incubations in ultrapure water of four minutes each at 37 °C and then a drying step of seven minutes at 50 °C.

[0142] After drying, the fluorescently labeled locus-specific hybridization probe, which was present in the hybridization solution, was applied. For this purpose, 130 µl of the hybridization solution was applied to each slide. Co-denaturation took place over a period of ten minutes at 80 °C, followed by hybridization for 120 minutes at 42 °C. Hybridization was accompanied by constant movement of the slides, thus creating wave-like movements of the hybridization solution. After hybridization, the slides were washed with wash buffer for four minutes at 37 °C.

[0143] The stringency wash was performed for 15 minutes at 45°C. A final wash was performed for four minutes at 37°C. Finally, the specimens were dried for five minutes at 45°C on the automated system. After these steps had been performed automatically, the specimens were removed from the machine. This was followed by an ascending ethanol series (70%, 90%, 96%) for one minute each at room temperature before the specimens were air-dried in the absence of light. Finally, the cell nuclei were stained using the dye 4',6-diamidine-2-phenylindole (DAPI) by applying DAPI DuraTect Solution (ZytoVision GmbH, Bremerhaven), and the specimen was coverslipped.

[0144] The evaluation was carried out using a fluorescence microscope (Axio Scope.A1 with illumination unit HXP 120V, Carl Zeiss Microscopy GmbH) and appropriate filter sets for the respective underlying absorption and emission ranges. Example A.3 ISH Procedure III (Automated FISH on the Pathcom Stainer)

[0145] The reagents used for automated FISH (on the Pathcom Stainer from PathCom Systems Corporation, California, USA) came from the ISH Detection Kit (PathCom Systems Corporation, Sierra CT, Dublin, USA). The kit contained all necessary reagents (Dewax Solutions, Retrieval Solution, Pepsin, diH2O) for automated FISH. The PathCom Systems Wash Buffer for IHC and ISH was also required. DAPI / DuraTect™ Solution (ZytoVision GmbH, Bremerhaven, Germany) was used for mounting the specimens and staining the nuclei.

[0146] The automated FISH was performed on 3 to 5 µm thick sections of formalin-fixed and paraffin-embedded (FFPE) human tissues, e.g. from breast carcinomas, lungs, lymph nodes, kidneys, prostate and / or placenta, which were mounted on coated slides and baked overnight at 58 °C.

[0147] To remove the paraffin, the specimens were first treated with an initial Dewax Solution for six minutes at 65°C. This was followed by treatment with four additional Dewax Solutions for six minutes each at 62°C, and finally with a final Dewax Solution for six minutes at 50°C. This was followed by heat pretreatment with the Retrieval Solution, initially for 15 minutes at 98°C and then for eight minutes at 65°C, so that the total heat pretreatment lasted approximately 23 minutes. After two incubation steps with a wash buffer (“Wash Buffer”) for six minutes each at 40°C, the specimens were dried for ten minutes at 45°C. Drying was followed by proteolytic pretreatment. This was carried out by incubating the specimens in pepsin for a period of between 5 and 40 minutes at 37°C.The digestion was followed by three incubations in ultrapure water for three minutes each at 37 °C and then a drying step for ten minutes at 50 °C.

[0148] After drying, 110 µl of hybridization solution containing the fluorescently labeled locus-specific hybridization probes was applied to the specimens. Co-denaturation of the sample and hybridization solution was carried out over a period of 20 minutes at 75 °C. Following denaturation, hybridization or renaturation was carried out over a period of 120 minutes at 45 °C with continuous agitation of the hybridization solution by induced agitation of the reaction chamber in the automated system.

[0149] After hybridization, the specimens were washed with wash buffer for four minutes at 37°C. The stringency wash was performed for 15 minutes at 45°C. A final wash was performed for four minutes at 37°C. Finally, they were dried for five minutes at 45°C on the automated system. After these steps were completed automatically, the specimens were removed from the system. This was followed by an ascending ethanol series (70%, 90%, 96%) for one minute each at room temperature before the specimens were air-dried in the absence of light. Finally, the nuclei were stained with the dye 4',6-diamidine-2-phenylindole (DAPI) by applying "DAPI DuraTect™< Solution" (ZytoVision GmbH, Bremerhaven), and the specimen was coverslipped.

[0150] The evaluation was carried out using a fluorescence microscope (Axio Scope.A1 with illumination unit HXP 120V, Carl Zeiss Microscopy GmbH) and appropriate filter sets for the respective underlying absorption and emission ranges.

[0151] B) In the following, particularly preferred embodiments of the present invention as well as comparative in situ hybridizations are described: Example B.1 FISH for the detection of gene-specific HER2 signals in a "flexible" rapid FISH procedure

[0152] Using the locus-specific hybridization probe "Flexible HER2 / CEN17 Probe," gene-specific HER2 signals were generated or detected using a "flexible" rapid FISH procedure, which can be performed with both short hybridization times of two hours and overnight hybridization of 16 hours. Hybridization itself was performed according to the ISH procedure I described above (Flexible FISH with hybridization times of two hours or 16 hours).

[0153] The hybridization probe consisted of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against the 17q11.2-q12 region of the HER2 gene and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against the alpha satellite centromere region of chromosome 17 (D17Z1).

[0154] Hybridization solution I: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 12 wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA and 5 ng / µl locus-specific hybridization probe, each based on the composition.

[0155] Hybridization solution II: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5% wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA, and 5 ng / µl locus-specific hybridization probe, each based on the composition.

[0156] Both hybridization solutions I and II produced equally excellent results, i.e., very strong signals. Very strong gene-specific HER2 signals were observed alongside equally strong CEN17-specific signals with very low background and well-preserved cell and tissue structure, both with short hybridization times of two hours and with long hybridization times of 16 hours. This could be observed both with normal cells / tissues or cells / tissues without aberrations on chromosome 17 as well as with cells / tissues, particularly from breast carcinomas, with HER2 amplifications. Thus, the HER2 amplifications to be identified with the hybridization probe, which were identified in the signal pattern based on the occurrence of multiple HER2-specific signals or HER2 signal clusters (cf. Fig. 4 ), can be clearly detected. Example B.2 Influence of formamide concentration on flexible rapid FISH methods

[0157] Using the locus-specific hybridization probe "Flexible HER2 / CEN17 Probe" for the ISH procedure I described above, the influence of the formamide concentration in the hybridization solutions on the signal intensity was investigated.

[0158] The hybridization probe consisted of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against the 17q11.2-q12 region of the HER2 gene and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against the alpha satellite centromere region of chromosome 17 (D17Z1).

[0159] Hybridization solution I: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA and 5 ng / µl locus-specific hybridization probe, each based on the composition.

[0160] Compared to hybridization solution I, hybridization solutions II to V have different formamide concentrations of 14 vol% (II), 11 vol% (III), 7 wt% (IV) and 0 vol% (V).

[0161] Hybridization solutions I to V were each used within the framework of the ISH procedure I described above, both with short hybridization times of two hours and with long hybridization times of 16 hours, using the "Flexible HER2 / CEN17 Probe" hybridization probe. The resulting signal patterns were compared, particularly with regard to the signal intensity achieved.

[0162] With hybridization solution I (27 vol% formamide), very strong gene-specific HER2 signals were obtained alongside very strong CEN17-specific signals with very low background and well-preserved cell and tissue structure at both short and long hybridization times.

[0163] Lower formamide concentrations in the hybridization solutions resulted in weaker or no HER2-specific signals after the two-hour hybridization period. Strong signals were generated with 14 vol% formamide (Solution II), which were still very well evaluated. In contrast, 11 vol% formamide (Solution III) produced only moderately strong signals in the signal patterns. Hybridization solutions with 7 vol% formamide (Solution IV) resulted in only weak signals, which no longer allowed evaluation. No signals were obtained with hybridization solutions without formamide (Solution V).

[0164] Even with long hybridization times of 16 hours, decreasing formamide concentrations in the hybridization solutions led to decreasing signal strengths for the HER2-specific signals. Very strong signals were generated with 14 vol% formamide (solution II), which were still very well evaluated. Strong signals were also generated with 11 vol% formamide (solution III), which were still easily evaluated. Hybridization solutions with 7 vol% formamide (solution IV) resulted in only weak signals, which no longer allowed evaluation. No signals were obtained with hybridization solutions without formamide (solution V). Example B.3 Influence of ethylene carbonate concentration on flexible rapid FISH methods

[0165] Using the locus-specific hybridization probe "Flexible HER2 / CEN17 Probe" for the ISH procedure I described above, the influence of the ethylene carbonate concentration in the hybridization solutions on the signal intensity was investigated.

[0166] The hybridization probe consisted of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against the 17q11.2-q12 region of the HER2 gene and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against the alpha satellite centromere region of chromosome 17 (D17Z1).

[0167] Hybridization solution I: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA and 5 ng / µl probe DNA, each based on the composition.

[0168] Compared to hybridization solution I, hybridization solutions II to IV had different ethylene carbonate concentrations of 7 wt% (II), 5 wt% (III) and 0 wt% (IV).

[0169] Hybridization solutions I to IV were each used within the framework of the ISH procedure I described above, both with short hybridization times of two hours and with long hybridization times of 16 hours, using the hybridization probe "Flexible HER2 / CEN17 Probe." The resulting signal patterns were compared, particularly with regard to the signal intensity achieved.

[0170] With hybridization solution I (10.5 wt% ethylene carbonate), very strong gene-specific HER2 signals were obtained alongside very strong CEN17-specific signals with very low background and well-preserved cell and tissue structure, both with short and long hybridization times.

[0171] Lower ethylene carbonate concentrations in the hybridization solutions resulted in weaker or no HER2-specific signals after the two-hour hybridization period. Strong signals were generated with 7 wt% ethylene carbonate (Solution II), which were still very well interpretable. In contrast, 5 wt% ethylene carbonate (Solution III) produced only moderately strong signals in the signal patterns. Hybridization solutions without ethylene carbonate (Solution IV) produced only very weak and no longer interpretable signals.

[0172] Even with long hybridization times of 16 hours, decreasing ethylene carbonate concentrations in the hybridization solutions led to decreasing signal strengths for the HER2-specific signals. Very strong signals were generated with 7 wt% ethylene carbonate (solution II). Very strong signals were also generated with 5 vol% ethylene carbonate (solution III). Only moderately strong signals were obtained with hybridization solutions without ethylene carbonate (solution IV). Example B.4 Influence of dextran sulfate on flexible rapid FISH methods

[0173] Using the locus-specific hybridization probe "Flexible HER2 / CEN17 Probe" for the ISH procedure I described above, the influence of dextran sulfate in the hybridization solutions on signal intensity was investigated. Regarding the hybridization procedure and the specificity of the probes, reference is made to the above explanations.

[0174] Hybridization solution I: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA and 5 ng / µl probe DNA, each based on the composition.

[0175] Hybridization solution II: 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate, 2 µg / µl blocking and stabilizing DNA and 5 ng / µl probe DNA, each based on the composition.

[0176] The signal patterns obtained with hybridization solutions I and II were compared, particularly with regard to the signal strength achieved.

[0177] The signal patterns obtained with Hybridization Solution I showed very strong gene-specific HER2 signals alongside equally strong CEN17-specific signals with very low background and well-preserved cell and tissue structure, both with short and long hybridization times. Hybridization Solution II without dextran sulfate resulted in no visible or detectable signals with short hybridization times. With long hybridization times, only very weak or no visible or detectable signals were obtained with Hybridization Solution II. Example B.5 Influence of different polar protic or polar aprotic solvents with cyclic molecular structure on flexible rapid FISH methods

[0178] Using the locus-specific hybridization probe "Flexible HER2 / CEN17 Probe" for the ISH procedure I described above, the influence of various polar protic and polar aprotic solvents with cyclic molecular structures in the hybridization solutions on signal intensity was investigated. Regarding the hybridization procedure and the specificity of the probes, reference is made to the above explanations.

[0179] Hybridization solution I: 15 wt% dextran sulfate, 500 mM NaCl, 27 vol% formamide, 1-fold concentrated SSC buffer, 10.5 wt% ethylene carbonate (I), 2 µg / µl blocking and stabilizing DNA and 5 ng / µl probe DNA, each based on the composition. Hybridization solution II: instead of ethylene carbonate, 10.5 wt% 2-piperidone (valerolactam) Hybridization solution III: instead of ethylene carbonate, 10.5 vol% 2-pyrrolidone (γ-butyrolactam) Hybridization solution IV: instead of ethylene carbonate, 10.5 wt% 3-sulfolene (butadiene sulfone) Hybridization solution V: instead of ethylene carbonate, 10.5 vol% γ-butyrolactone

[0180] The signal patterns obtained with hybridization solutions I to V were compared with each other, particularly with regard to the signal strength achieved in each case.

[0181] With all hybridization solutions I to V, i.e., with all tested polar protic or polar aprotic solvents with a cyclic molecular structure, very strong gene-specific HER2 signals were obtained alongside equally strong CEN17-specific signals with very low background and well-preserved cell and tissue structure, both with short and long hybridization times. The best results were achieved with 2-pyrrolidone (γ-butyrolactam), γ-butyrolactone, and ethylene carbonate, although very good results were also obtained with 2-piperidone (valerolactam) and 3-sulfolene ("butadiene sulfone").

[0182] These results were observed both in normal cells / tissues or cells / tissues without aberrations on chromosome 17, as well as in cells / tissues with HER2 amplifications, particularly from breast cancer. Thus, the HER2 amplifications to be identified could be clearly identified even with all polar protic or polar aprotic solvents with a cyclic molecular structure used individually. Example B.6 FISH for the detection of gene-specific ALK signals in automated FISH procedures

[0183] To detect ALK-specific signals in automated FISH procedures, hybridization solutions based on formamide and ethylene carbonate were used, containing the "Auto ALK Break Apart Probe" as a locus-specific hybridization probe. FISH was performed according to the ISH procedure II described above (automated FISH on the Celerus Wave RPD system).

[0184] The locus-specific hybridization probe consisted of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against sequences in 2p23 proximal to the ALK breakpoint region and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against sequences in 2p23 distal to the ALK breakpoint region.

[0185] Hybridization solution: 18 wt% dextran sulfate, 600 mM NaCl, 22 vol% formamide, 1-fold concentrated SSC buffer, 12.75 wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl locus-specific hybridization probe, each based on the composition.

[0186] The resulting signal pattern showed very strong ALK-specific signals with very low background and well-preserved cell and tissue structure. This was observed in both normal cells / tissues or cells / tissues without aberrations on chromosome 2, as well as in cells / tissues with ALK aberrations. Thus, the ALK aberrations (i.e., break-apart events of green-orange fusion signals) identified with the hybridization probe could be clearly identified. Example B.7 Influence of formamide concentration on automated FISH procedures

[0187] Using the locus-specific hybridization probe "Auto HER2 / CEN17 Probe" for the ISH procedure III described above, the influence of the formamide concentration in the hybridization solutions on the signal strength was investigated.

[0188] The locus-specific hybridization probe consisted of orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against the 17q11.2-q12 region of the HER2 gene and green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against the alpha satellite centromere region of chromosome 17 (D17Z1).

[0189] Hybridization solution I: 18 wt% dextran sulfate, 600 mM NaCl, 21 vol% formamide, 1-fold concentrated SSC buffer, 12.75% wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl hybridization probe, each based on the composition.

[0190] Compared to hybridization solution I, hybridization solutions II to V had different formamide concentrations of 17 vol% (II), 13 vol% (III), 9 vol% (IV) and 0 vol% (V).

[0191] With Hybridization Solution I, very strong gene-specific HER2 signals (along with equally strong CEN17-specific signals) were detected with very low background and well-preserved cell and tissue structure. Very strong signals were also obtained with Hybridization Solution II (17 vol.% formamide). Hybridization Solution III with 13 vol.% formamide still produced strong signals, whereas with 9 vol.% (Hybridization Solution IV) only moderately strong signals were obtained. If the hybridization solutions (Hybridization Solution V) did not contain formamide, no signals were obtained. Example B.8 Influence of ethylene carbonate concentration on automated FISH procedures

[0192] Using the locus-specific hybridization probe "Auto HER2 / CEN17 Probe" for the ISH procedure III described above, the influence of the ethylene carbonate concentration in the hybridization solutions on signal intensity was investigated. Regarding the hybridization procedure and the specificity of the probes, reference is made to the above discussion regarding the hybridization probe "Auto HER2 / CEN17 Probe."

[0193] Hybridization solution I: 18 wt% dextran sulfate, 600 mM NaCl, 21 vol% formamide, 1-fold concentrated SSC buffer, 12.75 wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl hybridization probe, each based on the composition.

[0194] Compared to hybridization solution I, hybridization solutions II to IV had different ethylene carbonate concentrations of 9 wt% (II), 6 wt% (III) and 0 wt% (IV).

[0195] Hybridization Solution I detected very strong gene-specific HER2 signals (along with equally strong CEN17-specific signals) with very low background and well-preserved cell and tissue structure. Hybridization Solution II (9 wt% ethylene carbonate) yielded only moderately strong signals. Hybridization Solutions III and IV, with 6 vol% ethylene carbonate and without ethylene carbonate, respectively, resulted in weak and unmeasurable signals. Example B.9 Influence of dextran sulfate on automated FISH procedures

[0196] Using the locus-specific hybridization probe "Auto ROS1 Break Apart Probe" for the ISH procedure II described above (automated FISH on the Celerus Wave RPD system), the influence of dextran sulfate in the hybridization solutions on signal intensity was investigated. Regarding the hybridization procedure, please refer to the above explanations regarding procedure II.

[0197] The hybridization probe consisted of green-labeled polynucleotides (absorption at 503 nm and emission at 528 nm) directed against sequences located proximal to the ROS1 breakpoint region in 6q22 and orange-labeled polynucleotides (absorption at 547 nm and emission at 572 nm) directed against sequences located distal to the ROS1 breakpoint region in 6q22.

[0198] Hybridization solution I: 18 wt% dextran sulfate, 600 mM NaCl, 22 vol% formamide, 1-fold concentrated SSC buffer, 12.75 wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl locus-specific hybridization probes, each based on the composition.

[0199] Compared to Hybridization Solution I, Hybridization Solution II did not contain dextran sulfate.

[0200] Hybridization Solution I yielded very strong gene-specific ROS1 signals with very low background and well-preserved cell and tissue structure. Hybridization Solution II, which contains the hybridization probe in a composition without dextran sulfate, produced no visible or detectable signals. Example B.10 Influence of various selected polar protic or polar aprotic solvents with cyclic molecular structure on automated FISH methods

[0201] Using the locus-specific hybridization probe "Auto HER2 / CEN17 Probe" for the ISH procedure III described above (automated FISH on the Pathcom Stainer), the influence of various polar protic and polar aprotic solvents with a cyclic structure in the hybridization solutions on signal intensity was investigated. ISH procedure III (see above for ISH procedure III) was performed using the Pathcom Stainer. Regarding the specificity and labeling of the hybridization probe "Auto HER2 / CEN17 Probe," reference is made to the above statements to avoid unnecessary repetition.

[0202] Hybridization solutions I to V: 18 wt% dextran sulfate, 600 mM NaCl, 22 vol% formamide, 1-fold concentrated SSC buffer, 12.75 wt% or vol% (depending on the substance used) of a polar protic or polar aprotic solvent with a cyclic structure, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl hybridization probe, each based on the composition, whereby the polar protic or polar aprotic solvent with a cyclic structure is either ethylene carbonate (I, wt%), 2-piperidone (valerolactam) (II, wt%), 2-pyrrolidone (γ-butyrolactam) (III, vol%), 3-sulfolene ("butadiene sulfone") (IV, wt%) and γ-butyrolactone (V, Vol.-%) were used.

[0203] With all polar protic and polar aprotic solvents with a cyclic structure tested, strong to very strong gene-specific HER2 signals (along with equally strong to very strong CEN17-specific signals) were obtained with very low background and well-preserved cell and tissue structures during in situ hybridization. Only minor differences in the intensity of the signals were observed, ranging from very strong to slightly weaker: γ-butyrolactone, 2-pyrrolidone (γ-butyrolactam), ethylene carbonate, 2-piperidone (valerolactam), and 3-sulfolene ("butadiene sulfone"). These results were observed both in normal cells / tissues or cells / tissues without aberrations on chromosome 17, as well as in cells / tissues, particularly breast carcinomas, with HER2 amplifications. Thus, all polar protic orpolar aprotic solvents with a cyclic structure, the HER2 amplifications to be detected (ie either multiple HER2-specific signals or HER2 signal clusters) can be clearly identified. Example B.11 Influence of movement on hybridization in automated FISH procedures

[0204] Using the locus-specific hybridization probe "Auto HER2 / CEN17 Probe" for the ISH procedure III described above, the influence of wave-like movements during the hybridization step of automated in situ hybridization on the resulting signal patterns was investigated. For this purpose, the ISH procedure III described above was performed on the automated "Pathcom Stainer" using the hybridization probe "Auto HER2 / CEN17 Probe."

[0205] Regarding the specificity of the probes, reference is made to the above statements regarding the hybridization probe "Auto HER2 / CEN17 Probe".

[0206] The hybridization solutions contained 18 wt% dextran sulfate, 600 mM NaCl, 21 vol% formamide, 1-fold concentrated SSC buffer, 12.75 wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA, and 2 ng / µl locus-specific hybridization probes.

[0207] For comparison purposes, ISH procedure III was performed with agitation, specifically wave-like agitation, of the samples and hybridization composition or solution, and without agitation of the samples and hybridization composition or solution during hybridization. Hybridization was carried out for 120 minutes at 45 °C.

[0208] The movement of the samples or hybridization solutions was carried out continuously by moving the reaction chamber of the machine. The movement of the reaction chamber was induced at intervals of 15 seconds, two minutes, and 10 minutes, with the hybridization chamber cover opened for one second each time. For comparison purposes, the hybridization of the samples was performed without movement or without opening the hybridization chamber.

[0209] With all assays performed with movement or with the hybridization chamber open, very strong gene-specific HER2 signals (along with equally strong CEN17-specific signals) were obtained with very low background and well-preserved cell and tissue morphology. The excellent results were equally obtained for movement intervals of 15 seconds, two minutes, and 10 minutes. Without movement of the hybridization solutions or samples, well-evaluable signals and signal patterns were also obtained, although the signals were weaker than in hybridizations performed with movement of the hybridization solutions or samples. Example B.12 Influence of detergents on hybridization in automated FISH procedures

[0210] Using the locus-specific hybridization probe "Auto ROS1 Break Apart Probe" for the above-described ISH procedure III (automated FISH on the Pathcom Stainer), the influence of the polyalkylene glycol ether Brij®<-35 (synonymously also referred to as Brij®<-L23 or polyoxyethylene(23) lauryl ether) in the hybridization solutions on signal intensity was investigated. Regarding the performance of hybridization according to ISH procedure III and the specificity of the hybridization probe "Auto ROS1 Break Apart Probe," reference is made to the above explanations.

[0211] Hybridization solution I: 18 wt% dextran sulfate, 600 mM NaCl, 22 vol% formamide, 1-fold concentrated SSC buffer, 9.95 wt% ethylene carbonate, 0.1 µg / µl blocking and stabilizing DNA and 2 ng / µl locus-specific hybridization probes, each based on the composition.

[0212] Compared to hybridization solution I, hybridization solutions II to VI had different Brij ®< -35 concentrations of 0.125 wt% (II), 0.2 wt% (III), 0.4 wt% (IV), 0.9 wt% (V) and 1.8 wt% (VI), each based on the composition.

[0213] With hybridization solution I (without Brij ®< -35), strong gene-specific ROS1 signals were obtained with low background and well-preserved structure of cells and tissue.

[0214] Very strong signals were obtained with hybridization solutions II to V (Brij ®<-35 concentrations from 0.125 wt% to 0.9 wt%). These stronger signals compared to hybridization solution I (without Brij ®<-35) could be due to the weaker nuclear staining and thus better contrast (signal to background) associated with the use of the detergent—without wishing to be limited to this theory.

[0215] If the hybridization solutions contain the detergent Brij ®< -35 in a concentration of 1.8 wt.% (hybridization solution VI), based on the composition, only moderately strong signals were obtained.

Claims

1. Composition for use in in situ hybridization, wherein the composition contains: (a) at least one hybridization probe ("component (a)"), said hybridization probe being DNA, RNA or locked nucleic acids; (b) at least one polar protic or polar aprotic solvent ("component (b)") having a cyclic molecular structure in an amount of at least 1 vol.% or wt.%; and (c) at least one carboxylic acid amide and / or salts thereof ("component (c)") in an amount of more than 10 vol.% based on the composition, wherein the composition contains the at least one polar protic or polar aprotic solvent having a cyclic molecular structure and the at least one carboxylic acid amide and / or salts thereof in a volume-based ratio ranging from 1:5 to 1:1.

2. Composition according to Claim 1, wherein the at least one polar protic or polar aprotic solvent is selected from the group consisting of solvents having lactone, sulfone, carbonate and / or amide functionality.

3. Composition according to Claim 1 or 2, wherein the cyclic molecular structure comprises a heterocycle, preferably a heterocycle having one or more ring-forming nitrogen, sulfur and / or oxygen atoms.

4. Composition according to any of the preceding claims, wherein the composition contains the at least one polar protic or polar aprotic solvent in an amount which does not result in denaturation of nucleic acids.

5. Composition according to any of the preceding claims, wherein the composition contains the at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount ranging from 15 to 50 vol.% based on the composition.

6. Composition according to any of the preceding claims, wherein the composition contains at least one polysaccharide ("component (d)"), preferably dextran, and / or derivatives or salts thereof, particularly preferably dextran sulfate, in particular wherein the composition contains component (d) in an amount ranging from 0.1 to 50 wt.% based on the composition.

7. Composition according to any of the preceding claims, wherein the composition contains at least one detergent and / or surfactant.

8. Composition for use in in situ hybridization, preferably composition according to any of the preceding claims, wherein the composition contains: (a) at least one hybridization probe ("component (a)"), said hybridization probe being DNA, RNA or locked nucleic acids; (b) at least one polar protic or polar aprotic solvent ("component (b)") having a cyclic molecular structure in an amount of at least 1 vol.% or wt.%; (c) at least one carboxylic acid amide and / or salts thereof ("component (c) "), in particular formamide and / or salts thereof, in particular in an amount ranging from 15 to 50 vol.% based on the composition; (d) optionally at least one polysaccharide ("component (d) ") ; (e) optionally at least one chemical buffer system ("component (e)"); (f) optionally at least one blocking and / or stabilizing agent ("component (f)"); (g) optionally at least one inorganic salt ("component (g)"); (h) optionally at least one detergent and / or surfactant ("component (h)"), wherein the composition contains the at least one polar protic or polar aprotic solvent having a cyclic molecular structure and the at least one carboxylic acid amide and / or salts thereof in a volume-based ratio ranging from 1:5 to 1:1.

9. Composition for use in in situ hybridization, preferably composition according to any of the preceding claims, wherein the composition contains: (a) at least one hybridization probe ("component (a)"), said hybridization probe being DNA, RNA or locked nucleic acids; (b) at least one polar protic or polar aprotic solvent ("component (b)") having a cyclic molecular structure in an amount of at least 1 vol.% or wt.%; (c) formamide and / or salts thereof ("component (c)"); (d) optionally dextran and / or derivatives or salts thereof ("component (d)"); (e) optionally at least one chemical buffer system; (f) optionally at least one blocking and / or stabilizing agent ("component (f)"); (g) optionally at least one inorganic salt ("component (g)"); and (h) optionally at least one polyalkylene glycol ether ("component (h)"), wherein the composition contains the at least one polar protic or polar aprotic solvent having a cyclic molecular structure and formamide and / or salts thereof in a volume-based ratio ranging from 1:5 to 1:1.

10. Use of a composition according to any of the preceding claims in in situ hybridization.

11. Method for detecting nucleic acids and / or chromosome aberrations in a biological sample by means of in situ hybridization using a composition according to any of Claims 1 to 9.

12. Method for detecting nucleic acids and / or chromosome aberrations in a biological sample, in particular method according to Claim 11, wherein the method comprises the following method steps: (a) providing a biological sample based on one or more cells and / or one or more cell nuclei for in situ hybridization; (b) providing a composition for use in in situ hybridization, the composition containing at least one hybridization probe ("component (a) "), at least one polar protic or polar aprotic solvent having a cyclic molecular structure in an amount of at least 1 vol.% or wt.% ("component (b) "), and at least one carboxylic acid amide and / or salts thereof, in particular formamide and / or salts thereof, in an amount of more than 10 vol.% based on the composition ("component (c)"), wherein the composition contains the at least one polar protic or polar aprotic solvent having a cyclic molecular structure and the at least one carboxylic acid amide and / or salts thereof in a volume-based ratio ranging from 1:5 to 1:1, wherein the at least one hybridization probe ("component (a)") is DNA, RNA or locked nucleic acids; in particular providing a composition according to any of Claims 1 to 9; (c) bringing the biological sample from method step (a) into contact with the composition from method step (b); (d) denaturing the biological sample from method step (a) and the composition from method step (b), the biological sample and the composition being denatured separately from one another, in particular before carrying out method step (c), or together, in particular after carrying out method step (c); (e) subsequently hybridizing the at least one hybridization probe contained in the composition and the nucleic acids contained in the biological samples; (f) subsequently detecting the hybridized hybridization probes and / or the nucleic acids to be detected in the biological sample.

13. Method according to Claim 12, wherein the method is carried out on an automatic machine and / or automatically and / or wherein method step (e) is carried out over a period ranging from 10 min to 240 min.

14. Kit for detecting nucleic acids and / or chromosome aberrations in a biological sample, preferably in one or more cells and / or in one or more cell nuclei, by means of in situ hybridization, wherein the kit contains a composition according to any of Claims 1 to 9, wherein the kit is intended and / or is used for carrying out a method according to any of Claims 11 to 13.