Compositions and methods for hybridisation. RU patent 2505609.

FIELD: biotechnologies.

SUBSTANCE: there proposed is the method, composition and application of polar aprotic solvent with cyclic main structure for hybridisation of nucleic acid sequences.

EFFECT: invention can be used in hybridisation analysis.

19 cl, 25 tbl, 1 dwg, 22 ex

FIELD OF THE INVENTION

The present invention relates to water compositions for use in hybridization, for example, for the purpose of in situ hybridization (ISH).

One form of the implementation of the present invention relates to the field of molecular study of DNA and RNA. In particular, the invention relates to the areas of Cytology, histology and molecular biology. One aspect of the present invention relates to energy (for example, the incubation time and heating)which are necessary during hybridization between nucleic acids, for example, in situ hybridization aimed at DNA and RNA.

PRIOR ART

The structure of the DNA double helix is stable formation of hydrogen bonds between the bases on opposite strands, when the grounds are paired the only concrete way (A+T or G+C). This pairing of complementary bases (hybridization) is Central to all processes are involved in nucleic acids.

Mainly example hybridization fragments or nucleic acid sequences associated with complementary fragment or a sequence of nucleic acid. For example, when hybridization can be used -acid probes, designed to link or "hybridization" with a target, such as DNA or RNA. One type of hybridization, in situ hybridization (ISH), includes hybridization with the target in the sample, where the sample may be in vivo or, for example, to be fixed or attached on a glass slide. Probes can be tagged to allow identification of the hybrid probe target through the use of a fluorescent microscope or microscope with a bright field/scanner. Fragment or sequence typically represents or single-stranded acid, such as DNA, RNA or analogues. In some forms of carrying out the selection or the sequence can be a probe that can be labeled with the use of radioactive labels, such as 31 P 33 P or 32 S, not radioactive labels, such as and Biotin, or fluorescent labels. Such labelled probes can be used to identify genetic anomalies in the target sequence, providing valuable information, for example, about prenatal disorders, cancer and other genetic or infectious diseases.

The efficiency and accuracy of hybridization analysis of nucleic acids, mainly depends on at least one of three factors: a) the conditions of denaturation (that is, separation of, for example, two threads of the nucleic acid), b) the conditions for the renaturation (i.e. re-annealing, for example, two threads of the nucleic acid) and C) conditions of washing after hybridization.

In traditional hybridization experiments, such as analyses ISH, use formamide-containing solution for denaturation double nucleic acid. Formamide is solvent, which has a destabilizing effect on the state of the spiral, for example, DNA, RNA, and their analogues, at the expense of the weak and uniformly associated molecules of the hydrate. In addition, formamide stabilizes spiral state of DNA, RNA, and their analogues by '' binding sites of the grounds of Watson-Crick. However, formamide is toxic, hazardous substance, subject to strict use and waste.

In addition, the use , although it adopted as the standard technique of hybridization, difficult long time required for the completion of hybridization, depending on the conditions and -acid fragments or sequences. For example, after the stage denaturation should be long, consuming stage of hybridization, which, for example, in the traditional Protocol fluorescence in situ hybridization (FISH) takes 14-24 hours and may even take up to 72 hours. Examples of time traditional hybridization are shown in figures 1 and 2.

Stage renaturation (i.e. hybridization) of two complementary strands chains nucleic acid is undoubtedly the most time-consuming aspect of the analysis using hybridization. So far considered that the use of agents, such as formamide, hydrogen and urea, which interact with the binding sites of the bases of nucleic acid Watson-Scream and thereby destroy the hydrogen bonds between complementary bases of nucleic acids, is the only way to reduce the melting temperature (TM) of the complementary circuits. However, while the use agents reduces the TM, these agents turns out to be significantly prolong the time hybridization compared with hybridisation in a water solution without agent. In addition, besides the lack associated with long processing time, the high concentration of , it turns out, entails morphological cell destruction, nuclear and/or chromosomal structure. Finally, formamide is considered toxic and hazardous chemical for people.

In the present invention offered some potential advantages over prior art, such as faster time hybridization, lower temperature hybridization and less toxic solvents for hybridization.

BRIEF SUMMARY OF THE INVENTION

The present invention is development of compositions, which results in at least one of the following advantages: highly sensitive, technically simple, flexible, reliable techniques of hybridization and quick tests. In some forms of implementation, for example, one of the advantages can be the ability to regulation time hybridization by varying the reaction temperature hybridization to a much greater extent than is available using the methods of prior art. For example, hybridization may be possible at room temperature.

In one form of compositions and methods of the invention reduce the energy needed for hybridization. Compositions and methods of the invention is applicable to any method of hybridization. Compositions and methods of the invention also apply to any molecular system, which or communicates using mating grounds, such as DNA, RNA, PNK ( acid), (locked nucleic acid) and their synthetic and natural counterparts.

Following the aim of the invention is development of methods and compositions for hybridization, which keep the morphology of a biological sample. Another aim of the invention is development of non-toxic composition and methods of hybridization. One other aim of the invention is development of the method of hybridization with low evaporation. The next objective of the invention is development of the method of hybridization, detectable 20x lens. One other aim of the invention is development of a song with a low concentration of the probe. Another objective of the invention is to reduce and/or eliminate the need for blocking the nonspecific binding. Compositions and methods of the invention may also allow the use of a heterogeneous probes without the need to block, remove, or otherwise damage linking, for example, repeated sequences in a biological sample.

One form of the implementation of the method and composition for hybridization of nucleic acids according to the present invention, useful for analysis of in vivo or in vitro genomic DNA, chromosome, chromosome fragments of genes and chromosome aberrations, such as translocations, deletions, amplification, insertions, mutations or inversion associated with normal condition or disease. In addition, these methods and compositions are useful for detection of infectious agents, as well as changes in the levels of expression of RNA, such as mRNA and its complementary DNA (cDNA).

Other applications include the analysis of in vivo or in vitro messenger-RNA (mRNA), viral RNA viral DNA, small interfering RNA (siPHK), small nuclear RNA (snPHK), non-coding RNA (, for example, tRNA and rRNA), transport messenger-RNA (tmPHK), microRNA (miPHK), piwi-interacting RNAS (piRNA), long non-coding RNA, a small nucleolar RNA (snoPHK), antisense RNA, double RNA (dsPHK), and other modifications grounds, single nucleotide polymorphism (SNP), variations in the number of copies (CNV) and nucleic acids, labeled, for example, radioactive isotopes, fluorescent molecules, Biotin, (DIG) or antigens, alone or in combination with nucleic acids.

Method and composition for hybridization of nucleic acids according to the present invention, useful for analysis of in vivo or in vitro nucleic acids, using techniques such as PCR, PCR, in situ -blotting, southern blotting, flow cytometry, , fluorescence microscopy, chemiluminescence, immunohistochemistry, virtual karyotype, analysis of the gene, DNA chips (for example, comparative genomic hybridization on microchips (CGH on microchips)), profiling of gene expression, Gene ID, analysis of mosaic , gel electrophoresis, capillary electrophoresis and in situ hybridization, such as FISH, SISH, CISH. Methods and composition according to the invention can be used on the samples in vitro and in vivo, such as such as strokes bone marrow, blood smear, prisoners in paraffin drugs tissues, enzymatically samples of tissue, bone marrow, , drugs, etc.

In one form of implementation of the invention of the offered methods and compositions for the hybridization of at least one molecule with the target. The invention can, for example, to exclude the use of or reduce dependence on him. For example modalities and composition according to the invention can reduce the energy barrier for hybridization without the use of . Lower energy barrier can reduce the time and/or temperature, necessary for hybridization. For example, the invention can give the possibility of hybridization at lower temperatures or may allow a quick hybridization at higher temperatures. Thus, some aspects of the present invention allows to overcome the main long period in hybridization assays.

One aspect of the invention is a composition or solution for use in hybridization. Compositions for application in the invention include water composition containing at least one -acid sequence and at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences. Number, effective for denaturation of nucleotide sequences, is the number that provides hybridization. For example, one way of testing whether the number of polar solvent effective hybridization, is to determine whether the polar non-Protic solvent used in the methods and compositions for hybridization described in this application, such as example 1, detectable signal and/or product nucleic acid.

Not limiting examples of effective amounts of polar aprotic solvents include, for example, from about 1% to about 95% (about/about). In some forms of the implementation of the concentration of polar solvent is from 5% to 60% (about/about). Other forms of exercise of concentration of polar solvent is from 10% to 60% (about/about). Other forms of exercise of concentration of polar solvent is from 30% to 50% (about/about). Concentration of 1% to 5%, from 5% to 10%, 10%, 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50% or 50% to 60% (about/about) are also suitable. In some forms of implementation of polar non-Protic solvent will be present at a concentration of 0,1%, 0,25%, 0,5%, 1%, 2%, 3%, 4% or 5% (about/about). Other forms of implementation of polar non-Protic solvent will be present at a concentration of 7%, 7,5%, 8%, 8,5%, 9%, 9,5%, 10%, 10,5%, 11%, 11,5%, 12%, 12,5%, 13%, 13,5%, 14%, 14,5%, 15%, 15,5%, 16%, 16,5%, 17%, 17,5%, 18%, 18,5%, 19%, 19,5% or 20% (about/about).

In accordance with another aspect of the present invention water composition, containing polar non-Protic solvent, has lowered toxicity. For example, the composition, less toxic than traditional solutions for hybridization may include composition, provided that this song does not contain formamide, or provided that the composition contains less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% . Less toxic composition may also include composition, provided that this song does not contain dimethyl sulfoxide (DMSO), or provided that the composition contains less than 10%, 5%, 2% or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% DMSO.

One aspect of the invention suitable polar aprotic solvents for use in the invention can be selected on the basis of their Hansen solubility parameters. For example, a suitable polar aprotic solvents can have the dispersive parameter solubility from 17.7 to 22.0 MPa 1/2 , polar parameter solubility from 13 to 23 MPa 1/2 and parameter solubility due to hydrogen bonds from 3 to 13 MPa 1/2 .

In accordance with one aspect of the present invention suitable polar aprotic solvents for use in the invention are cyclic compounds. Circular connection has a cyclic basic structure. Examples include cyclic compounds, mentioned in the given application. Other forms of implementation of polar non-Protic solvent can be selected from the following formulas 1-4:

where X represents About and R 1 is a .

In accordance with another aspect of the invention suitable polar aprotic solvents for use in the invention can be selected from the following formula 5:

where X is optional and, if present, the selected of O, S;

where Z is optional and, if present, the selected of O, S;

where A and b independently represent About or N or S or part or primary amine;

where R is the ; and

where Y is On or S or S.

Examples of suitable aprotic solvents in accordance with the formula 5 below formulas 6-9:

X does not exist;

Z and X represent O;

X does not exist;

X does not exist;

A, B and Z represent O;

A and form part of the ;

It is a part of ;

It is a part of ;

Y is S; and R is the ethane-1,2-;

Y is S; and R is the butane-1,4-;

In and Z represent O; and R is the propane-1,3-;

Represents a methylamine;

Z is an O; and

R is a propane-1,3-;

In accordance with one another aspect of the invention polar non-Protic solvent has , sulfonic, , or carbonate functional group. Such compounds are characterised by their relatively high dielectric constant, high dipole moments and water solubility.

In accordance with another aspect of the invention polar non-Protic solvent, having functional group is a γ-butyrolactone (GBL), polar non-Protic solvent, having sulfonic functional group is a (SL), polar non-Protic solvent, having functional group is a acetonitrile (AN), polar non-Protic solvent, having functional group is a / (GS), and polar non-Protic solvent, having carbonate functional group is a (EU), propylene carbonate (PC) or (ETC).

In accordance with some other aspect of the invention disclosed way hybridization -acid sequences, including:

- first -acid sequence,

- getting a second -acid sequence,

- obtaining a water composition, containing at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences, and

- unification of the first and second -acid sequence and water composition for at least a period of time sufficient for the hybridization of the first and second -acid sequence.

In one form of realization provide enough energy for the hybridization of the first and second nucleic acids.

In one form of hybridization first -acid sequence from the second -acid sequence occurs in less than 8 hours, as, for example, less than 6 hours less than 5 hours-less than 4 hours less than 3 hours, less than 2 hours, or less than 1 hour.

Way could for example include:

- first -acid sequence, and

- application of water composition, containing the second -acid sequence and at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences, the first -acid sequence for at least a period of time sufficient for the hybridization of the first and second -acid sequence.

In one form of realization provide enough energy for the hybridization of the first and second nucleic acids.

In one form of hybridization first -acid sequence from the second -acid sequence occurs in less than 8 hours, as, for example, less than 6 hours less than 5 hours-less than 4 hours less than 3 hours, less than 2 hours, or less than 1 hour.

In accordance with some other aspect of the present invention energy hybridization provide by heating water composition and -acid sequence. Thus, the stage of hybridization may include stage heating and cooling water composition and -acid sequences.

In accordance with another aspect of the invention stage denaturation and hybridization may occur separately. For example, the sample can be a solution without the probe, and then with the probe.

Another aspect of the invention includes a way where the stage of ensuring a sufficient amount of energy for the hybridization of nucleic acids involved stage heating carried out through the use of microwaves, hot baths, hot plates, a heating coil, Peltier element, induction heating or heating lamps.

In accordance with another aspect of the present invention relates to a method where hybridization takes less than 1 hour. Other forms of implementation of hybridization takes less than 30 minutes. Other forms of implementation of hybridization in less than 15 minutes. Other forms of implementation of hybridization takes less than 5 minutes.

In accordance with the following aspect of the invention refers to the application of composition containing from 1 to 95% (on/of) at least one polar solvent in hybridization assays.

In accordance with one another aspect of the invention refers to the application of composition containing water composition, as described in the present invention, for use in hybridization assays.

SHORT DESCRIPTION OF GRAPHIC MATERIALS

Figure 2 shows the typical periodization define a single locus initially labelled FISH probes on cytological samples. The stripes represent the hybridization carried out using traditional solution (top)and the typical periodization hybridization, carried out with the use of the composition according to the invention (NCDs). The first band on the left in each of periodization is a stage of fixation; the second strip is a stage of denaturation and hybridization; the third band is a stage rigid washing; and the fourth strip represents a stage fill in the environment.

DETAILED DESCRIPTION

A. Definitions

In the context of the present invention, the following terms should be understood as described below.

"Biological specimen" must be understood as any sample in vivo, in vitro or in situ one or more than one cell or cell fragments. He may for example consist of single-celled or multicellular organism, tissue slice, cytological sample, chromosomal drug, peeled -acid sequence, artificially obtained -acid sequences received, for example, through a system of on biological basis or by chemical synthesis, microchip or other form of acid chip. In one form of a sample is a sample from a mammal, such as, for example, the sample from the human, mouse, rat, cat or horse.

"Nucleic acid", "-acid chain" and "-acid sequence" means all that is bound or using mating grounds, including oligomers or polymers having framework formed by natural nucleotides, and/or -acid analogues containing custom nucleic bases and/or non-standard frames (for example, the peptide acid (NCP) or closed acid ()), or any form of derivative nucleic acid.

As used in this application, the term "peptide nucleic acid" or "NCP" means synthetic polymer, has polyamide frame with side nucleic bases (natural or modified), including, but not limited by them, any of oligomer and polymer segments, called or declared as peptide nucleic acids, for example, in the U.S. patents№№5539082, 5527675, 5623049, 5714331, 5718262, 5736336, 5773571, 5766855, 5786461, 5837459, 5891625, 5972610, 5986053, 6107470, 6201103, 6228982 and 6357163, WO 96/04000, where these documents are included in this proposal by reference, or any of the links, quoted in them. Side basis, such as, for example, or pyrimidine base, the NCP may be connected with the frame by means of the linker, such as, for example, one of the declared in the PCT/US02/30573 or in any of the references cited there. One form of the implementation of the NCP has frame N-(2-aminoethyl)-glycine). The NCP may be synthesized (and optional outline), as stated in the PCT/US02/30573 or in any of the references cited there. PNK hybridized firmly and with highly specific sequences of DNA and RNA, because the frame of the NCP is unloaded. Therefore, short PNK probes can be comparable specificity with longer DNA or RNA probes. PNK probes can also be higher specificity when linking with a complementary DNA or RNA.

As used in this application, the term "closed nucleic acid" or "" means oligomer or polymer containing at least one or more than one subunit . As used in this application, the term "subunit " means containing methylene bridge, which connects the 2'-oxygen ribose with 4'-carbon. Cm. General description in Kurreck, Eur. J. Biochem, 270:1628-44(2003).

Examples of nucleic acids and -acid analogues also include polymers of nucleotide monomers, including bilateral and (DNA), ribonucleotides (RNA), their α- forms, their synthetic and natural counterparts and the like. -acid chain may consist entirely of , , peptide nucleic acids (NCP), closed nucleic acids (), their synthetic or natural analogues or their mixtures. DNA, RNA, or other nucleic acids, as defined in this application, you can use the method and compositions of the invention.

"Polar non-Protic solvent" refers to the organic solvent, having dipole moment about 2 units of Peter Debye or more, solubility in water at least approximately 5% (volume) at ambient temperatures or close to it, that is about 20 C, and which does not undergo significant hydrogen currency at about neutral pH, i.e. in the range from 5 to 9 or in the range of 6 to 8. Polar aprotic solvents include solvents, defined in accordance with Hansen solubility parameters, discussed below.

"Hybridization" should be understood as including the stage as a denaturation and renaturation of hybridization techniques, unless otherwise specified.

"Composition for hybridization" refers to a water solution of the invention for the implementation of hybridization techniques, for example, to associate a probe with -acid sequence. Compositions for hybridization may contain, for example, at least one polar non-Protic solvent at least one -acid sequence and the solution for hybridization. Compositions for hybridization not contain enzymes or other components, such as (dNTP), amplification of nucleic acids in a biological sample.

"The solution for hybridization" refers to a water solution for use in a composition for hybridization the invention. Solutions for hybridization discussed in detail below and may contain, for example, buffer agents, catalysts, chelating agents, salts, detergents and blocking agents.

"The composition of PCR" refers to a water solution of the invention for the implementation of hybridization techniques to amplify -acid sequence. Song PCR may contain, for example, at least one polar non-Protic solvent at least one enzyme for nucleic acid amplification, set -acid oligonucleotide primers, dNTP mix and solution PCR.

"Solution of PCR" refers to a water solution for use in a composition according to the invention of PCR. Solutions PCR may contain, for example, buffer agents, catalysts, chelating agents, salts and detergents.

"Hansen solubility parameters" and "HSP" refer to the following parameters of energy cohesion (solubility): (1) parameter solubility (d D , "parameter-D"), which measures non-polar interactions, formed as a result of nuclear forces; (2) polar parameter solubility (b P "parameter R"), which measures the interaction of permanent dipoles with permanent dipoles; and (3) parameter solubility due to hydrogen bonds (b H "parameter N")which measures the currency electrons. Hansen solubility parameters are further defined below.

"Repeating sequences" should be understood as relating to components of mammalian genomes, undergoing fast (approximately 25%) and/or secondary (about 30%). Quickly components contain small (the length of a few nucleotides) highly repetitive sequences, usually found in tandem (for example, satellite DNA), while medium components contain scattered repetitive DNA. Scattered repeated sequences are classified either as a SINE (short scattered recurring sequence), or as a LINE (long scattered repeated sequences), where they are classified as retrotransposons turn to primates. SINE and LINE items include, but not limited to, Alu-repetitions, Kpn replays, consisted of dinucleotide repeats, repetitions, repetitions, replays and repetitions. Alu repeats constitute a majority of the SINE elements of man and are characterized by consensus sequence from about 280 to 300 BP, which consists of two similar sequences organized as a dimer of the head to the tail. In addition to the SINE and LINE elements of recurrent sequences also exist in chromosomes on the ends of chromosomes and chromosomes, which contain different repeating sequences that exist only in the Central part of the chromosome. However, in contrast to the SINE and LINE, which randomly distributed throughout the genome, repeating sequences telomeres and localized in the Central part of the chromosome.

"Non-toxic" and "reduced toxicity determine labelling toxicity in accordance with the Directive 1999/45/EC of the European Parliament and of the Council of 3.1 may 1999, bringing changes in laws, regulations and administrative acts of the member States relating to the classification, packaging and labelling of dangerous substances, with a view to bringing them into conformity with the Regulations" (ecb.jrc.it/legislation/1999L0045EC.pdf) (the"Directive"). In accordance with the Directive toxicity determined using the following procedure for classification: T+very toxic"; T "toxic", With a "corrosive", XII "harmful", Xi irritant". Risk phrases (R-phrases," describe the risks classified toxicity. Formamide is described as T (toxic) and R61 (may cause harm to the newborn child). All of the following chemical substances classified as less toxic than formamide: acetonitrile (Xn R11, R20, R21, R22, R36); (Xn, R22); γ-butyrolactone (Xn, R22, R32) and (Xi, R36, R37, R38). At the time of filing of this application and currently not labeled.

B. Choice of solvent

Suitable polar aprotic solvents for use in the invention can be selected on the basis of their Hansen solubility parameters. Methods of experimental determination and/or calculation HSP solvent-known in the art, and HSP reported more than 1,200 chemicals.

For example, the parameter-D can be computed with reasonable accuracy on the basis of the refractive index or it can be inferred from table by comparison with known solvents such size, shape and composition after the establishment of the critical temperature and molar volume. Parameter P can be estimated on the basis of the known dipole moments (see, for example, McClellan A.L., Tables of Experimental Dipole Moments (W.H. Freeman 1963))using equation 1:

Equation 1:δ P =37,4(dipole moment)/V 1/2 ,

where V is the molar volume. Does not exist equations to calculate the parameter N. Instead, the parameter N is usually determined on the basis of group contributions.

Characteristics of HSP is conveniently visualize using the circular image, where HSP experimentally measured, appropriate reference solvent is located in the center of the circle. The radius of the circle (R) shows the maximum allowed deviation from HSP reference solvent, which still gives the opportunity to was "good" interaction. Good solvents are inside the circle, and the bad are outside the circle. Distance, Ra, between the two solvents on the basis of their respective connotations, HSP can be determined using equation 2:

Equation 2:(e a ) 2 =4(d D1-Delta D2 ) 2 +(d P1-Delta P2 ) 2 (b H1-Delta H2 ) 2 ,

where the subscript index 1 shows the reference sample, subscript index 2 shows the chemical, and all values are expressed in MPa 1/2 . For good solubility you want to e a was less than the experimentally determined radius of the circle solubility R o . The relative difference in energy between the two solvents, i.e. the number of RED, can be calculated by taking the ratio R a to R o as shown in equation 3.

Equation 3: RED=R (a /R o .

The number of RED less than 1.0 indicate a high affinity; the number of RED, equal or close to 1.0, show borderline state; and progressively increasing the number of RED indicate progressively declining affinity.

In some forms of exercise of options D polar aprotic solvents according to the invention, are from 17.7 to 22.0 MPa 1/2 . These relatively high parameters D usually associated with solvents with cyclic structure and/or the structure of the atoms of sulphur or Halogens. Linear connection, probably, are not among the most suitable polar aprotic solvents for use in the invention, but can be considered if their parameters P and N are within intervals, as discussed below. Since the parameter D is multiplied by 4 in equation 2, the limits represent half of the R o . In addition, it should be noted that the values of D about 21 or above are often characteristic of a solid substance.

In some forms of implementation of the P polar aprotic solvents according to the invention range from 13 to 23 MPa 1/2 . Such exceptionally high parameters of P are usually associated with solvents with high dipole moment, and mainly the relatively low molecular volume. For example, to V of about 60 cm 3 /mol dipole moment must be between 4.5 to 3.1. For V of about 90 cm 3 /mol dipole moment should be from 5.6 to 3.9.

In some forms of exercise of options N polar aprotic solvents according to the invention is from 3 to 13 MPa 1/2 . As a rule, polar aprotic solvents having alcohol group, are not applicable in the compositions and methods of the invention, as the parameters of N such solvents would be too high.

Molar volume of the polar solvent may also be relevant, as it is included in the assessment of all three Hansen solubility parameters. As the molar volume becomes less liquid prone to rapid evaporation. As the molar volume becomes more fluid prone to joining the firm region in the range of parameters D and P above. Thus, polar aprotic solvents invention is likely close to the boundary liquid/solid in the range of HSP.

Molar volume (cm3 /mol)

The correlation(R =3,9)

n/a=not available

Other appropriate polar solvents that can be used in the invention, are cyclic compounds, such as, for example, e-. In addition, substituted and related structures with a nitrogen atom in the 5 - or 6- ring and cyclic structures with two groups or one atom of bromine and one group may also be suitable for use in an invention. For example, N- (shown below) may be a suitable polar aprotic solvent for use in the methods and compositions of the invention.

Other suitable polar aprotic solvents may contain ring group (NHCOO-). However, not all such compounds are suitable, because of 1,3-dimethyl-2- produces signals when used in compositions for hybridization the invention. Specialist in a given field of technology may carry out screening compounds useful in the compositions and methods of the invention as described in the application. Approximate chemicals that can be suitable for use in the invention, are presented below in tables 2 and 3.

Acetanilide

N-

4-Aminopyridine

Б

Benzimidazole

1,2,3-Б

Б

2,3-Б

(Epsilon)

Maleic acid chloride

2-

Chloronitromethane

anhydride

Dimethyl sulfoxide

1,2-Dinitrobenzene

2,4-Dinitrotoluene

1,2-Dinitrobenzene

2,4-Dinitrotoluene

1,2-Dinitrobenzene

2,4-Dinitrotoluene

Epsilon-caprolactam

2-

Isoxazole

Maleic anhydride

4-

1-Methoxy-2-nitrobenzene

3-

N--N-oxide

Methyl-4-

3-Nitroaniline

2-

9,10-

Phthalic anhydride

1,3-

beta-

2-Pyrrolidone

Saccharin

Sulfanilamide

2,2,6,6-

3,3,3-

1,1,2-

1,2,3-

Table 2 provides a sample list of potential chemicals for use in the compositions and methods of the invention on the basis of their Hansen solubility parameters. Other connections may of course also meet these requirements. Some of these chemicals used in solutions for hybridization and/or PCR prior art (for example, dimethyl sulfoxide (DMSO) is used in solutions for hybridization and/or PCR, and (SL) is used in solutions PCR), but most of it was not used. However, at the level of technology has not been recognized that these connections can be used advantageously to reduce the time and/or temperature hybridization, as disclosed in the application.

Chemical (dipole moment)

Melting point °

(4,02)

2-Oxazolidinone (5,07)

2-Imidazol

1,5- (5,3)

about 1.5

M- (5,46)

about 1.5

dioxide (4,49)

(3,63)

1,3-Dimethyl-5- (4,02)

(3,97)

2- (4,21)

N-Mei (6,2)

1-Nitroso-2-

(3,51)

5-Cyano-2- (5,19)

4H-PYRAN-4-tion (4,08)

4H-PYRAN-4-one=gamma Piron (4,08)

Boiling Point (BP) 80

2- (4,41)

Methyl alpha- (6,24)

oxide (4,19)

(2-cyanopyridine) (5,23)

26-28 (BP 212-215)

(6,0)

Isatin (USD 5.76)

N- (At 6.55)

(Ethylene glycol) note: insoluble at 40%

Not all of the chemicals listed in tables 2 and 3, are suitable for use in the compositions and methods of the invention. For example, although DMSO listed in table 2 in connection with the fact that his Hansen solubility parameters (HSP) fall within the above intervals, DMSO is not functioning as reducing time and/or temperature hybridization in the compositions and methods of the invention. Thus, in some forms of the implementation of the water composition contains DMSO as polar solvent. However, within the competence of the ordinary skilled in the art is screening suitable connection is presented in the application guidelines, including testing the connection in one example. For example, some forms of carrying out appropriate polar aprotic solvents will have HSP within the above intervals and structure shown above formulas 1-9.

Century Compositions, buffers and solutions

(1) Solutions for hybridization

Traditional solutions for hybridization known in the art. Such solutions may contain, for example, buffer agents, catalysts, chelating agents, salts, detergents and blocking agents.

For example, buffer agents may include SSC, HEPES, SSPE, PIPES, , TRIS, SET, citric acid, phosphate buffer, such as, for example, phosphate potassium or sodium pyrophosphate, etc. Buffer agents may be present in concentrations ranging from 0,5x to 50x. Typical buffer agents are present in concentrations from 2x to 10x.

Catalysts may include polymers such as , PVP, heparin, , proteins, such as BSA, glycols, such as ethylene glycol, glycerin, 1,3-propandiol, propylene glycol or diethylene glycol, their combinations, such as the solution and BLOTTO, and organic solvents, such as formamide, dimethylformamide, DMSO, etc. Catalyst may be present in concentrations ranging from 1% to 80% or 0,1x to 10x. Typically formamide is present in concentrations ranging from 25% to 75%, while the DHS and glycol are present in concentrations ranging from 5% to 10%.

Chelating agents may include EDTA, etc. Chelating agents may be present in concentrations ranging from 0.1 mm up to 10 mm. Typically chelating agents are present in concentrations from 0.5 mm to 5 mm.

Salt may include sodium chloride, sodium phosphate, magnesium phosphate, etc. Salt may be present in concentrations ranging from 1 mm to 750 mm. Typically salt present in concentrations ranging from 10 mm to 500 mm.

Detergents may include twin, LTOs, Triton, CHAPS, acid Detergent etc. may be present in concentrations ranging from 0.01% to 10%. Typical detergents are present in concentrations ranging from 0.1% to 1%.

-acid-blocking agents may include yeast tRNA, DNA, sperm DNA salmon sperm DNA herring, total human DNA, DNA 1 etc. Blocking nucleic acids may be present in concentrations ranging from 0.05 mg/ml up to 100 mg/ml

In the literature there are large differences in relation to traditional solutions for hybridization. For example, the traditional solution for hybridization may contain 5x or 6x SSC, 0.01 M EDTA, 5x solution , 0.5% of LTOs and 100 mg/ml degraded as a result of hydrodynamic shift, denatured DNA salmon sperm. Another traditional solution for hybridization may contain 50 mm HEPES, 0.5 M NaCl and 0.2 mm EDTA. A typical solution for hybridization to FISH on biological samples for the determination of RNA can contain, for example, 2x SSC, 10% , 2 mm complex -, 50% formamide, 0,02% BSA, free of RNase, and 1 mg/ml tRNA E. Li. A typical solution for hybridization to FISH on biological samples for DNA identification may contain, for example, 2x SSC, 10% , 50% formamide and, for example, 0.3 mg/ml salmon sperm DNA or 0.1 mg/ml DNA 1. Other typical solutions for hybridization can contain 40% formamide, 10% , 30 mm NaCl, 5 mm phosphate buffer, Alu-PNK (blocking NCP) or DNA HUNDRED-1 and in some cases, 0.1 mg/l of total DNA rights (THD).

The composition of the invention may include a solution for the hybridization of containing any of the components of traditional solutions for the hybridization of the above, in combination with at least one polar aprotic solvent. Traditional components may be present in such concentrations, as used in traditional solutions for hybridization, or may be present in higher or lower concentrations, or may be completely excluded.

For example, if the composition of the invention contain salts, such as NaCl, and/or phosphate buffer, salt may be present in concentrations 0-1200 mm NaCl and/or 0-200 mm phosphate buffer. In some forms of concentration of salts can be, for example, 300 mm NaCl and 5 mm phosphate buffer or 600 mm NaCl and 10 mm phosphate buffer.

If the composition of the invention contain catalysts, such as , glycol or DMSO, may be present in concentrations ranging from 5% to 40%, glycol may be present in concentrations ranging from 0.1% to 10%, and DMSO can constitute from 0,1% to 10%. In some forms of exercise of concentration can be 10% or 20% and the concentration of ethylene, 1,3- or glycerin can be from 1% to 10%. In some forms of implementation of DMSO concentration may be 1%. In some forms of the implementation of the water composition contains DMSO as a catalyst. In some forms of the implementation of the water composition does not contain formamide as a catalyst or contains formamide, provided that the composition contains less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01%.

If the composition of the invention contain lemon acid, the concentration can be in the range from 1 mm up to 50 mm pH can be in the range 5.0 to 8.0. In some forms of the implementation of the concentration of a citric acid can be 10 mm, and pH can be 6,2.

The composition of the invention may contain agents that reduce nonspecific binding, for example, with the cell membrane, such as salmon sperm DNA or small amounts of total DNA of a person, or, for example, they may contain blocking agents for blocking the binding, such as recurrent sequences with a target, such as a large number of the total human DNA or DNA enriched with repetitions, or specific blocking agents, such as parts and sequence NCP or . These agents may be present in concentrations ranging from 0.01 to 100 mcg/ml or 0.01 to 100 microns. For example, some forms of implementation of these agents will be 0.1 mcg/ml total DNA of a person, or 0.1 mcg/ml human DNA, such as sperm DNA herring, salmon sperm or calf thymus, or 5 microns blocking the NCP.

One aspect of the invention is a composition or solution for use in hybridization. Compositions for application in the invention include water composition containing -acid sequence and at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences. Number, effective for denaturation of nucleotide sequences, represents the amount that provides hybridization. For example, one way of testing whether or not the number of polar solvent to ensure hybridization, is to determine whether the polar non-Protic solvent use in the methods and compositions for hybridization described in this application, such as example 1, detectable signal and/or product nucleic acid.

Not limiting examples of effective amounts of polar aprotic solvents include, for example, from about 1% to about 95% (about/about). In some forms of the implementation of the concentration of polar solvent is from 5% to 60% (about/about). Other forms of exercise of concentration of polar solvent is from 10% to 60% (about/about). Other forms of exercise of concentration of polar solvent is from 30% to 50% (about/about). Concentration of 1% to 5%, from 5% to 10%, 10%, 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50% or 50% to 60% (about/about) are also suitable. In some forms of implementation of polar non-Protic solvent will be present at a concentration of 0,1%, 0,25%, 0,5%, 1%, 2%, 3%, 4% or 5% (about/about). Other forms of implementation of polar non-Protic solvent will be present at a concentration of 7%, 7,5%, 8%, 8,5%, 9%, 9,5%, 10%, 10,5%, 11%, 11,5%, 12%, 12,5%, 13%, 13,5%, 14%, 14,5%, 15%, 15,5%, 16%, 16,5%, 17%, 17,5%, 18%, 18,5%, 19%, 19,5% or 20% (about/about).

If the composition of the invention is used in analysis, they can optionally contain one or more than one acid probe. These probes can be directly or indirectly tagged findable substances such as enzymes, chromophores, and . DNA probes may be present in concentrations from 0.1 to 100 ng/ml. For example, some forms of implementation of the probes can be present in concentrations ranging from 1 to 10 ng/ml. PNK probes can be present in concentrations ranging from 0.5 to 5000 nm. For example, some forms of implementation of the probes can be present in concentrations ranging from 5 to 1000 nm.

One form of the implementation of the composition according to the invention, contains a mixture of 40% of the polar solvent (on/of) (for example, , "EU"), 10% , 300 mm NaCl, 5 mm phosphate buffer and 1-10 ng/ml probe. Another approximate composition of the present invention comprises a mixture of 15% EU, 20% , 600 mm NaCl, 10 mm phosphate buffer and 0.1 mcg/ml total human DNA. Another another approximate composition comprises 15% of the EU, 20% , 600 mm NaCl, 10 mm citric acid pH of 6.2 and 0.1 mcg/ml human DNA (for example, sperm herring, salmon sperm or calf thymus), or 0.5% formamide or 1% glycol (for example, ethylene, 1,3-propandiol or glycerol).

(2) Polar non-Protic solvent(and)

Various polar aprotic solvents can give various properties of the compositions of the invention. For example, the choice of the polar solvent can contribute to the stability of the composition, because some polar aprotic solvents may decompose over time. For example, polar non-Protic solvent breaks up ethylene glycol, which is relatively stable molecule, and carbon dioxide, which can interact with water to form carbonic acid, changing the acidity of the compositions of the invention. Contacting theory believe that the change in pH when decay makes the composition of the invention less effective for hybridization. However, stability can be improved by lowering the pH of the composition, by adding citric acid-as a buffer at pH 6,2 instead of the traditional phosphate buffer, which typically use about at pH 7.4, and/or by the addition of ethylene glycol in concentrations, for example, from 0.1% to 10% or 0.5% to 5%, as, for example, 1%, 2%, 3% etc. For example, with a 10 mm citrate buffer composition according to the invention stable at 2-8oC for about 8 months. Stability can be improved, if you store songs at low temperatures such as -20 degrees).

In addition, some polar aprotic solvents may cause division of compositions of the invention on multiphase systems in certain conditions. The conditions under which receive multiphase systems, can be different for different polar aprotic solvents. As a rule, however, as soon as the concentration of polar solvent increase, the number of phases. For example, the compositions containing low concentrations of (i.e. less than 20%)can exist in the form of one phase, while the compositions containing higher concentrations of , can be divided into two or even three phases. For example, the compositions containing 15% , exist in the form of single phase at room temperature, while the compositions containing 40% consist of a viscous lower phase (approximately 25% of the total) and less viscous top phase (about 75% of total amount) at room temperature.

On the other hand, some polar aprotic solvents can exist in two phases at room temperature even at low concentrations. For example, , , , and propylene carbonate exist in the form of two phases at concentrations of 10, 15, 20 or 25% (20% dextran sulfate, 600 mm NaCl, 10 mm buffer) at room temperature.

It may also be possible to change the number of phases by regulating the temperature of the compositions of the invention. As a rule, as soon as the temperature rises, the number of phases is reduced. For example, at 2-8oC compositions, containing 40% , can be divided on three step system.

It may also be possible to change the number of phases by regulation of concentration and/or salt composition. Generally speaking, lower concentration (traditional concentration is 10%) and/or the concentration of salt can reduce the number of phases. However, depending on the polar solvent and its concentration in the composition of uniform phases can be obtained even at the higher concentrations of salt and . For example, a composition containing a low quantity of the EU (for example, 15%, 10% or 5%), can work well by increasing concentrations and salt, still holding on in a single-phase system. In the specific form of implementation of the compositions containing DNA probe of HER2 gene, PNK probe CENY, 15% EU, 20% , 600 mm NaCl and 10 mm phosphate buffer, freeze at -20 degrees C. other forms of implementation of the songs are liquid at -20 degrees C.

Some polar aprotic solvents may give stronger signals in one phase or another. For example, 40% gives strong signals in the lower phase and gives signals in the upper phase. Similarly, some types of probes can give stronger signals in one phase or another. For example, PNK probes tend to show a stronger signals in the lower phase than in the upper phase.

Accordingly, multiphase systems according to the invention can be used to explore the different aspects of the sample. For example, a two-phase system can be used for the separation of samples labeled PNK probes, from the samples labeled DNA probes. Other applications include the selection of a specific phase, manifesting, for example, certain advantages hybridization, so isolated phase can be used as a single-phase system. Probe and/or sample can be added before or after the selection of the particular phase.

The composition of the invention can be varied to optimize results for a particular application. For example, the concentration of polar solvent, salt, catalyst, blocking agent and/or hydrogen ions (i.e. pH) can be varied to improve the results for a particular application.

For example, the concentration of polar solvent can be varied to improve the intensity of the signal and background staining. As a rule, as soon as the concentration of polar solvent increases, the intensity of the signal increases and background staining decreases. For example, the compositions containing 15% EU, tend to display a stronger signals and less background than the compositions containing 5% of the EU. However, the intensity of the signal can be improved for the songs that have low concentrations of polar solvent (for example, from 0% to 20%), if you increase the concentration of salts and/or . For example, strong signals can be observed in 5%-10% of the EU when the salt concentration rises approximately 8-16 times of the traditional salt concentrations (approximately 1200 mm NaCl, 20 mm phosphate buffer). Similarly, when the use lower concentrations of polar solvent, high concentration are typically required to maintain a good signal intensity and background.

Accordingly, the concentration of salts and can also vary to improve the intensity of the signal and background staining. As a rule, as soon as the salt concentration increases, increases the intensity of the signal and reduces background. For example, the concentration of salt, which is about two to four times above the traditional concentrations (that is 300 mm NaCl, 5 mm phosphate buffer), give strong signals and low background. Unexpectedly, however, hybridization occurs when the use of compositions according to the invention, even in the absence of salt. Signal strength can be improved in conditions by increasing the concentration of the catalyst and/or polar solvent.

Similarly, the intensity of the signal increases as the concentration of increases from 0% to 20%. However, good signals can be observed even at concentrations 0%. The intensity of the signal can be improved in conditions of low by increasing concentrations of polar solvent and/or salt.

Furthermore, the types of probes used in the compositions of the invention can be varied to improve the results. For example, some aspects of the invention combination of DNA/DNA probes can show less of the background than the combination of DNA/PNK probes in the compositions of the invention or Vice versa. On the other hand, the NCP probes tend to show a stronger signal than the DNA probes, low salt and/or low concentrations of polar solvent. Indeed, the NCP probes also show signals, when the polar non-Protic solvent missing when DNA probes show weak or do not show any signals without polar solvent.

, The field of application, methods and application

(1) Analytical samples

Methods and composition according to the invention can be used fully or partially in all types of fields of application of hybridization in the field of Cytology, histology and molecular biology. In accordance with one form of implementation of the first or the second -acid sequence of the methods of the present invention in a biological sample. Examples of such specimens include, for example, tissue, cellular medicines fragments and isolated or fortified drugs cellular components. The sample may have the origin from various fabrics, such as, for example, mammary gland, lung, rectum and the colon, prostate, head and neck, stomach, pancreas, esophagus, liver, urinary bladder, or other relevant tissues and tumors of any cell suspension, blood sample, aspiration diagnostic puncture, Erlich's ascites fluid, sputum, lavage of the abdominal cavity, lung lavage, urine, faeces, scraping cells, smear cells, cells obtained using , or cytological preparations.

A sample can be selected and processed using standard protocols. Drugs fragments can be, for example, obtained by homogenization cells, processing, freezing-defrosting or cell lysis. Isolated sample can be processed in many different ways depending on the purpose of receiving the sample, based on the usual methods adopted in this place. Often the sample treated with various reagents to save the cloth for further analysis of the sample, alternatively, the sample can be analyzed directly. Examples of commonly used ways to preserve the samples is formalin fixation with the subsequent formation of paraffin and cryopreservation.

For drugs cell cultures usually handle or other suitable agent intercepting spindle pole division to stop the cell cycle in metaphase. Then the cells record and placed on glass slides treated with formaldehyde, washed and dehydrate in ethanol. Then add probes and samples are analyzed by any of the methods discussed below.

Cytology includes the study of individual cells and/or chromosomal drugs from a biological sample. Cytological examination of the sample begins with a sample of cells, which typically can be performed by scraping, smear or brush biopsy area, as in the case of cervical samples, or through the collection of body fluids, such as those obtained from the thoracic cavity, bladder or spine, or by aspiration diagnostic puncture or fine-needle puncture biopsy, as in the case of internal tumors. When receiving conventional Cytology preparation manually sample transfer in liquid suspending material, and then the cells in the fluid is transferred directly or through processing stages by centrifugation on the microscope slide for visualization. The typical automated receipt of cytological filtering device is placed in a liquid slurry, and it's filtering device and distributes cells, and captures the cells on the filter. The filter is then removed and placed in contact with the subject glass. Then the cells are fixed on a glass slide, and then analyzed by any of the methods discussed below.

In a typical experiment on hybridization using a cytological sample glass containing sample, immersed in formaldehyde buffer, washed, and then dewatered in ethanol. Then add probes and sample cover cover slip. Glass incubated at a temperature sufficient for denaturation of any nucleic acid in the sample (for example, 5 minutes at 82 degrees C), and then incubated at a temperature sufficient to enable hybridization (for example, during the night at 45 C). After hybridization cover glasses are removed, and the samples subjected to washing of high hardness (for example, 10 minutes at 65 C), followed by a series cleaning low hardness (for example, 2 x 3 minutes at room temperature). Then the samples dehydrate and pour in the environment for analysis.

Histology includes the study of cells in thin tissue sections. For the preparation of a sample of tissue for histological examination pieces of cloth fixed in a suitable latch, typically in , such as formaldehyde or , and then sign in the melted paraffin wax. Then the wax block, containing a sample of tissue is cut on the microtome for thin slices of paraffin-containing tissue, typically from 2 to 10 microns thickness. Then cut the sample is put on a glass slide, dried on air and heated to cause gluing sample to a substantive glass. Then the residual wax dissolved suitable solvent, typically with xylene, toluene or other. Then the so-called solvents remove reagent leaching - dehydrating type, then analyzed by any of the methods discussed below. Alternatively slices can be cooked from frozen samples, briefly fixed in 10% formalin, or other suitable place, and then reagent sample prior to analysis.

In a typical experiment on hybridization using histological sample formalin fixed, prisoners in paraffin samples of tissue is cut on slices 2-6 microns and collect on the glass. Paraffin wax is melted (for example, 30-60 minutes at 60 C), and then removed () by washing with xylene (or a substitute xylene), for example, 2 x 5 minutes. Samples , washed, and then pre-treated (for example, 10 minutes at 95-100 Celsius). Glass washed, and then treated pepsin or other suitable amplifier permeability, for example, 3-15 minutes at 37 deg C. Glass washed (for example, 2 x 3 minutes), dehydrated and put a probe. The samples cover the chosen glass, and glass incubated at a temperature sufficient for denaturation of any nucleic acid in the sample (for example, 5 minutes at 82 degrees C), and incubated at a temperature sufficient to enable hybridization (for example, during the night at 45 C). After hybridization cover glasses are removed, and the samples subjected to washing of high hardness (for example, 10 minutes at 65 C), followed by a series cleaning low hardness (for example, 2 x 3 minutes at room temperature). Then the samples dehydrate and pour in the environment for analysis.

(2) hybridization Techniques

Molecular probes, which are suitable for use in the invention described, for example, U.S. patent no 2005/0266459 included in this application by reference. As a rule, probes can be obtained by chemical synthesis or by amplification of a specific DNA sequence using cloning, embedding DNA vector and amplification of the vector with the insert in the appropriate cells of the host. Usually used vectors include bacterial plasmids, , bacterial artificial chromosome (BAC), artificial chromosomes phage P1 (RAS) or yeast artificial chromosome (YAC). Then amplified DNA is extracted and purified for use as a probe. Methods of obtaining and/or fusion probes are known in the art, for example, as disclosed in the PCT/US02/30573.

As a rule, probe type specifies the type of character, which you can define in analysis. For example, probes based on total nuclear or genomic DNA can be used as a species-specific probe. Chromosomal prints are collections of DNA sequences, originating from one type of chromosomes, and may allow identification of the specific type of chromosomes in and interphase nuclei, count the number of chromosome, show translocation or identify fragments of chromatin. Various types of chromosomal unique recurring sequences that can be targeted for hybridization probe to identify and quantify specific chromosomes. Probes, which represent insert a large size, you can use to direct the unique sequence. When these probes large size hybridization efficiency inversely proportional to the size of the probe. Probes smaller you can also use it to determine aberrations, such as deletions, amplification, inversion, duplications and aneuploidy. For example, differently colored locus-specific probes can be used to determine translocations by in situ hybridization with separate signal.

As a rule, the ability to distinguish between closely related sequences inversely proportional to the length of the probe for hybridization, since the difference in thermal stability decreases between the complexes of wild-type and mutant complexes as increasing the length of the probe. Probes more than 10 BP in length are typically required to obtain differences sequence required to correctly identify a unique organism or of interest to clinical condition. On the other hand, so small differences sequence as one base pair (point mutation)in a very short (<10 base pairs) can be sufficient to ensure the discernment of hybridization with complementary -acid sequences target compared with sequences of non-targets.

In one form, the implementation of at least one series of probes for in situ hybridization may include one or more than one PNK probe as defined above and as described in U.S. patent №7105294, which is included in this application by reference. Methods of synthesis of PNK probes are described in the PCT/US02/30573. Alternatively or additionally at least one series of probes for hybridization in any of the techniques discussed above, may contain one or more than one probe, which is a closed acid (), as described in the WO 99/14226 included in this application by reference. Due to the additional bridge of communication between the 2' and 4' carbon atoms frame pre-arranged for hybridization. Interaction /DNA and /RNA is more powerful than the corresponding interaction of DNA/DNA or DNA/RNA, which is indicated by a higher melting point. Thus, composition and methods of the invention that reduce the energy required for hybridization, especially useful for with probes.

One form of the implementation of the probes can contain label (a molecule that provides analytically distinguishable signal, which provides the capability to detect hybrid probe target)as disclosed in the publication of a patent, US no 2005/0266459 included in this application by reference. label can be directly attached to the probe or attached to the probe indirectly, for example by using the linker. Any way of tagging, known to specialists in the field of technology, including enzymatic and chemical methods can be used for marking is probes used in the methods and compositions of the invention. Other forms of implementation of the probes are .

As a rule, in hybridization techniques, such as CGH, FISH, CISH and SISH use large, mainly non-specific, -acid probes, which hybridize with varying rigidity with genes or fragments of genes in the chromosomes of the cells. The use of large probes makes the technique in situ hybridization very sensitive. Successful exploitation of the large genomic probes in the traditional hybridization assays depends on blocking unwanted background of the painting, which is the result of, for example, repeating sequences, which are present throughout the genome. Such stages blocking time-consuming and expensive. As discussed below, methods and composition according to the invention profitable to reduce and/or eliminate the need for such stages of the block. However, one form of the implementation of the recurring sequences can be in accordance with the methods, known in the art, for example, as disclosed in the PCT/US02/30573.

Related probes can be found in the cytological and histological samples, either directly or indirectly with (e.g., FISH), organic (for example, CISH), with silver particles (for example, SISH) or other particles of metal (for example, fluorescent in situ hybridization, reinforced gold, GOLDFISH). Thus, depending on the method of detection, the population of cells obtained from a sample for testing, can be visualized by means of fluorescence microscopy or conventional light microscopy with a light field.

analyses on cytological and histological samples are important tools to determine the number, size and/or localization of specific DNA sequences. For example, when CGH the entire genomes of paint and compared with normal reference genomes to identify areas with aberrant number of copies. Typical DNA of the fabric of the subject and of normal tissue tests have been labelled probes of different colors. Pula DNA mix and add to drug normal chromosomes (or to a microchip for CGH on microchips or matrix). Then the ratio of colors compare to identify areas with aberrant number of copies.

FISH typically used when you want multiple color visualization and/or when the Protocol requires the quantification of signals. This method usually includes the preparation of a cytological sample, tagging probes, denaturation of chromosome damage and probe hybridization probe with a sequence of target detection and signal. Typical reaction hybridization fluorescent paints the target sequence in such a way that their localization, size or number can be determined using fluorescent microscopy, flow cytometry or other suitable devices. DNA sequence in the range of entire genomes to several you can explore using FISH. FISH can also be used on drugs and interphase nuclei.

The composition of the invention can also be used fully or partially in all types of molecular biological techniques, which involve hybridization, including blotting and hybridization with a probe (for example, southern, etc), in microchips and amplification techniques, including traditional PCR, RT-PCR, mutant PCR, PCR, PCR with hot running inverse PCR, PCR, nested PCR, quantitative PCR and PCR in situ. PCR, in situ is a polymerase chain reaction, which goes inside the cell or on the glass, for example, on the above cytological and histological samples. Typically after the adhesion of the sample to a substantive glass cells and , and then combine it with the appropriate blend of PCR reagents, including polymerase, dNTP and primers. PCR can be performed in a specially designed device, such as GeneAmp In situ PCR System 1000 (Perkin Elmer Biosystems, Foster City, CA), and product can be defined using labelled probes or by including labeled dNTP during amplification. The composition of the invention will improve the effectiveness of traditional and in situ PCR analysis, for example, by reducing the temperature denaturation and hybridization and/or time required for the conduct of cycles of amplification.

(3) the Conditions hybridization

Method of the present invention comprises the application of a polar aprotic solvent-hybridization chains of nucleic acids. Compositions of the present invention are particularly useful in this method.

Ways of hybridization using composition according to the invention may include application of compositions on a sample containing -acid sequence of the target, most likely in double form. Typically to provide access probe for hybridization with the sequence of the target, the design and composition of the heat up for denaturation of nucleic acid targets. During the melting polar non-Protic solvent interacts with the sequence and contributes to the denaturation of the target and the renaturation of the probe with the target. Polar aprotic solvents, described in the present invention, significantly accelerate this process and reduce stiffness and toxicity hybridization conditions compared with shifted toward longer wavelengths.

Hybridization using the compositions of the invention can be done using the same methodology of the analysis, as to conducted with traditional compositions. However, the composition of the invention will allow more short periods of hybridization. For example, in the stages of pre-processing heating, splitting, denaturation, hybridization, washing and fill in the environment, you can use the same conditions in terms of volume, temperature, reagents and incubation periods, both for traditional compositions. In traditional hybridization protocols, known in the art, there is considerable variation. For example, in some protocols specify a separate stage of denaturation of potential double strand of nucleotides in the absence of a probe before the next stage of hybridization. The composition of the invention can be used in any of the traditional hybridization protocols, known in the art.

Alternatively analyses using the compositions of the invention can change and optimize compared to traditional methodologies, for example, by reducing the time hybridization, increase or decrease in temperature denaturation and/or hybridization and/or to increase or decrease the volume of hybridization.

For example, some forms of carrying out the composition of the invention will give strong signals when the denaturation temperature ranges from 60 to 100 C, and the temperature hybridization is from 20 to 60 degrees-C. other forms of implementation of the composition according to the invention will give strong signals when the denaturation temperature is from 60 to 70 deg C, 70 to 80 C, 80 to 90 Celsius or from 90 to 100 C, and the temperature hybridization is from 20 to 30 C and 30 to 40 degrees, from 40 to 50 degrees, or from 50 to 60 degrees-C. Other forms of implementation of the composition according to the invention will give strong signals when the denaturation temperature is 72, 82 or 92 OC, and temperature hybridization is 40, 45 or 50 degrees C.

Other forms of implementation of the composition according to the invention will give strong signals when the time denaturation is from 0 to 10 minutes, and time hybridization is from 0 minutes to 24 hours. Other forms of implementation of the composition according to the invention will give strong signals when the time denaturation is from 0 to 5 minutes, and time hybridization is 0 minutes to 8 hours. Other forms of implementation of the composition according to the invention will give strong signals when the time denaturation is 0, 1, 2, 3, 4 or 5 minutes, and time hybridization is 0 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes 180 minutes or 240 minutes. The specialists in this field of technology it is clear that in some cases, for example, RNA, stage of denaturation is not required.

Accordingly, hybridization using the compositions of the invention can be performed in less than 8 hours. Other forms of implementation of hybridization are less than 6 hours. Still other forms of implementation of hybridization are less than 4 hours. Other forms of implementation of hybridization within 3 hours. Still other forms of implementation of hybridization within 2 hours. Other forms of implementation of hybridization within 1 hour. Still other forms of implementation of hybridization within 30 minutes. Other forms of implementation of hybridization may take place within 15 minutes. Hybridization may even take place within 10 minutes or less than 5 minutes. Figures 1 and 2 illustrate the typical periodization for held on cytological and histological samples, respectively, using the compositions of the invention compared with using traditional compositions.

When the time changes of hybridization, the concentration of the probe can be also vary with the purpose of getting strong signals and/or reduce the background. For example, when time hybridization decreases, the number of the probe can be increased to improve the strength of the signal. On the other hand, when the time hybridization decreases, the number of the probe can be reduced to improve the background staining.

The composition of the invention unexpectedly eliminate the need to stage blocking during hybridization by improving the intensity of the signal and the background by blocking the binding of, for example, repeating sequences of DNA target. Thus, there is no need to use a total of human DNA, blocking the NCP, HONEYCOMB-1 DNA or DNA from any other source as a blocking agent. However, the background levels could be reduced further by adding agents that reduce nonspecific binding, for example, with the cell membrane, such as small amounts of the total human DNA or DNA is not of human origin (for example, salmon sperm DNA), in response hybridization using the compositions of the invention.

Water composition according to the invention, in addition, provide an opportunity to significantly reduce the concentration of -acid sequences included in the composition. As a rule, the concentration of probes can be reduced in 2-8 times in comparison with traditional concentrations. For example, if probes HER2 DNA and probes CEN17 NCP used in the compositions of the invention, their concentrations can be reduced by? and?, respectively, compared with concentrations in the traditional compositions for hybridization. This characteristic, in parallel with the lack of any requirement of a blocking DNA, such as blocking the NCP or 1, allows to increase the volume of probe systems, devices than traditional volume of 10 ml used in traditional systems, compositions, which reduces the loss due to evaporation, as discussed in more detail below.

A decrease in the concentration of the probe also allows to reduce the background. However, the decrease in the concentration of the probe was inversely associated with time hybridization, that is, the lower the concentration, the more time hybridization required. However, even when using the extremely low concentrations of the probe with water compositions of the invention, the time hybridization still shorter than with traditional compositions.

The composition of the invention enable best values of the signal and background than the traditional compositions for hybridization. For example, some of the probes hybridization with compositions of the invention will give similar backgrounds and stronger signals than hybridization during the night in traditional compositions. The background is not visible when the probe is added.

Traditional methods of analysis, you can also edit and optimize when using the compositions of the invention, depending on whether the system is a manual, semi-automatic or automatic. For example, for semi-automatic or automatic system will be useful for short periods of hybridization obtained with compositions of the invention. A short time hybridization can reduce the difficulties encountered when used in such systems of traditional compositions. For example, one of the problems with semi-automatic or automatic systems is that during hybridization can be considerable evaporation of the sample, since such systems require small amounts of sample (for example, 10-150 ml), elevated temperatures and extended periods hybridization (for example, 14 hours). Thus, the share of components in the traditional compositions for hybridization virtually unchanged. However, since the composition of the invention will allow more rapid , evaporation is reduced, which gives the possibility of increased flexibility in the proportions of the components in compositions for hybridization used in semi-automatic and automatic systems.

Another problem for automated analysis of visualization is required as of images, a huge number of required storage space and the time necessary for removing the images. The composition of the invention can solve this problem by getting very strong signals than traditional compositions. Due to very strong signals, which give the composition according to the invention, visualization can be done with a smaller increase than that required for traditional compositions, and you can still detect and analyze, for example, using the algorithms. Since the focus plane becomes broader with a smaller increase, the composition of the invention reduce or eliminate the need for the withdrawal of serial sections of the sample. In result, the total rendering is much faster, because the composition of the invention require less or do not require a serial sections, and each image covers a much larger area. In addition, the total analysis time is quicker, since the General image files much smaller.

Thus, the composition and the ways in which the invention can solve many of the problems associated with traditional songs and ways of hybridization.

Description can be more clearly understood by the following non-limiting examples of which are the preferred form of the compositions described in the following. Aside from the examples, or, where not otherwise indicated, all numbers expressing the number of ingredients, reaction conditions, etc. used in the description and claims to be understood as a modifiable in all cases, the term "approximately". Respectively, if not otherwise stated to the contrary, numeric parameters shown in the following description and the accompanying claims are approximate, which may vary depending on the desired characteristics that would like to get in this regard. At least, not as an attempt to restrict the application of the doctrine of equivalents to the volume of claims, each numeric parameter should be interpreted in the light of the number of significant figures and ordinary rounding methods.

Despite the fact that the numerical ranges and parameters set forth in the wide volume represent approximate numeric values contained in the specific example, are specified as precisely as possible. Any numerical value, however, in itself contains some errors, which are the inevitable result of a standard deviation, found in the respective control measurements. The following examples illustrate the present invention, and they cannot be considered as limiting the invention.

Now will be made detailed reference to specific forms of carrying out the invention. Although the invention described in connection with these forms of implementation, it should be clear that they are not intended to limit the invention these forms of implementation. On the contrary, imply that the invention covers alternatives, modification and equivalents that may be included in the invention, as defined by the attached by the claims.

Reagents used in the following examples represent the reagents from a set of Dako's Histology FISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (Dako Denmark A/S, Glostrup, Denmark). These kits contain all key reagents except the probe necessary to perform the techniques of FISH for formalin fixed, prisoners in paraffin tissue sections of specimens. All samples were prepared in accordance with the description of the manufacturer. The device Dako Hybridizer (S2450, Dako) used for stages of splitting, denaturation and hybridization.

Assessment of FISH preparations were conducted during the week after hybridization, using a fluorescent microscope Leica DM6000B equipped with separate filters for , FITZ, Texas Red and dual filter FITZ/Texas Red, under 10x, 20x, 40x and 100x oil immersion lens.

Assessment CISH drugs was performed using a light microscope Olympus BX51, under 4x, 10x, 20x, 40x and 60x lens.

In the following Examples, "" refers to sodium salt (D8906, Sigma), which has a molecular mass MW more than 500000. All concentrations of polar aprotic solvents are given in percentage about/about. Phosphate buffer refers to phosphate buffer solution containing NaH 2 PO 4 x 2H 2 O (dwuhosnovny sodium phosphate dihydrate) and Na 2 HPO 4 x H 2 O ( sodium phosphate monohydrate). buffer refers to buffer solution containing sodium citrate (Na 3 6 N 5 OF 7 X 2H 2 O; 1.06448, Merck) and citric acid monohydrate (6 N 8 7 X N 2 O; 1.00244, Merck).

Total histological methods of FISH/CISH (Examples 1-20)

Drugs with sections of formalin fixed, prisoners in paraffin (FFPE) multiple tissue ordered slices of people (tonsils, carcinoma of the breast, kidney and colon intestine) baked at 60 OC for 30 to 60 minutes, in trays with with xylene, in trays with ethanol, then transferred in buffer. Then the samples preliminarily processed in the solution of preliminary processing of at least 95 degrees C for 10 min, and washed the 2 x 3 minutes Then the samples digested pepsin RTU at 37 C for 3 min, washed, 2 x 3 min, in a series of ethanol and dried in air. Then the samples were incubated with 10 MKL FISH probe, as described for individual experiments. Then the samples were washed rigid laundering at 65 C for 10 minutes, then washed the 2 x 3 minutes, then in a series of ethanol and dried in air. Finally, drugs poured 15 ul pouring environment Antifade. When the painting was completed, observers, trained assessing the intensity of the signal, morphology and background of stained smears, assessed.

Total cytological method FISH (Examples 21-22)

Glass with drugs metaphases recorded a 3.7% formaldehyde for 2 min, washed, 2 x 5 min, in a series of ethanol and dried in air. Then the samples were incubated with 10 MKL FISH probe, as described for individual experiments. Then the samples were washed rigid laundering at 65 C for 10 minutes, then washed the 2 x 3 minutes, then in a series of ethanol and dried in air. Finally, drugs poured 15 ul pouring environment Antifade. When the painting was completed, observers, trained assessing the intensity of the signal, morphology and background of stained smears, assessed, as described in the manual on assessment of histological sections.

Guidance on the assessment of tissue slices

Signal intensity was assessed on a scale of 0 to 3, where 0 means no signal, and 3 correspond to a strong signal. Cellular/tissue structures evaluate on a scale of 0 to 3, where 0 means no patterns and lack of boundaries nuclei, and 3 corresponds to the intact structure and clear boundaries nuclei. Between 0 and 3, there are additional gradation of 0.5, beyond which an observer can evaluate the intensity of the signal, tissue structure and background.

The intensity of the signal is evaluated according to a graduated system on the scale 0-3.

0 the Signal is not visible.

1 signal strength is weak.

2 the Intensity of the signal reasonable.

3 the Intensity of the signal is strong. This evaluation system allows the use of gradation?.

Tissue and nuclear structure is evaluated according to a graduated system on the scale 0-3.

0 Tissue structures and boundaries of nuclei completely destroyed.

1 Tissue structures and/or border nuclei weak. This classification includes situations where some areas have empty kernel.

2 Tissue structures and/or border nuclei are visible, but the boundaries of the nuclei fuzzy. This classification includes situations where multiple cores are empty.

3 Tissue structures and boundaries of the nuclei are intact and legible.

This evaluation system allows the use of gradation?.

Background assessed on a graduated system on the scale 0-3.

0 Von small or not visible.

1 Some background.

2 Moderate background.

3 High background.

This evaluation system allows the use of gradation?.

Example 1

In this example, compare the intensity of the signal and cell morphology of the samples processed compositions of the invention or traditional solutions for hybridization, depending on the temperature denaturation.

FISH Composition probe I: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% formamide (15515-026, Invitrogen), 5 microns blocking the NCP (see Kirsten Vang Nielsen et al., PNA Suppression Method Combined with Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006)), 10 ng/ml of tracer Texas Red DNA probe gene CCND1 (RP11-114320, the size of 192 KB).

FISH Composition probe II: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% (03519, Fluka), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1 (RP11-1143E20, the size of 192 KB).

Phase different viscosity, if they were present, mixed before use. FISH probes , as indicated, for 5 minutes and hybridized at 45 C for 60 minutes.

Denaturation temperature

Cell morphology

(I) Formamide

(II) THE EU

Formamide

82 o With

Signals ranked as "3", were clearly visible in 20x lens.

Example 2

In this example, compare the intensity of the signal and background staining of samples processed compositions of the invention or traditional solutions for hybridization, depending on the temperature denaturation.

FISH Composition probe I: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% formamide, 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe II: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% , 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for up to 14 hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes 0 minutes.

FISH Composition probe II: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% (EU), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe III: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% propylene carbonate (PC) (540013, Aldrich), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe IV: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% (SL) (T22209, Aldrich), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe V: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% acetonitrile (AN) (C02CIIX, Lab-Scan), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe VI: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% γ-butyrolactone (GBL) (B103608, Aldrich), 5 microns blocking the NCP, to 7.5 ng/ml of tracer Texas Red DNA probe gene CCND1.

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

(1) Formamide

(II)THE EU

Signals ranked as "3", were clearly visible in 20x lens.

Example 4

In this example, compare the intensity of the signal from the samples processed compositions of the invention, with different concentrations of polar solvent.

FISH Song probe: 10% , 300 mm NaCl, 5 mm phosphate buffer, 10-60% (as stated), 5 microns blocking the NCP, to 7.5 ng/ml of tracer Texas Red DNA probe gene /GK-constant field (CTD-3050E15, RP11-1083E8; size 227 KB) and 7.5 ng/ml of tracer FITZ DNA probe gene /GK-variable field (CTD-2575M21, RP11-122B6, RP11-316G9; size 350 and 429 KB).

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

(EU)

The intensity of the signal

Texas Red

Signals ranked as "3", were clearly visible in 20x lens.

Example 5

In this example, compare the intensity of the signal and the intensity of the background of samples processed compositions with blocking the NCP and without them.

FISH Song probe: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% of 7.5 ng/ml of tracer Texas Red DNA probe gene CCNDI.

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

(EU)

PNK-blocking

Without blocking PNK

The intensity of the signal

The intensity of the background

Signals ranked as "3", were clearly visible in 20x lens.

Example 6

In this example, compare the intensity of the signal from the samples processed compositions of the invention, depending on the concentration of the probe and the time hybridization.

FISH Song probe: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% and 10 7,5 5 or 2.5 ng/ml of tracer Texas Red DNA probe gene CCND1 (as indicated).

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 3 hours, 2 hours and 1 hour.

Time hybridization

The intensity of the signal

(I)10 ng/ml

(II)to 7.5 ng/ml

(III)5 ng/ml

(IV)2.5 ng/ml

Signals ranked as "3", were clearly visible in 20x lens.

Example 7

In this example, compare the intensity of the signal from the samples processed compositions of the invention, depending on the concentrations of salt, phosphate buffer.

FISH Song probe: 10% , ([NaCl], [phosphate buffer], [Tris-buffer], as indicated in the Results), 40% of 7.5 ng/ml of tracer Texas Red DNA probe gene CCND1.

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

The intensity of the signal phosphate [0 mm]

The intensity of the signal phosphate [5 mm]

The intensity of the signal phosphate [35 mm]

The intensity of the signal Tris [40 mm]

Signals ranked as "3", were clearly visible in 20x lens.

Example 8

In this example, compare the intensity of the signal from the samples processed compositions of the invention, depending on the concentration .

FISH Song probe: 0, 1, 2, 5 or 10% dextran sulfate (as stated), 300 mm NaCl, 5 mm phosphate buffer, 40% , 5 ng/ml of tracer Texas Red DNA probe gene SIL-TAL1 (RP1-278013; size 67 KB) and 6 ng/ml FITZ-SIL-TAL1 (ICRFc112-112C1794, RP11-184J23, RP11-8J9, CTD-2007B18, 133 B9; size 560 KB).

Phase different viscosity, if they were present, mixed before use. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes. Without blocking.

%

The intensity of the signal

Probe Texas Red

Probe FITZ

NOTE: in this experiment, the results ranked as "3", as the probe SIL-TAL1, tagged Texas Red, has a size of only 67 KB and is taken from a non-optimized drug.

Example 9

In this example, compare the intensity of the signal from the samples processed compositions of the invention, depending on the concentrations of , salt, phosphate and polar solvent.

FISH Composition probe la: 34% , 0 mm NaCl, 0 mm phosphate buffer, 0% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe Ib: 34% , 300 mm NaCl, 5 mm phosphate buffer, 0% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe Ic: 34% , 600 mm NaCl, 10 mm phosphate buffer, 0% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe ll: 32% , 0 mm NaCl, 0 mm phosphate buffer, 5% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe llb: 32% , 300 mm NaCl, 5 mm phosphate buffer, 5% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe llc: 32% , 600 mm NaCl, 10 mm phosphate buffer, 5% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe llla: 30% , 0 mm NaCl, 0 mm phosphate buffer, 10% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe lllb: 30% , 300 mm NaCl, 5 mm phosphate buffer, 10% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe lllc: 30% , 600 mm NaCl, 10 mm phosphate buffer, 10% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe IVa: 28% , 0 mm NaCl, 0 mm phosphate buffer, 15% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe IVb: 28% , 300 mm NaCl, 5 mm phosphate buffer, 15% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Composition probe IVc: 28% , 600 mm NaCl, 10 mm phosphate buffer, 15% , 10 ng/ml of tracer Texas Red DNA probe of HER2 gene (size 218 KB) and 50 nm FITZ-labeled PNK probe CEN-7.

FISH Reference probe V: Standard a commercially bottle mixture probe HER2 PharmDx (K5331, Dako)containing blocking the NCP. Night hybridization for 20 hours.

All songs were present in the form of one phase. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes without blocking except FISH of the Reference probe V, which was blocking the NCP and the for 20 hours.

Signal strength

DNA probes

PNK probes

Composition Ia

Composition Ib

Composition Ic

Composition IIa

Composition IIb

Composition IIc

Composition IIIa

Composition IIIb

Composition IIIc

Composition IVa

Composition IVb

Composition IVc

Reference V

NOTE: the Composition IVa give strong signals DNA without salt. This is not possible with standard FISH compositions, where the DNA binding is dependent on the salt.

Example 10

In this example, compare the intensity of the signal from the samples processed compositions of the invention, depending on the concentration of polar solvent and in conditions of high (4x normal) salt concentration.

FISH Composition probe I: 0% , 29% , 1200 mm NaCl, 20 mm phosphate buffer, 10 ng/ml of tracer Texas Red DNA probe of HER2 gene and 50 nm FITZ-labeled PNK probe CEN-7. The composition represents one phase.

FISH Composition probe II: 5% , 27% , 1200 mm NaCl, 20 mm phosphate buffer, 10 ng/ml of tracer Texas Red DNA probe of HER2 gene and 50 nm FITZ-labeled PNK probe CEN-7. The composition represents one phase.

FISH Composition probe III: 10% , 25% , 1200 mm NaCl, 20 mm phosphate buffer, 10 ng/ml of tracer Texas Red DNA probe of HER2 gene and 50 nm FITZ-labeled PNK probe CEN-7. The composition represents one phase.

FISH Composition probe IV (not tested): 20% , 21% , 1200 mm NaCl, 20 mm phosphate buffer, 10 ng/ml of tracer Texas Red DNA probe of HER2 gene and 50 nm FITZ-labeled PNK probe CEN-7. Composition had two phases.

Signal strength

DNA probes

PNK probes

Composition I

Composition II

Composition IV

Note: Composition II gave good signals DNA with only 5% of the EU and the strong signals DNA with 10% of the EU.

Example 11

In this example, compare the intensity of the signal and the noise from the samples processed by various phases of the compositions of the invention.

FISH Composition probe: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% , 8 ng/ml of tracer Texas Red DNA probe of HER2 gene and 600 nm FITZ-labeled PNK probe CEN-17. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes. Without blocking.

This example is similar to the previous example, but it uses a different DNA probe and GBL instead of the EU.

FISH Composition probe: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% GBL, 10 ng/ml of tracer Texas Red DNA probe gene CCND1 and 600 nm FITZ-labeled PNK probe CEN-17.

FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes. Without blocking.

Signal strength

Background

DNA probe

PNK probe

The upper phase

0 is half

The lower phase

Mixed phase

Example 13

In this example, examine the number of phases in the compositions of the invention depending on the concentration of polar solvent and .

FISH Song probe: 10 or 20% ; 300 mm NaCl, 5 mm phosphate buffer; 0, 5, 10, 15, 20, 25, 30% EU; 10 ng/ml probe.

Number of phases of 10% Dextran

Number of phases of 20% Dextran

NOTE: 15% EU, 20% forms the best available high signal intensity of the above single-phase solution. Biphasic 20% of the EU is even higher intensity than 15%. (Data not shown).

Example 14

In this example, compare the intensity of the signal and the noise from the samples processed various compositions of the invention, depending on the focus of the probe and the time hybridization.

FISH Composition probe I: 10 ng/ml with HER2 TxRed the labeled DNA probes (standard concentration), and the standard concentration CEN7 FITZ-labeled PNK probe (50 nm); 15% of the EU; 20% ; 600 mm NaCl; 10 mm phosphate buffer.

FISH Composition probe II: 5 ng/ml with HER2 TxRed the labeled DNA probes (1/2 standard concentration) and standard concentration CEN7 FITZ-labeled PNK probes (50 nm); 15% of the EU; 20% ; 600 mm NaCl; 10 mm phosphate buffer.

FISH Composition probe III: 2.5 ng/ml with HER2 TxRed labeled DNA probe (1/4 standard concentration) and threaten the standard concentration (25 nm) CEN7 PNK probes; 15% of the EU; 20% ; 600 mm NaCl; 10 mm phosphate buffer.

Songs of I-III existed as a single phase. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 3 hours, 2 hours and 1 hour.

Time hybridization

The intensity of the signal

Signals ranked as "3", were clearly visible in 20x lens. F: Background.

Example 15

In this example, compare the intensity of the signal and the noise from the samples processed compositions of the invention, depending on the blocking agent.

FISH Song probe: 15% of the EU; 20% ; 600 mm NaCl; 10 mm phosphate buffer; 2.5 ng/ml with HER2 TxRed labeled DNA probe (1/4 standard concentration) and? standard concentration (300 nm) FITZ-labeled CEN17 PNK probe. Samples blocked: (a) not blocked; (b) 0.1 mg/ml 1 (15279-011, Invitrogen); (in) 0.3 ug/ml 1; or (d) 0.1 mg/l of total DNA of a person before hybridization using the compositions of the invention.

All samples were present in the form of one phase. FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

Blocking agent

Background

The intensity of the signal

Without blocking

0.1 ug/ml 1

0.3 ug/ml 1

0.1 ug/l total human DNA

NOTE: background Levels without blocking significantly lower than usually observed by standard FISH without blocking. On the contrary, if the standard composition of FISH does not contain a blocking agent, signals are usually impossible to read.

Example 16

In this experiment compare different ways of removal of background staining using the compositions of the invention.

All songs contained 15% EU, 20% , 600 mm NaCl, 10 mm phosphate buffer, 2.5 ng/ml with HER2 DNA probes (1/4 standard concentration), 300 nm CEN17 PNK probe (1/2 standard concentration) and one of the following agents that reduce background:

A) 5 microns blocking the NCP (see Kirsten Vang Nielsen et al., PNA Suppression Method Combined with Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006))

B) 0.1 mg/ml HUNDRED-1 DNA

B) 0.1 mg/l of total DNA rights (THD) (treated with ultrasound, THD)

G) 0.1 mg/ml degraded as a result of hydrodynamic shift salmon sperm DNA (9680, Ambion)

E) 0.1 mg/ml calf (D8661, Sigma)

E) 0.1 mg/ml sperm DNA herring (D7290, Sigma)

G) 0.5% of formamide

3) 2% formamide

And) 1% ethylene glycol (1.09621, Merck)

R) 1% glycerin (1.04095, Merck)

L) 1% 1,3-propandiol (533734, Aldrich)

M) 1% N 2 O (control)

All samples were present in the form of one phase. The probes were incubated at 82 degree for 5 minutes, and then at 45 C for FFPE tissue sections within 60 and 120 minutes.

Blocking background

Hybridization/min

Background

The intensity of the signal

Blocking PNK

Blocking PNK

HUNDRED-1

HUNDRED-1

+0 is half

Salmon sperm DNA

Salmon sperm DNA

Calf thymus DNA from

Calf thymus DNA from

Sperm DNA herring

Sperm DNA herring

0,5% formamide

0,5% formamide

2% formamide

2% formamide

1% ethylene glycol

1% ethylene glycol

1% glycerin

1% glycerin

1% 1,3-propandiol

1% 1,3-propandiol

Without blocking

Without blocking

NOTE: all reagents, reduce background, except for blocking the NCP, showed effect in reducing the background. Thus, specific blocking against repetitive DNA is not required.

Example 17

In this experiment compared the intensity of the signal from the top and bottom of phases by using two different polar aprotic solvents.

FISH Composition probe I: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% (ET) (27750, Aldrich), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH Composition probe II: 10% , 300 mm NaCl, 5 mm phosphate buffer, 40% (GS) (G7208, Aldrich), 5 microns blocking the NCP, 10 ng/ml of tracer Texas Red DNA probe gene CCND1.

FISH probes were incubated at 82 degree for 5 min, and then at 45 C for 60 minutes.

The intensity of the signal

The upper phase

The lower phase

A mixture of upper and lower phases

Example 18.

In this experiment examined the capacity of different polar aprotic solvents to the formation of a single-phase system.

All songs contained: 20% , 600 mm NaCl, 10 mm phosphate buffer and 10, 15, 20 or 25% of one of the following polar aprotic solvents:

γ-Butyrolactone

Propylene carbonate

Results: all polar aprotic solvents for all studied concentrations formed by at least two-phase system used in compositions. However, this does not exclude that these compounds may form a single-phase system, under other compositions.

Example 19

In this experiment, explore the use of compositions of the invention in the analysis chromogenic in situ hybridization (CISH) on multiple FFPE tissue sections.

FISH Composition probe 1: 4.5 ng/ml FITZ-labeled DNA probe gene TCRAD (1/4 standard concentration) (RP11-654A2, RP11-246A2, CTP 2355L21, RP11-158G6, RP11-780M2, RP11-481C14; size 1018 KB); 15% of the EU; 20% ; 600 mm NaCl; 10 mm buffer pH 6.0.

FISH Composition probe II: 4.5 ng/ml FITZ-labeled DNA probe gene TCRAD (1/4 standard concentration) (RP11-654A2, RP11-246A2, CTP 2355L21, RP11-158G6, RP11-780M2, RP11-481C14; size 1018 KB); 15% of the EU; 20% ; 600 mm NaCl; 10 mm buffer pH 6.0; 0.1 mg/ml degraded as a result of hydrodynamic shift salmon sperm DNA.

FISH Composition probe III: 300 nm each individual FITZ-labeled PNK probe CEN17 (1/2 standard concentration); 15% of the EU; 20% ; 600 mm NaCl; 10 mm buffer pH 6.0.

All samples were analyzed using the Protocol Dako DuoCISH (SK108) and compositions for separated probes except that tough washing was conducted for 20 minutes instead of 10 minutes, and without the use stage of the red Chromogen DuoCISH.

Signal strength

Composition

Note: the Intensity of the signal is very strong. Due to high levels of background it is impossible to distinguish, does the background adding DNA salmon in the Composition II. Signals were clearly visible when using a 10x zoom lens, for example, the tonsils, which generally have less background. If the tissue were of a high background signals were clearly visible when using 20x lens.

Example 20

In this example, compare the intensity of the signal and the background of FFPE tissue slices, processed compositions of the invention with two DNA probes.

FISH Composition probe I: 9 ng/ml FITZ-labeled DNA probe gene IGH (RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23, P-308718; size 612 KB); 6,4 ng/ml TX Red labeled DNA probe MYC gene (CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 KB); 15% of the EU; 20% ; 600 mm NaCl; 10 mm buffer pH 6.0.

FISH Composition probe II: 9 ng/ml FITZ-labeled DNA probe gene IGH; 6,4 ng/ml TX Red labeled DNA probe MYC gene; 15% EU, 20% ; 600 mm NaCl; 10 mm buffer pH 6.0; 0.1 mg/ml degraded as a result of hydrodynamic shift salmon sperm DNA.

Signal strength

DNA salmon

FITZ probe

Texas Red probe

Background

NOTE: high background was probably due to the fact that used standard concentration probe.

Example 21

In this experiment investigated the application of the compositions of the invention on cytological samples.

FISH Composition probe: 15% of the EU; 20% ; 600 mm NaCl; 10 mm phosphate buffer; 5 ng/ml with HER2 TxRed labeled DNA probe (1/2 standard concentration) and Vi standard concentration CEN7 (25 nm).

FISH Composition probe 1: 6 ng/ITUC labeled Texas Red DNA probe gene TCRAD (standard concentration) (P-3166620, CTP 2373N7; size 301 KB) and 4.5 ng/ml FITZ-labeled DNA probe (1/4 standard concentration); 15% EU, 20% ; 600 mm NaCl; 10 mm buffer pH 6.0.

FISH Composition probe II: 6 ng/ml of tracer Texas Red DNA probe gene TCRAD (standard concentration) (301 KB) and 4.5 ng/ml FITZ-labeled DNA probe (1/4 standard concentration); 15% EU, 20% ; 600 mm NaCl; 10 mm buffer pH 6.0; 0.1 mg/ml degraded as a result of hydrodynamic shift salmon sperm DNA.

FISH probes were incubated in a preparations at 82 degree for 5 minutes, then at 45 C for 60 minutes

Blocking agent

Background

The intensity of the signal

0.1 ug/ml DNA salmon

Again no chromosomes (R-nature ) were observed with compositions of the invention. In addition, the observed background staining interphase nuclei or chromosomes.

ADDITIONAL FORMS OF

Form of implementation 1. Composition for hybridization with at least one -acid sequence of at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences, and solution for hybridization, where polar non-Protic solvent is not dimethyl sulfoxide (DMSO).

Form of implementation 2. Composition for hybridization in accordance with the form of the 1, where the concentration of polar solvent is about 1% to 95% (about/about).

Form of implementation of the 3. Composition for hybridization in accordance with the form of 1 or 2, where the concentration of polar solvent is from 5% to 10% (on/of).

Form of implementation of the 4. Composition for hybridization in accordance with the form of 1 or 2, where the concentration of polar solvent is from 10% to 20% (about/about).

Form of implementation 5. Composition for hybridization in accordance with the form of 1 or 2, where the concentration of polar solvent is from 20% to 30% (about/about).

Form 6. Composition for hybridization in accordance with any form of 1-5, where polar non-Protic solvent is non-toxic.

Form of implementation 7. Composition for hybridization in accordance with any form of exercise 1-6, provided that the composition for hybridization does not contain formamide.

Form of implementation 8. Composition for hybridization in accordance with the form 6, provided that the composition for hybridization contains less than 10% .

Form of implementation 9. Composition for hybridization in accordance with the form of implementation 8, provided that the composition for hybridization contains less than 2% .

Form of implementation 10. Composition for hybridization in accordance with the form of implementation 9, provided that the composition for hybridization contains less than 1% .

Form of implementation 11. Composition for hybridization in accordance with any form of 1-10, where polar non-Protic solvent has , sulfonic, , and/or carbonate functional group.

Form of implementation of the 12. Composition for hybridization in accordance with any form of exercise 1-11, where polar non-Protic solvent has dispersive parameter solubility in the interval from 17.7 MPa 1/2 to 22.0 MPa 1/2 , polar parameter solubility in the interval from 13 MPa 1/2 to 23 MPa 1/2 and parameter solubility due to the hydrogen bonds in the range of 3 MPa 1/2 to 13 MPa 1/2 .

Form of implementation 13. Composition for hybridization in accordance with any form of exercise 1-12, where polar non-Protic solvent has a cyclic basic structure.

Form of implementation 14. Composition for hybridization in accordance with any form of exercise 1-13, where polar non-Protic solvent is selected from the group consisting of:

where X represents the O and the R 1 is a , and

where X is optional and, if present, the selected of O, S;

where Z is optional and, if present, the selected of O, S;

where A and b independently represent About or N or S or part or primary amine;

where R is the ; and

where Y is On or S or S.

Form of implementation 15. Composition for hybridization in accordance with any form of exercise 1-14, where polar non-Protic solvent is selected from the group consisting of: , acetonitrile, N-, 4-aminopyridine, benzamida, benzimidazole, 1,2,3-benzotriazole series, , 2,3-, gamma-butyrolactone, (Epsilon)maleic acetyl chloride, 2-, , chloronitromethane, anhydride, , 5-cyano-2-, , , , 1,3-dimethyl-5-, 1,5-, 1,2-dinitrobenzene, 2,4-dinitrotoluene, , 1,2-dinitrobenzene, 2,4-dinitrotoluene, , Epsilon-caprolactam, , , N-, , , , , , 2-, 2-imidazole, term administration of isatin, , , 4-, 1-methoxy-2-nitrobenzene, methyl-alpha-, 1-methylimidazole, N-methylimidazole, 3-, nmethylmorpholine-N-oxide, , N-, , methyl-4-, 3-nitroaniline, , 2-, 1-nitroso-2-, 2-, 2-oxazolidinone, 9,10-, N-, phthalic anhydride, (2-), 1,3-, b-, , 4H-PYRAN-4-thione, 4H-PYRAN-4-it (gamma-pyrone derivatives), , 2-, saccharin, , , sulfolane, 2,2,6,6-, oxide, (sulfolane), thiazole, 2-, 3,3,3-, 1,1,2-, 1,2,3-, trimethylene sulfide radical cations dioxide and .

Form of implementation 16. Composition for hybridization in accordance with any form of exercise 1-14, where polar non-Protic solvent is selected from the group consisting of:

, , , and .

Form of implementation 17. Composition for hybridization in accordance with any form of exercise 1-14, where polar non-Protic solvent is:

Form of implementation 18. Composition for hybridization in accordance with any form of exercise 1-17, additionally contains at least one additional component selected from the group consisting of: buffer agents, salts, catalysts, chelating agents, detergents and blocking agents.

Form of implementation 19. Composition for hybridization in accordance with the form of 18, where the catalyst is a , and salt are sodium chloride and/or phosphate buffer.

Form of implementation 20. Composition for hybridization in accordance with the form of implementation 19, where is present in a concentration of 5% to 40%, sodium chloride present in concentrations from 0 mm to 1200 mm and/or phosphate buffer is present in concentrations from 0 mm up to 50 mm.

Form of implementation 21. Composition for hybridization in accordance with the form of 20, where present in concentrations ranging from 10% to 30%, sodium chloride is present in a concentration of 300 mm to 1200 mm and/or phosphate buffer is present in a concentration of 5 mm up to 20 mm.

Form of implementation 22. Composition for hybridization in accordance with the form of 18, where the catalyst is selected from the group consisting of:

, glycerin, propylene glycol 1,2-propandiol levels, diethylene glycol, glycol, glycol and 1,3-propandiol levels, a buffering agent is a buffer.

Form of implementation 23. Composition for hybridization in accordance with the form of implementation 22, where formamide is present in a concentration of 0,1-5%, glycerin, propylene glycol, 1,2-propandiol, diethylene glycol, glycol, glycol and 1,3-propandiol are present in a concentration of 0,1% to 10%, and buffer is present in a concentration of 1 mm up to 50 mm.

Form of implementation 24. Composition for hybridization in accordance with the form of 18, where blocking agent selected from the group consisting of: total human DNA, the DNA of sperm herring, salmon sperm DNA and calf thymus DNA from.

Form of implementation 25. Composition for hybridization in accordance with the form of implementation 24, where the total human DNA, the DNA of sperm herring, salmon sperm DNA and calf thymus DNA from present in concentrations ranging from 0.01 to 10 mcg/ml.

Form of implementation 26. Composition for hybridization in accordance with any form of exercise 1-25, containing 40% at least one polar solvent, 10% , 300 mm sodium chloride and 5 mm phosphate buffer.

Form of implementation 27. Composition for hybridization in accordance with any form of exercise 1-25, containing 15% at least one polar solvent, 20% , 600 mm sodium chloride, 10 mm phosphate buffer and 0.1 mcg/ml total human DNA.

Form of implementation 28. Composition for hybridization in accordance with any form of exercise 1-25, containing 15% at least one polar solvent, 20% , 600 mm sodium chloride, 10 mm buffer pH 6,2 and 0.1 mcg/ml sperm DNA of herring and salmon sperm DNA or calf thymus DNA from, or 0.5% formamide, or 1% ethylene glycol, or 1% 1,3-propandiol.

- unification of the first and second -acid sequence and composition for hybridization for at least a period of time sufficient for the hybridization of the first and second -acid sequences,

where polar non-Protic solvent is not dimethyl sulfoxide (DMSO).

Form of implementation 33. Method of hybridization -acid sequences, including:

- first -acid sequence in a biological sample, in situ

- application compositions for hybridization, containing the second -acid sequence and at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences, the first -acid sequence for at least a period of time sufficient for the hybridization of the first and second -acid sequences,

where polar non-Protic solvent is not dimethyl sulfoxide (DMSO).

Form of implementation 34. Method of hybridization -acid sequences, including:

- first -acid sequence,

- getting a second -acid sequence,

- a composition for hybridization in accordance with any form of exercise 1-31, and

- unification of the first and second -acid sequence and composition for hybridization for at least a period of time sufficient for the hybridization of the first and second -acid sequences.

Form of implementation 35. Method of hybridization -acid sequences, including:

- first -acid sequence, and

- application compositions for hybridization in accordance with any form of exercise 1-31 the first -acid sequence for at least a period of time sufficient for the hybridization of the first and second -acid sequences.

Form of implementation 36. Way in accordance with the forms of implementation of 30 or 31, where polar non-Protic solvent determined in accordance with any form of exercise 2-6 or 11-17.

Form of implementation 37. Method in accordance with any form of exercise 30-36, where I provide enough energy for the hybridization of the first and second nucleic acids.

Form of implementation 38. Way in accordance with the form of the implementation of 37, where the energy is provided by heating songs for hybridization and -acid sequence.

Form of implementation 39. Way in accordance with the form of the implementation of 38, where warming up phase is performed by the use of microwaves, hot baths, hot plates, a heating coil, Peltier element, induction heating or heating lamps.

Form of implementation 40. Method in accordance with any form of exercise 32-39, where the first -acid sequence is a double and the second nucleic acid is a single-stranded.

Form of implementation 41. Method in accordance with any form of exercise 32-40, where the stage of denaturation and hybridization are held separately.

Form of implementation 42. Method in accordance with any form of exercise 32-41, where phase hybridization includes stages of heating and cooling songs for hybridization and -acid sequences.

Form of implementation 43. Method in accordance with any form of exercise 32-42, where phase hybridization takes less than 8 hours.

Form of implementation 44. Way in accordance with the form of the implementation of 43, where the stage of hybridization is less than 1 hour.

Form of implementation 45. Way in accordance with the form of 44, where the phase hybridization takes less than 30 minutes.

Form of implementation 46. Way in accordance with the form of the implementation of the 45 where phase hybridization takes less than 15 minutes.

Form of implementation of the 47. Way in accordance with the form of the implementation of 46, where the phase hybridization takes less than 5 minutes.

Form of implementation of the 48. Method in accordance with any form of exercise 32-47, where the stage of cooling takes less than 1 hour.

Form of implementation 49. Way in accordance with the form of 48, where the stage of cooling takes less than 30 minutes.

Form of implementation 50. Way in accordance with the form of implementation 49, where the stage of cooling takes less than 15 minutes.

Form of implementation 51. Way in accordance with the form of implementation 50. where the stage of cooling takes less than 5 minutes.

Form of implementation of the 52. Method in accordance with any form of exercise 32-51 where -acid sequence is a biological sample.

Form of implementation of 53. Way in accordance with the form of 52, where the biological sample is a cytological or histological sample.

Form of implementation of the 54. Method in accordance with any form of exercise 32-53, where the composition for hybridization contains one phase at room temperature.

Form of implementation 55. Method in accordance with any form of exercise 32-53, where the composition for hybridization contains multiple phase at room temperature.

Form of 57. Way in accordance with the form of 55 or 56, where the phase composition for the hybridization of the mix.

Form of implementation 58. Method in accordance with any form of exercise 32-57, additionally includes the stage of blocking.

Form of implementation 59. Application songs for hybridization, containing from 1 to 95% (on/of) at least one polar solvent, in a hybridization assays.

Form of implementation 60. Application of the arrangement in accordance with a form of 59, where the composition for the hybridization of the matches any form of exercise 1-31.

1. Method of hybridization -acid sequences, including: - first -acid sequence cytological or histological sample - getting a second -acid sequence - a composition for hybridization, containing at least one polar non-Protic solvent in the amount effective for denaturation of nucleotide sequences, and - the Union of the first and second -acid sequence and composition for hybridization for at least a period of time sufficient for the hybridization of the first and second -acid sequences, where polar non-Protic solvent has a cyclic basic structure and is not dimethylsulfoxide (DMSO).

2. The method according to claim 1, where the specified composition for hybridization contains the second -acid sequence.

3. The method according to claim 1, where polar non-Protic solvent is non-toxic.

4. The method according to claim 1, where polar non-Protic solvent has , sulfonic, , and/or carbonate functional group.

5. The method according to claim 1, where polar non-Protic solvent has the dispersion of solubility in the interval from 17.7 MPa 1/2 to 22.0 MPa 1/2 , setting polar solubility in the interval from 13 MPa 1/2 to 23 MPa 1/2 and parameter solubility due to the hydrogen bonds in the range of 3 MPa 1/2 to 13 MPa 1/2 .

6. The method according to claim 1, where polar non-Protic solvent is selected from the group consisting of:

where X represents About and R 1 is a , and

where X is optional and, if present, the selected of O, S; where Z is optional and, if present, the selected of O, S; where A and b independently represent About, or N or S, or part or primary amine; where R is the ; and where Y represents About, or S, or C.

7. The method according to claim 1, where polar non-Protic solvent is selected from the group consisting of:

, , , and .

8. The method according to any one of claims 1 to 7, where the stage of hybridization is less than 8 hours, less than 1 hour, less than 30 min, less than 15 min or less than 5 minutes

9. Composition for hybridization of nucleic acids, containing from 1% to 95% (about./about.) at least one polar solvent, having circular basic structure, in an amount effective for denaturation of nucleotide sequences, and solution for hybridization, where polar non-Protic solvent is not dimethylsulfoxide (DMSO).

10. Composition for hybridization of claim 9, characterized by the fact that the polar non-Protic solvent is non-toxic.

11. Composition for hybridization of claim 9, characterized in that the composition contains less than 10% , preferably not contain formamide.

12. Composition for hybridization of claim 9, where polar non-Protic solvent has , sulfonic, , and/or carbonate functional group.

13. Composition for hybridization of claim 9, where polar non-Protic solvent has the dispersion of solubility in the interval from 17.7 MPa 1/2 to 22.0 MPa 1/2 , setting polar solubility in the interval from 13 MPa 1/2 to 23 MPa 1/2 and parameter solubility due to the hydrogen bonds in the range of 3 MPa 1/2 to 13 MPa 1/2 .

14. Composition for hybridization of claim 9, where polar non-Protic solvent is selected from the group consisting of:

where X represents About and R 1 is a , and

where X is optional and, if present, the selected of O, S; where Z is optional and, if present, the selected of O, S; where A and b independently represent About, or N or S, or part or primary amine; where R is the ; and where Y represents About, or S, or C.

15. Composition for hybridization of claim 9, where polar non-Protic solvent is selected from the group consisting of:

, , , and

16. Composition for hybridization of claim 9, additionally contains a catalyst and at least one salt, the catalyst is a , and at least one salt is a sodium chloride and/or phosphate buffer.

17. Composition for hybridization to P16, where present in concentrations ranging from 10% to 30%, sodium chloride is present in a concentration of 300 mm up to 600 mm and/or phosphate buffer is present in a concentration of 5 mm up to 20 mm.

18. Composition for hybridization of claim 9, containing 15% at least one polar solvent, 20% , 600 mm sodium chloride, 10 mm citrate buffer pH of 6.2.

19. Application songs for hybridization on any of the sub-clause 9-18 in hybridization assays.