ES2845073T3 - Hybridization compositions and methods - Google Patents

Abstract

In situ hybridization composition comprising at least one nucleic acid sequence, a polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences, and a hybridization solution, wherein the polar aprotic solvent is not dimethylsulfoxide (DMSO) , wherein the polar aprotic solvent is: ** (See formula) ** and wherein said composition further comprises at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents and blocking agents, in which the sulfolane concentration is 10% to 95% (v / v).

Description

DESCRIPTION

Hybridization compositions and methods

Field of the invention

The present invention relates to aqueous compositions for use in in situ hybridization (ISH).

In one embodiment, the present invention relates to the field of DNA and RNA molecular examination. In particular, the invention relates to the fields of cytology, histology and molecular biology. In one aspect, the present disclosure is concerned with the energy (eg, incubation time and heat) required during hybridization between nucleic acids, eg, in situ hybridization that targets DNA and RNA.

Background and description

The double helix structure of DNA is stabilized by hydrogen bonding between bases on opposite strands when the bases pair in a particular way (A + T or G + C). This complementary base pairing (hybridization) is essential in all processes involving nucleic acids.

In a basic example of hybridization, nucleic acid sequences or fragments are joined to a complementary nucleic acid sequence or fragment. For example, hybridization can use nucleic acid probes, designed to bind, or "hybridize", to a target, eg, DNA or RNA. One type of hybridization, in situ hybridization (ISH), includes hybridization to a target in a sample, where the sample can be in vivo, or for example fixed or adhered to a glass slide. Probes can be labeled to enable identification of the probe-target hybrid by use of a brightfield or fluorescence microscope / scanner. The fragment or sequence is usually a double-stranded or single-stranded nucleic acid, such as DNA, RNA, or the like. In some embodiments, the fragment or sequence can be a probe that can be labeled using radioactive labels such as 31P, 33P, or 32S, non-radioactive labels such as digoxigenin and biotin, or fluorescent labels. Such labeled probes can be used to detect genetic abnormalities in a target sequence, providing valuable information about, for example, prenatal disorders, cancer, and other genetic or infectious diseases.

The efficiency and accuracy of nucleic acid hybridization assays depend primarily on at least one of three factors: a) denaturation conditions (i.e. separation of, for example, two nucleic acid strands), b) renaturation conditions ( ie, re-matching of, for example, two nucleic acid strands), and c) wash conditions after hybridization.

Traditional hybridization experiments, such as ISH assays, use a solution containing formamide to denature double-stranded nucleic acid. Formamide is a solvent that has a destabilizing effect on the helix state of, for example, DNA, RNA, and the like, displacing bound hydrated molecules loosely and uniformly. In addition, formamide stabilizes the helical state of DNA, RNA, and the like by "formamidation" of the Watson-Crick binding sites of the bases. However, formamide is a hazardous toxic material, subject to strict regulations for its use and disposal.

Furthermore, the use of formamide, although accepted as the conventional technique for hybridization, is hampered by the long time required to complete hybridization, depending on the conditions and the nucleic acid fragments or sequences used. For example, the denaturation step is followed by a hybridization step that takes much longer than, for example, a traditional fluorescent in situ hybridization (FISH) protocol takes 14-24 hours, and can take even up to 72 hours. Examples of traditional hybridization times are shown in Figures 1 and 2.

The step of re-pairing (ie, hybridizing) of two complementary strands of nucleic acid strands is by far the most time consuming aspect of an assay using hybridization. Until now it was believed that the use of chaotropic agents, such as formamide, guanidinium, hydrogen and urea, that interfere with the Watson-Crick binding sites of nucleic acid bases and thereby disrupt the hydrogen bonds between complementary bases of nucleic acid, was the only way to lower the melting temperature (Tm) of the complementary strands. However, although the use of chaotropic agents lowers Tf, these agents appear to significantly prolong the hybridization time, compared to hybridization in an aqueous solution without a chaotropic agent. Additionally, apart from the disadvantage of the long processing time, the use of a high concentration of formamide appears to incur the morphological destruction of the cellular, nuclear and / or chromosomal structure. Finally, formamide is considered a toxic and dangerous chemical for humans.

The present invention provides several possible advantages over the prior art, such as faster hybridization times, lower hybridization temperatures, and less toxic hybridization solvents.

Summary of the invention

An object of the present invention is to provide compositions that result in at least one of the following advantages: highly sensitive, technically easy, flexible and reliable hybridization procedures, and rapid assays. In some embodiments, for example, an advantage may be the ability to tailor hybridization time by varying the temperature of the hybridization reaction to a much greater extent than is available using prior art methods. For example, hybridization may be possible at room temperature.

In one embodiment, the compositions and methods of the invention lower the energy required for hybridization. The compositions and methods of the invention are applicable to in situ hybridization. The compositions and methods are also applicable to any molecular system that hybridizes or binds using base pairing, such as, for example, DNA, RNA, PNA, LNA, and natural and synthetic analogs thereof.

A further object of the invention is to provide in situ hybridization methods and compositions that preserve the morphology of a biological sample. Another object of the invention is to provide a non-toxic hybridization composition and method. Yet another object of the invention is to provide a low evaporation hybridization technique. A further object of the invention is to provide a detectable hybridization technique with a 20x objective. Still another object of the invention is to provide a composition with a low concentration of probe. Another object of the invention is to reduce and / or eliminate the need to block non-specific binding. The compositions and methods of the invention may also allow the use of heterogeneous probes without the need to block, eliminate, or otherwise disable the binding of, for example, repetitive sequences in a biological sample.

The nucleic acid in situ hybridization compositions and methods of the present invention are useful for the in vitro analysis of genomic DNA, chromosomes, chromosome fragments, genes, and chromosomal aberrations such as translocations, deletions, amplifications, insertions, mutations, or inversions. associated with a normal state or disease. Furthermore, the methods and compositions are useful for the detection of infectious agents as well as changes in the expression levels of RNA, eg, mRNA and its complementary DNA (cDNA).

Other uses include in vitro analysis of messenger RNA (mRNA), viral RNA, viral DNA, small interfering RNA (siRNA), small nuclear RNA (npRNA), non-coding RNA (cRNA, e.g. tRNA and rRNA), RNA messenger transfer (mtRNA), micro RNA (miRNA), Piwi interacting RNA (piRNA), long noncoding RNA, small nucleolar RNA (pRNA), antisense RNA, double-stranded RNA (dsRNA), methylations and other modifications of bases, single nucleotide polymorphisms (SNPs), copy number variations (VNC) and nucleic acids labeled with, for example, radioisotopes, fluorescent molecules, biotin, digoxigenin (DIG) or antigens, alone or in combination with unlabeled nucleic acids. The compositions and nucleic acid hybridization method disclosed herein are useful for in vivo or in vitro analysis of nucleic acids using techniques such as PCR, in situ PCR, Northern blotting, Southern blotting, flow cytometry, autoradiography, fluorescence microscopy, chemiluminescence, immunohistochemistry, virtual karyotype, gene assay, DNA microalignment (e.g. comparative genomic hybridization with alignments (CGH with alignments)), gene expression profiling, gene ID, alignment Tiling, gel electrophoresis, capillary electrophoresis, and in situ hybridizations such as FISH, SISH, CISH. In situ methods and compositions of the invention can be used on in vitro samples such as bone marrow smears, blood smears, paraffin embedded tissue preparations, enzymatically dissociated tissue samples, bone marrow, amniocytes, Cytospin preparations, impressions, etc. .

In one embodiment, the invention provides in situ methods and compositions for hybridizing at least one molecule to a target. The invention can, for example, eliminate the use of, or reduce dependence on, formamide. For example, the methods and compositions of the invention can lower the energy barrier to hybridization without the use of formamide.

The lower energy barrier can reduce the time and / or temperature required for hybridization. For example, the invention may allow hybridization at lower temperatures, or it may allow rapid hybridization at higher temperatures. Thus, in some respects, the present invention overcomes a major time-consuming step in hybridization assays.

One aspect of the invention is a composition or solution for use in in situ hybridization.

Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and the polar aprotic solvent sulfolane in an amount effective to denature double-stranded nucleotide sequences. An amount effective to denature double-stranded nucleotide sequences is an amount that enables hybridization. For example, one way to test whether the amount of polar aprotic solvent is effective to enable hybridization is to determine whether the polar aprotic solvent, when used in the compositions and hybridization methods described herein, such as Example 1, produces a detectable signal and / or an amplified nucleic acid product.

Non-limiting examples of effective amounts of polar aprotic solvents include, for example, from about 1% to about 95% (v / v). In some embodiments, the polar aprotic solvent concentration is 5% to 60% (v / v). In other embodiments, the polar aprotic solvent concentration is 10% to 60% (v / v). In still other embodiments, the polar aprotic solvent concentration is 30% to 50% (v / v). Also suitable are concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or 50% to 60% (v / v). In some embodiments, the polar aprotic solvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5%. (v / v). In other embodiments, the polar aprotic 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% (v / v ).

According to another aspect of the present invention the aqueous composition comprising the polar aprotic solvent has reduced toxicity. For example, a composition less toxic than traditional hybridization solutions may comprise a composition provided that the composition does not contain formamide, or provided that the composition contains less than 10%, or less than 5%, or less. 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% of formamide. A less toxic composition may also comprise a composition provided that the composition does not contain dimethylsulfoxide (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% of DMSO.

In one aspect, polar aprotic solvents can be selected based on their Hansen solubility parameters. For example, suitable polar aprotic solvents may have a dispersion solubility parameter of between 17.7 and 22.0 MPa1 / 2, a polar solubility parameter of between 13 and 23 MPa1 / 2, and a solubility parameter by hydrogen bonds between 3 and 13 MPa1 / 2.

According to one aspect, suitable polar aprotic solvents are cyclic compounds. A cyclic compound has a cyclic base structure. Examples include the cyclic compound sulfolane. In other aspects, the polar aprotic solvent can be chosen from formulas 1-4 below:

According to another aspect, suitable polar aprotic solvents can be chosen from formula 5 below:

Formula 5

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

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

wherein A and B are independently O, N, or S or part of the alkyldiyl or a primary amine;

where R is alkyldiyl; Y

where Y is O, S, or C.

Examples of suitable polar aprotic solvents according to Formula 5 are provided in Formulas 6-9 below:

Formula 6 Formula 7 Formula 8 Formula 9

in which: in which: in which:

X does not exist; X does not exist; X does not exist;

A, B and Z are O; A is part of A is part of Y is C; and alkyldiyl; alkyldiyl;

R is ethane-1,2-diyl Y is C; Yes c;

B y Z son O; y B es metilamina;

B and Z are O; and B is methylamine;

R is propane-1,3-diyl Z is O; Y

R is propane-1,3-diyl. According to yet another aspect, the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, or carbonate functionality. Such compounds are distinguished by their relatively high dielectric constants, high dipole moments, and solubility in water.

According to one aspect of the invention, the polar aprotic solvent having sulfone functionality is sulfolane (SL). According to yet another aspect, the invention provides a method of in situ nucleic acid sequence hybridization comprising:

- provide a first nucleic acid sequence,

- provide a second nucleic acid sequence,

- providing an aqueous composition that comprises at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences, as further defined in the appended claims, and

- combining the first and second nucleic acid sequences and the aqueous composition at least for a period of time sufficient to hybridize the first and second nucleic acid sequences.

In one embodiment, a sufficient amount of energy is provided to hybridize the first and second nucleic acids.

In one embodiment, hybridization of the first nucleic acid sequence to the second nucleic acid sequence occurs in less than 8 hours, such 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.

The method may comprise, for example:

- provide a first nucleic acid sequence,

- applying an aqueous composition comprising a second nucleic acid sequence and at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences to said first nucleic acid sequence at least for a period of time sufficient to hybridize the first and second nucleic acid sequences.

In one embodiment, a sufficient amount of energy is provided to hybridize the first and second nucleic acids.

In one embodiment, hybridization of the first nucleic acid sequence to the second nucleic acid sequence occurs in less than 8 hours, such 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.

According to yet another aspect of the present invention, the hybridization energy is provided by heating the aqueous composition and the nucleic acid sequence. Thus, the step of hybridizing can include the steps of heating and cooling the aqueous composition and nucleic acid sequences.

According to another aspect of the invention, the denaturation and hybridization steps can occur separately. For example, the sample can be denatured with a probeless solution and then probe-hybridized.

A further aspect of the invention comprises a method in which the step of providing a sufficient amount of energy to hybridize the nucleic acids involves a heating step performed using microwaves, hot baths, hot plates, hot wire, Peltier element , induction heating or heat lamps.

According to another aspect, the present invention relates to a method in which hybridization takes less than 1 hour. In other embodiments, hybridization takes less than 30 minutes. In still other embodiments, hybridization takes less than 15 minutes. In other embodiments, hybridization takes less than 5 minutes.

According to a further aspect, the invention relates to the use of a composition comprising between 10 and 95% (v / v) of sulfolane in in situ hybridization assays.

According to yet another aspect, the invention relates to the use of a hybridization composition comprising an aqueous composition as described for use in in situ hybridization assays.

Brief description of the drawings

Figure 1 depicts a typical time course for single locus detection with primary labeled FISH probes in paraffin-embedded, formaldehyde-fixed tissue sections (histological specimens). The bars represent a hybridization performed using a traditional solution (upper part) and a typical time course for a hybridization performed using a composition of the invention (lower part). The first bar on the left in each time course represents the dewaxing stage; the second bar represents the heat pretreatment stage; the third bar represents the digestion stage; the fourth bar represents the denaturation and hybridization step; the fifth bar represents the stringency wash stage; and the sixth bar represents the assembly stage.

Figure 2 depicts a typical time course for single locus detection with primary labeled FISH probes with cytological specimens. The bars represent a hybridization performed using a traditional solution (upper part) and a typical time course for a hybridization performed using a composition of the invention (lower part). The first bar on the left in each time course represents the fixation stage; the second bar represents the denaturation and hybridization step; the third bar represents the stringency wash stage; and the fourth bar represents the assembly stage.

Detailed description

A. Definitions

In the context of the present invention the following terms are to be understood as follows:

"Biological sample" is to be understood as any in vivo, in vitro or in situ sample of one or more cells or cell fragments. This can, for example, be a unicellular or multicellular organism, tissue section, cytological sample, chromosome extension, purified nucleic acid sequences, artificially produced nucleic acid sequences produced, for example, by a biologically based system or by synthesis chemistry, microalignment, or other form of nucleic acid chip. In one embodiment, a sample is a mammalian sample, such as, for example, a human, murine animal, rat, feline, or canine sample.

"Nucleic acid", "nucleic acid chain", and "nucleic acid sequence" mean anything that binds or hybridizes using base pairing including, oligomers or polymers that have a backbone made up of nucleotides that are produced in such a manner natural and / or nucleic acid analogs comprising unconventional nucleobases and / or unconventional backbones (e.g., a peptide nucleic acid (PNA) or locked nucleic acid (LNA)), or any derivatized form of a nucleic acid.

As used herein, the term "peptide nucleic acid" or "PNA" means a synthetic polymer having a polyamide backbone with pendant nucleobases (naturally occurring and modified), including, but not limited to a, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in, for example, US Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470, 6,201,103, 6,228,982, and 6,357. 163, WO 96/04000. The pendant nucleobase, such as, for example, a purine or pyrimidine base in PNA can be connected to the backbone by a linker such as, for example, one of the linkers taught in WO03 / 027328 or in any of the references cited therein. In one embodiment, the PNA has a backbone of N- (2-aminoethyl) -glycine). PNAs can be synthesized (and optionally labeled) as taught in WO03 / 027328 or any of the references cited therein. PNAs hybridize strongly and with high sequence specificity, with DNA and RNA, because the backbone of PNA remains unaltered. Thus, short PNA probes can exhibit specificity comparable to longer DNA or RNA probes. PNA probes can also show greater specificity in binding to complementary DNA or RNA.

As used herein, the term "blocked nucleic acid" or "LNA" means an oligomer or polymer comprising at least one or more LNA subunits. As used herein, the term "LNA subunit" means a ribonucleotide containing a methylene bridge that connects the 2 'oxygen of the ribose to the 4' carbon. See generally, Kurreck, Eur. J. Biochem., 270: 1628-44 (2003).

Examples of nucleic acids and nucleic acid analogs also include polymers of nucleotide monomers, including double and single-stranded deoxyribonucleotides (DNA), ribonucleotides (RNA), α-anomeric forms thereof, natural and synthetic analogs thereof. , and the like. The nucleic acid chain can be composed entirely of deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNAs), blocked nucleic acids (LNAs), natural or synthetic analogs thereof, or mixtures thereof. DNA, RNA, or other nucleic acids as defined herein can be used in the method and compositions of the invention.

"Polar aprotic solvent" refers to an organic solvent having a dipole moment of about 2 debye units or more, a solubility in water of at least about 5% (volume) at or near room temperature, that is, about 20 ° C, and does not undergo significant hydrogen exchange at approximately neutral pH, that is, in the range 5 to 9, or in the range 6 to 8. Polar aprotic solvents include those defined by Hansen's solubility parameters discussed below. "Alkyldiyl" refers to a saturated or unsaturated, branched, straight-chain or cyclic hydrocarbon radical having two monovalent radical centers derived by removing one hydrogen atom from each of two different carbon atoms of an alkane, alkene, or original alkyne.

"Aqueous solution" is to be understood as a solution containing water, even small amounts of water. For example, a solution containing 1% water is to be understood as an aqueous solution. "Hybridization" is to be understood as incorporating both the denaturation and re-matching steps of the hybridization procedure unless otherwise specified.

"Hybridization composition" refers to an aqueous solution for performing a hybridization procedure, for example, to bind a probe to a nucleic acid sequence. Hybridization compositions can comprise, for example, at least one polar aprotic solvent, at least one nucleic acid sequence, and a hybridization solution. Hybridization compositions do not comprise enzymes or other components, such as deoxynucleoside triphosphate (dNTP), to amplify nucleic acids in a biological sample.

"Hybridization solution" refers to an aqueous solution for use in a hybridization composition. Hybridization solutions are discussed in detail below and may comprise, for example, buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents.

"PCR composition" refers to an aqueous solution for performing a hybridization procedure to amplify a nucleic acid sequence. The PCR compositions can comprise, for example, at least one polar aprotic solvent, at least one enzyme for amplifying nucleic acids, a set of oligonucleotide nucleic acid primers, a dNTP mix and a PCR solution.

"PCR solution" refers to an aqueous solution for use in a PCR composition. PCR solutions can comprise, for example, buffering agents, accelerating agents, chelating agents, salts, and detergents.

"Hansen solubility parameters" and "HSP" refer to the following cohesion energy (solubility) parameters: (1) the dispersion solubility parameter ( ^8d , "D parameter"), which measures apolar interactions derived from the atomic forces; (2) the solubility parameter in polar medium ( ^8p , "P parameter"), which measures permanent dipole-permanent dipole interactions; and (3) the hydrogen bond solubility parameter ( ^8h , "H parameter"), which measures electron exchange. Hansen's solubility parameters are further defined below.

"Repetitive sequences" is to be understood as referring to the rapid (approximately 25%) and / or intermediate (approximately 30%) re-pairing of components of mammalian genomes. Fast-pairing components contain small highly repetitive sequences (a few nucleotides long) that are commonly found in tandem (eg, satellite DNA), while intermediate-pairing components contain sparse repetitive DNA. Sparse repeat sequences are classified as either SINE (short sparse repeat sequences) or LINE (long sparse repeat sequences), both of which are classified as primate retrotransposons. SINE and LINE include, but are not limited to, Alu repeats, Kpn repeats, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats, and hexanucleotide repeats. Alu repeats make up the majority of human SINEs and are characterized by a consensus sequence of approximately 280 to 300 bp consisting of two similar sequences arranged as a head-to-tail dimer. In addition to SINE and LINE, there are also repeat sequences in chromosome telomeres at the terminal ends of chromosomes and chromosome centromeres, which contain different repeat sequences that only exist in the chromosome. central region of a chromosome. However, unlike SINE and LINE, which are randomly dispersed throughout the entire genome, telomere and centromere repeat sequences are located in a particular region of the chromosome.

“Non-toxic” and “reduced toxicity” are defined with respect to the toxicity labeling of formamide according to “Directive 1999/45 / EC of the European Parliament and of the Council of 31 May 1999 concerning the approximation of the laws, regulations and administrative provisions of the Member States relating to the classification, packaging, and labeling of dangerous preparations ”(ecb.jrc.it/legislation/1999L0045EC.pdf) (“ Directive ”). According to the Directive, toxicity is defined using the following order of classification: T + "very toxic"; T "toxic", C "corrosive", Xn "harmful", .Xi "irritant". Risk phrases ("R phrases") describe the hazards of classified toxicity. Formamide is listed as T (toxic) and R61 (may harm the fetus). All of the following chemicals are classified as less toxic than formamide: acetonitrile (Xn, R11, R20, R21, R22, R36); sulfolane (Xn, R22); ybutyrolactone (Xn, R22, R32); and ethylene carbonate (Xi, R36, R37, R38). At the time of filing this application, ethylene trithiocarbonate and glycol sulfite are not currently labeled.

B. Solvent selection

Suitable polar aprotic solvents can be selected based on their Hansen solubility parameters. Methods for experimentally determining and / or calculating HSP for a solvent are known in the art, and HSPs have been reported for more than 1200 chemicals.

For example, the parameter D can be calculated with reasonable accuracy based on the refractive index, or it can be derived from tables by comparison with known solvents of similar size, shape and composition after establishment of a critical temperature and molar volume. The parameter P can be estimated from known dipole moments (see, for example, McClellan A.L., Tables of Experimental Dipole Moments (W.H. Freeman 1963)) using Equation 1:

Equation 1: 5 ^p = 37.4 (dipole moment Ji / V1.12

where V is the molar volume. There are no equations to calculate the H parameter. Instead, the H parameter is usually determined based on group contributions.

HSP characterizations are conveniently visualized using a spherical plot, with HSPs from a suitable reference solvent experimentally determined in the center of the sphere. The radius of the sphere (R) indicates the maximum tolerable variation from the HSPs of the reference solvent that still allows a "good" interaction to take place. The good solvents are in the sphere and the bad ones are outside. The distance, Ra, between two solvents based on their respective HSP values can be determined using equation 2:

where subscript 1 indicates reference sample, subscript 2 indicates test chemical, and all values are in MPa1 / 2. Good solubility requires that Ra be less than the experimentally determined radius of the solubility sphere Ro. The relative energy difference between two solvents, that is, the RED number, can be calculated by taking the ratio of Ra to Ro, as shown in equation 3.

Equation 3: RED = Ra / Ro

RED numbers less than 1.0 indicate high affinity; RED numbers equal to or close to 1.0 indicate boundary conditions; and progressively higher RED numbers indicate progressively lower affinities.

In some embodiments, the D parameters of polar aprotic solvents are between 17.7 and 22.0 MPa1 / 2. Such relatively high D parameters are generally associated with solvents having cyclic structures and / or structures with sulfur or halogens. Linear compounds are not likely to be among the most suitable polar aprotic solvents, but can be considered if their P and H parameters are within the ranges discussed below. Since the parameter D is multiplied by 4 in equation 2, the limits are half of Ro. Furthermore, it should be noted that D values of about 21 or greater are often characteristic of a solid.

In some embodiments, the P parameters of polar aprotic solvents are between 13 and 23 MPa1 / 2. Such exceptionally high P parameters are generally associated with solvents that have a high dipole moment and presumably also a relatively low molecular volume. For example, for V near 60 cc / mol, the dipole moment should be between 4.5 and 3.1. For V near 90 cc / mol, the dipole moment should be between 5.6 and 3.9.

In some embodiments, the H parameters of polar aprotic solvents are between 3 and 13 MPa1 / 2.

Generally, polar aprotic solvents having an alcohol group are not useful in compositions and methods, since the H parameters of such solvents would be too high.

The molar volume of the polar aprotic solvent may also be relevant, since it participates in the evaluation of the three Hansen solubility parameters. As the molar volume becomes smaller, liquids tend to evaporate rapidly. As the molar volume becomes larger, liquids tend to enter the solid region in the range of parameters D and P cited above. Therefore, polar aprotic solvents, including but not limited to polar aprotic solvents are quite close to the liquid / solid limit in the HSP space.

In some aspects, polar aprotic solvents have lactone, sulfone, nitrile, sulfite, and / or carbonate functionality. Such compounds are distinguished by their relatively high dielectric constants, high dipole moments, and solubility in water. An exemplary polar aprotic solvent with lactone functionality is ybutyrolactone (GBL), an exemplary polar aprotic solvent with sulfone functionality is sulfolane (SL; tetramethylene dioxide sulfide), an exemplary polar aprotic solvent with nitrile functionality is acetonitrile ( AN), an exemplary polar aprotic solvent with sulfite functionality is glycol sulfite / ethylene sulfite (GS), and exemplary polar aprotic solvents with carbonate functionality are ethylene carbonate (EC), propylene carbonate (PC ) or ethylene trithiocarbonate (ETC). The structures of these solvents are provided below by way of example and their Hansen solubility parameters, RED numbers and molar volumes are given in Table 1.

Table 1

Other suitable polar aprotic solvents that can be used are cyclic compounds such as, for example, g-caprolactone. In addition, substituted pyrrolidinones and nitrogen-related structures on a 5- or 6-membered ring, and cyclic structures with two nitrile groups, or a bromo and a nitrile group, may also be suitable. For example, N-methyl-pyrrolidinone (shown below) can be a suitable polar aprotic solvent for use in the methods and compositions disclosed herein.

Other suitable polar aprotic solvents disclosed may contain a ring urethane (NHCOO-) group. However, not all of such compounds are suitable, since 1,3-dimethyl-2-imidazolidinone does not produce signals when used in hybridization compositions. One skilled in the art can select compounds useful in the compositions and methods of the invention as described herein. Chemicals are set forth in Tables 2 and 3 below by way of example.

Table 2

Table 2 sets forth an exemplary list of possible chemicals for use in the compositions and methods based on their Hansen solubility parameters. Other compounds, of course, can also satisfy these requirements. Some of these chemicals have been used in hybridization and / or PCR solutions in the prior art (for example, dimethylsulfoxide (DMSO) has been used in hybridization and PCR solutions, and sulfolane (SL) has been used in PCR solutions. ), but most do not. However, the prior art did not recognize that these compounds can be used to advantage to decrease hybridization times and / or temperatures, as disclosed in this application.

Table 3

Not all of the chemicals listed in Tables 2 and 3 are suitable for use in the disclosed compositions and methods. For example, although DMSO is listed in Table 2 because its Hansen solubility parameters (HSP) are within the ranges cited above, DMSO does not work by decreasing hybridization times and / or temperatures in compositions. and the methods of the invention. Thus, in some embodiments, the aqueous composition does not contain DMSO as a polar aprotic solvent. However, it is well within the skill of one of ordinary skill to examine suitable compounds using the guidance provided herein including testing a compound in one of the provided examples. For example, in some embodiments, suitable polar aprotic solvents will have hSp within the ranges listed above and a structure shown in formulas 1-9 above. C. Compositions, buffers, and solutions

(1) Hybridization solutions

Traditional hybridization solutions are known in the art. Such solutions can comprise, for example, buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents. For example, the buffering agents can include SSC, HEPES, SSPE, PIPES, TMAC, TRIS, SET, citric acid, a phosphate buffer, such as for example potassium phosphate or sodium pyrophosphate, etc. Buffering agents can be present at concentrations from 0.5x to 50x. Buffering agents are normally present at concentrations of from 2x to 10x.

Accelerating agents can include polymers such as FICOLL, PVP, heparin, dextran sulfate, proteins such as BSA, glycols such as ethylene glycol, glycerol, 1,3-propanediol, propylene glycol or diethylene glycol, combinations thereof such as Dernhardt's solution and BLOTTO and organic solvents such as formamide, dimethylformamide, DMSO, etc. The accelerating agent can be present at concentrations of from 1% to 80% or from 0.1x to 10x. Typically, formamide is present at concentrations of from 25% to 75%, while DMSO, dextran sulfate, and glycol are present at concentrations of from 5% to 10%.

Chelating agents can include EDTA, EGTA, etc. Chelating agents can be present at concentrations from 0.1 mM to 10 mM. Typically, chelating agents are present at concentrations of from 0.5 mM to 5 mM.

The salts can include sodium chloride, sodium phosphate, magnesium phosphate, etc. The salts can be present at concentrations from 1 mM to 750 mM. The salts are normally present at concentrations of from 10 mM to 500 mM.

Detergents can include Tween, SDS, Triton, CHAPS, deoxycholic acid, etc. The detergent can be present in concentrations from 0.01% to 10%. Typically, detergents are present in concentrations from 0.1% to 1%.

Nucleic acid blocking agents can include, yeast tRNA, homopolymeric DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA, COT1 DNA, etc. Blocking nucleic acids can be present at concentrations from 0.05 mg / ml to 100 mg / ml.

There is great variation in the literature regarding traditional hybridization solutions. For example, a traditional hybridization solution may comprise 5x or 6x SSC, 0.01 M EDTA, 5x Dernhardt's solution, 0.5% SDS, and 100 mg / ml fragmented, denatured salmon sperm DNA. Another traditional hybridization solution can comprise 50 mM HEPES, 0.5 M NaCl, and 0.2 mM EDTA. A typical hybridization solution for FISH with biological samples for RNA detection may comprise, for example, 2x SSC, 10% dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 50% formamide, 0 RNase-free BSA, 02% and E. coli tRNA 1 mg / ml. A typical hybridization solution for FISH with biological samples for DNA detection may comprise, for example, 2x SSC, 10% dextran sulfate, 50% formamide, and for example, 0.3 mg / salmon sperm DNA. ml or 0.1 mg / ml COT1 DNA. Other typical hybridization solutions may comprise 40% formamide, 10% dextran sulfate, 30 mM NaCl, 5 mM phosphate buffer, Alu-PNA (P ⁿ A blocking) or COT-1 DNA, and in some cases DNA total human (total human DNA, THD) 0.1 | ig / | yl.

The compositions of the invention may comprise a hybridization solution comprising any of the components of the traditional hybridization solutions mentioned above in combination with the polar aprotic solvent. Traditional components can be present at the same concentrations as used in traditional hybridization solutions, or they can be present at higher or lower concentrations, or they can be omitted altogether.

For example, if the compositions of the invention comprise salts such as NaCl and / or phosphate buffer, the salts may be present at concentrations of 0-1200 mM NaCl and / or 0-200 mM phosphate buffer. In some embodiments, the salt concentrations can be, for example, 300 mM NaCl and 5 mM phosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer.

If the compositions of the invention comprise accelerating agents such as dextran sulfate, glycol or DMSO, dextran sulfate can be present at concentrations from 5% to 40%, glycol can be present at concentrations from 0, 1% to 10%, and the DMSO can be from 0.1% to 10%. In some embodiments, the dextran sulfate concentration can be 10% or 20% and the ethylene glycol, 1,3-propanediol, or glycerol concentration can be 1% to 10%. In some embodiments, the DMSO concentration can be 1%. In some embodiments, the aqueous composition does not comprise DMSO as an accelerating agent. In some embodiments, the aqueous composition does not comprise formamide as an accelerating agent, or it does comprise formamide provided that the composition contains less than 10%, or less than 5%, or less than 2%, or less than 1%, or less. 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01%.

If the compositions of the invention comprise citric acid, the concentrations can range from 1mM to 50mM and the pH can range from 5.0 to 8.0. In some embodiments the citric acid concentration can be 10 mM and the pH can be 6.2.

Compositions of the invention may comprise agents that reduce non-specific binding to, for example, the cell membrane, such as salmon sperm or small amounts of total human DNA, or, for example, may comprise blocking agents to block the binding of , for example, target repeat sequences such as increased amounts of total human DNA or enriched repeat DNA or specific blocking agents such as PNA or LNA fragments and sequences. These agents can be present at concentrations from 0.01-100 µg / µl or 0.01-100 µM. For example, in some embodiments, these agents will be 0.1 | ig / | yl total human DNA, or 0.1 | ig / | yl non-human DNA, such as herring sperm, salmon sperm, or calf thymus DNA. , or 5 | iM blocking PNA.

One aspect of the invention is a composition or solution for use in in situ hybridization. Compositions for use in the invention include an aqueous composition comprising a nucleic acid sequence and sulfolane as a polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences. An amount effective to denature double-stranded nucleotide sequences is an amount that enables hybridization. For example, one way to test whether the amount of polar aprotic solvent is effective to enable hybridization is to determine whether the polar aprotic solvent, when used in the compositions and hybridization methods described herein, such as Example 1, produces a detectable signal and / or an amplified nucleic acid product.

Non-limiting examples of effective amounts of polar aprotic solvents include, for example, from about 1% to about 95% (v / v). In some embodiments, the polar aprotic solvent concentration is 5% to 60% (v / v). In other embodiments, the polar aprotic solvent concentration is 10% to 60% (v / v). In still other embodiments, the polar aprotic solvent concentration is 30% to 50%. (v / v). Also suitable are concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or 50% to 60% (v / v). In some embodiments, the polar aprotic solvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5%. (v / v). In other embodiments, the polar aprotic 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% (v / v ).

If the compositions of the invention are used in an in situ hybridization assay, they may further comprise one or more nucleic acid probes. Probes can be directly or indirectly labeled with detectable compounds such as enzymes, chromophores, fluorochromes, and haptens. DNA probes can be present at concentrations of 0.1 to 100 ng / µl. For example, in some embodiments, the probes may be present at concentrations of 1 to 10 ng / µl. PNA probes can be present at concentrations from 0.5 to 5000 nM. For example, in some embodiments, the probes may be present at concentrations of 5 to 1000 nM. In one embodiment, a composition of the invention comprises a mixture of 40% (v / v) polar aprotic solvent (ie, sulfolane, "SL"), 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer. and probe 1-10 ng / | yl. Another exemplary composition of the present invention comprises a mixture of 15% SL, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and 0.1 µg / µl total human DNA. Yet another exemplary composition comprises 15% SL, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid pH 6.2, and 0.1 | ig / | yl non-human DNA (eg, sperm herring, salmon sperm or calf thymus) OR 0.5% formamide OR 1% glycol (eg ethylene glycol or 1,3-propanediol or glycerol).

(2) Polar aprotic solvent (s)

Different polar aprotic solvents can confer different properties on the compositions. For example, the choice of polar aprotic solvent can contribute to the stability of the composition, since certain polar aprotic solvents can degrade over time. For example, the polar aprotic solvent ethylene carbonate decomposes into ethylene glycol, which is a relatively stable molecule, and carbon dioxide, which can interact with water to form carbonic acid, altering the acidity of the compositions. Without being bound by theory, it is believed that the change in pH upon decomposition of ethylene carbonate renders the compositions less effective for hybridization. However, stability can be improved by lowering the pH of the composition, adding citric acid as a buffer at pH 6.2 instead of the traditional phosphate buffer, which is normally used at about pH 7.4 and / or adding ethylene glycol at concentrations, for For example, 0.1% to 10%, or 0.5% to 5%, such as, for example, 1%, 2%, 3%, and so on. For example, with 10 mM citrate buffer, the compositions of the invention are stable at 2-8 ° C for approximately 8 months. Stability can also be improved if the compositions are stored at low temperatures (eg -20 ° C).

Furthermore, certain polar aprotic solvents can cause compositions to separate in multiphase systems under certain conditions. The conditions under which multiphase systems are obtained may be different for different polar aprotic solvents. Generally, however, as the concentration of polar aprotic solvent increases, the number of phases increases. For example, compositions comprising ethylene carbonate at low concentrations (ie, less than 20%) can exist as one phase, while compositions comprising higher concentrations of ethylene carbonate can separate into two or even three phases. For example, compositions comprising 15% ethylene carbonate exist as a single phase at room temperature, while compositions comprising 40% ethylene carbonate consist of a viscous lower phase (approximately 25% of the total volume) and a less viscous upper phase (approximately 75% of the total volume) 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, sulfolane, y-butyrolactone, ethylene trithiocarbonate, glycol sulfite, and propylene carbonate exist as two phases at concentrations of 10, 15, 20, or 25% (20% dextran sulfate, 600 mM NaCl, 10 mM citrate buffer) at room temperature.

It may also be possible to alter the number of phases by adjusting the temperature of the compositions. Generally, as the temperature increases, the number of phases decreases. For example, at 2-8 ° C, compositions comprising 40% ethylene carbonate can be separated in a triphasic system.

It may also be possible to alter the number of phases by adjusting the concentration of dextran sulfate and / or salt in the composition. Generally speaking, decreasing the dextran sulfate concentration (the traditional concentration is 10%) and / or the salt concentration can reduce the number of phases. However, depending on the particular polar aprotic solvent and its concentration in the composition, monophases can occur even with higher concentrations of salt and dextran sulfate. For example, a composition comprising low amounts of EC (eg, 15%, 10%, or 5%) can work well by increasing the concentrations of dextran sulfate and salt, while still maintaining a single phase system. In a particular embodiment, the compositions comprising a HER2 gene DNA probe, a CEN7 PNA probe, 15% EC, 20% dextran sulfate, 600 mM NaCl and 10 mM phosphate buffer are frozen at -20 ° C. In other embodiments, compositions are liquid at -20 ° C.

Some polar aprotic solvents can produce stronger signals in one phase or another. For example, 40% glycol sulfite produces strong signals in the lower phase and does not produce signals in the upper phase. Similarly, certain types of probes can produce stronger signals in one phase or another. For example, PNA probes tend to show stronger signals in the lower phase than in the upper phase. Consequently, multiphase systems can be used to conveniently examine different aspects of a sample. For example, a biphasic system could be used to separate samples labeled with PNA probes from samples labeled with DNA probes. Other uses include the isolation of a specific phase that has, for example, certain advantages in hybridization such that the isolated phase can be used as a single phase system. The probe and / or sample can be added before or after isolation of a particular phase.

Hybridizations can be performed with a monophasic composition of the invention, with individual phases of the multiphasic compositions, or with mixtures of any one or more of the phases in a multiphasic composition. For example, in a single-phase system, a volume of the sample can be drawn for use in hybridization. In a multiphase system, a sample volume of the phase of interest (eg, upper, lower or middle phase) can be drawn for use in hybridization. Alternatively, the phases in a multiphase system can be mixed prior to drawing a volume of the mixed sample for use in hybridization. However, the multiphase system can produce intense and uneven local background staining depending on the composition. Although the addition of low amounts of formamide will reduce the background in a single phase system, it has little effect on a multiphase system with high concentrations (eg 40%) of a polar aprotic solvent. Furthermore, as the formamide concentration increases, higher probe concentrations and / or longer hybridization times are required to maintain strong signal intensity.

(3) Optimization for particular applications

Compositions of the invention can be varied to optimize results for a particular application. For example, the concentration of polar aprotic solvent, salt, accelerating agent, blocking agent, and hydrogen ions (ie, pH) can be varied to improve results for a particular application.

For example, the concentration of polar aprotic solvent can be varied to improve signal intensity and background staining. Generally, as the concentration of polar aprotic solvent increases, the signal intensity decreases and the background staining decreases. For example, compositions comprising 15% EC tend to show stronger signals and less background than compositions comprising 5% EC. However, the signal intensity for compositions having low concentrations of polar aprotic solvent (eg, 0% to 20%) can be improved by increasing the concentrations of dextran salt and / or sulfate. For example, strong signals can be observed with EC at 5% to 10% when the salt concentration is raised approximately 8 to 16 times traditional salt concentrations (ie, approximately 1200 mM NaCl, 20 mM phosphate buffer). Also, as lower concentrations of polar aprotic solvent are used, higher concentrations of dextran sulfate are generally required to maintain good background and signal intensity.

Consequently, dextran sulfate and salt concentrations can also be varied to improve signal intensity and background staining. Generally, as salt and dextran sulfate concentrations increase, signal intensity increases and background decreases. For example, salt concentrations that are approximately two to four times the traditional concentrations (ie, 300 mM NaCl, 5 mM phosphate buffer) produce more intense signals and low background. However, surprisingly hybridization occurs using the compositions of the invention even in the complete absence of salt. Signal intensities under salt-free conditions can be improved by increasing concentrations of accelerating agent and / or polar aprotic solvent.

Also, the signal intensity increases as the dextran sulfate concentration increases from 0% to 20%. However, good signals can be seen even at sulfate concentrations of 0%. Signal intensity can be enhanced under conditions of low dextran sulfate concentration by increasing the concentrations of polar aprotic solvent and / or salt.

In addition, the type probes used in the compositions of the invention can be varied to improve results. For example, in some aspects of the invention, DNA / DNA probe combinations may show less background than DNA / PNA probe combinations in the compositions of the invention or vice versa. On the other hand, PNA probes tend to show stronger signals than DNA probes at low concentrations of salt and / or low concentrations of polar aprotic solvent. In fact, PNA probes also show signals when no polar aprotic solvent is present, while DNA probes show weak or no signals without polar aprotic solvent.

D. Applications, methods and uses

(1). Analytical samples

The methods and compositions of the invention can be used wholly or partially in in situ hybridization applications in the fields of cytology, histology or molecular biology.

According to one embodiment, the first or second nucleic acid sequence in the in situ methods of the invention is present in a biological sample. Examples of such samples include, for example, tissue samples, cell preparations, cell fragment preparations, and isolated or enriched cell component preparations. The sample can originate from various tissues such as, for example, breast, lung, colorectal tissue, prostate, lung, head and neck, stomach, pancreas, esophagus, liver and bladder, or other relevant tissues and neoplasms thereof, any suspension cell, blood sample, fine needle aspiration, ascites fluid, sputum, peritoneal lavage, lung lavage, urine, stool, cell scraping, cell smear, cytospun or cytoprepared cells.

The sample can be isolated and processed using standard protocols. Cell fragment preparations can be obtained, for example, by cell homogenization, freeze-thaw treatment, or cell lysate. The isolated sample can be treated in many different ways depending on the purpose of the sample collection and depending on the routine at the site. Often the sample is treated with various reagents to preserve the tissue for later analysis of the sample, alternatively the sample can be analyzed directly. Examples of widely used methods for preserving samples are formalin fixation followed by paraffin embedding and cryopreservation.

For metaphase extensions, cell cultures are generally treated with colcemide, or other suitable spindle pole disrupting agents, to arrest the metaphase cell cycle. The cells are then fixed and spotted on microscope slides, treated with formaldehyde, washed, and dehydrated in ethanol. Probes are then added and the samples analyzed by any of the techniques discussed below.

Cytology involves the examination of individual cells and / or extensions of chromosomes from a biological sample. Cytological examination of a sample begins with obtaining a sample of cells, which can usually be done by scraping, swabbing, or brushing an area, as in the case of cervical samples, or by collecting body fluids, such as those obtained from the thoracic cavity, bladder, or spine, or by fine needle aspiration or fine needle biopsy, as in the case of internal tumors. In a conventional manual cytological preparation, the sample is transferred to a liquid suspension material and the cells in the fluid are then transferred directly or by centrifugation-based processing steps onto a glass microscope slide for observation. In a typical automated cytological preparation, a filter assembly is placed in the liquid suspension and the filter assembly both disperses the cells and captures the cells on the filter. The filter is then removed and contacted with a microscope slide. The cells are then fixed on the microscope slide prior to analysis by any of the techniques discussed below.

In a traditional hybridization experiment using a cytological sample, slides containing the sample are immersed in a formaldehyde buffer, washed, and then dehydrated in ethanol. The probes are then added and the sample is covered with a coverslip. The slide is incubated at a temperature sufficient to denature any nucleic acid in the sample (for example, 5 minutes at 82 ° C) and then incubated at a temperature sufficient to allow hybridization (for example, overnight at 45 ° C ). Following hybridization, coverslips are removed and samples are subjected to a high stringency wash (eg, 10 minutes at 65 ° C) followed by a series of low stringency washes (eg, 2 x 3 minutes at room temperature ). The samples are then dehydrated and mounted for analysis.

Histology involves the examination of cells in thin sections of tissue. To prepare a tissue sample for histological examination, pieces of the tissue are fixed in a suitable fixative, usually an aldehyde such as formaldehyde or glutaraldehyde, and then embedded in molten paraffin wax. The wax block containing the tissue sample is then cut on a microtome to produce fine sections of paraffin containing the tissue, typically 2 to 10 microns thick. The sample slice is then applied to a microscope slide, air dried, and heated to cause the sample to adhere to the glass slide. The residual paraffin is then dissolved with a suitable solvent, usually xylene, toluene or others. These so-called dewaxing solvents are then removed with a dehydration wash type reagent prior to sample analysis by any of the techniques discussed below. Alternatively, sections can be prepared from frozen samples, briefly fixed in 10% formalin or other suitable fixatives, and then infused with dehydration reagent prior to sample analysis. In a traditional hybridization experiment using a histological sample, formalin-fixed, paraffin-embedded tissue samples are cut into 2-6 µm sections and collected on slides. The paraffin is melted (eg 30-60 minutes at 60 ° C) and then removed (dewaxed) by washing with xylene (or a xylene substitute), eg 2 x 5 minutes. Samples are rehydrated, washed, and then pretreated (eg, 10 minutes at 95-100 ° C). Slides are washed and then treated with pepsin or other suitable permeabilizer, eg 3-15 minutes at 37 ° C. The slides are washed (eg 2 x 3 minutes), dehydrated and the probe applied. The samples are covered with a coverslip and the slide is incubated at a temperature sufficient to denaturing any nucleic acid in the sample (eg, 5 minutes at 82 ° C), followed by incubation at a temperature sufficient to allow hybridization (eg, overnight at 45 ° C). Following hybridization, coverslips are removed and samples are subjected to a high stringency wash (eg, 10 minutes at 65 ° C) followed by a series of low stringency washes (eg, 2 x 3 minutes at room temperature ). The samples are then rehydrated and mounted for analysis.

(2). Hybridization techniques

The compositions and methods of the present invention can be used in whole or in part in nucleic acid in situ hybridization techniques known in the art for cytological and histological samples. Such techniques include, for example, in situ hybridization (ISH), fluorescence in situ hybridization (FISH; including multi-color FISH, Fiber-FISH, etc.), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH) , comparative genomic hybridization (CGH), chromosomal representations and in situ alignments.

Molecular probes that are suitable for use in the hybridizations of the invention are described, for example, in US Patent Publication No. 2005/0266459. In general, probes can be prepared by chemical synthesis or by amplifying a specific DNA sequence by cloning, inserting the DNA into a vector, and amplifying the vector and inserting it into appropriate host cells. Commonly used vectors include bacterial plasmids, cosmids, bacterial artificial chromosomes (BAC), P1 derived artificial chromosomes (PAC), or yeast artificial chromosomes (YAC). The amplified DNA is then extracted and purified for use as a probe. Methods for preparing and / or synthesizing probes are known in the art, for example, as disclosed in WO03 / 027328.

In general, the type of probe determines the type of feature that can be detected in a hybridization assay. For example, genomic or total nuclear DNA probes can be used as the species-specific probe. Chromosomal representations are collections of DNA sequences derived from a single type of chromosome and can identify that specific chromosome type in metaphase and interphase nuclei, count the number of a particular chromosome, show translocations, or identify extrachromosomal fragments of chromatin. Different chromosome types also have unique repeat sequences that can be targeted for probe hybridization to detect and count specific chromosomes. Large insertion probes can be used to target single copy sequences. With these large probes, the hybridization efficiency is inversely proportional to the size of the probe. Smaller probes can also be used to detect aberrations such as deletions, amplifications, inversions, duplications, and aneuploidy. For example, differently colored locus-specific probes can be used to detect translocations by hybridization of separate signals in situ.

In general, the ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild-type and mutant complexes as the length of the probe increases. Probes greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a single organism or clinical condition of interest. On the other hand, sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs) may be sufficient to enable discrimination of hybridization with complementary target nucleic acid sequences compared to non-sequences. Diana.

In one embodiment, at least one set of the in situ hybridization probes may comprise one or more PNA probes, as described in US Patent No. 7,105,294. Methods for synthesizing PNA probes are described in WO03 / 027328. Alternatively, or in addition, at least one set of the hybridization probes in any of the techniques discussed above may comprise one or more blocked nucleic acid (LNA) probes, as described in WO 99/14226. Due to the additional bridging between the 2 'and 4' carbons, the LNA backbone is pre-arranged for hybridization. LNA / DNA and LNA / RNA interactions are stronger than the corresponding DNA / DNA and DNA / RNA interactions, as indicated by a higher melting temperature. Thus, the methods and compositions of the invention, which lower the energy required for hybridization, are particularly useful for hybridizations with LNA probes.

In one embodiment, the probes may comprise a detectable marker (a molecule that provides an analytically identifiable signal that allows detection of the probe-target hybrid), as described in US Patent Publication No. 2005/0266459. The detectable label can be linked directly to a probe, or can be indirectly linked to a probe, for example, through the use of a linker. Any labeling method known to those of skill in the art, including enzymatic and chemical processes, can be used for labeling probes used in the methods and compositions of the invention. In other embodiments, the probes are unmarked.

In general, in situ hybridization techniques such as CGH, FISH, CISH, and SISH employ large, primarily unspecified nucleic acid probes that hybridize with varying stringency to genes or gene fragments on the chromosomes of cells. The use of large probes makes the in situ hybridization technique very sensitive. However, the successful use of large genomic probes in traditional hybridization assays depends on blocking unwanted background staining derived from, for example, repetitive sequences that are present throughout the genome. Such blocking stages are time consuming and expensive. As discussed herein, the methods and compositions of the invention advantageously reduce and / or eliminate the need for such blocking steps. However, in one embodiment, repetitive sequences can be deleted according to methods known in the art, for example, as disclosed in WO03 / 027328.

Bound probes can be detected in cytological and histological specimens either directly or indirectly with fluorochromes (eg, FISH), organic chromogens (eg, CISH), silver particles (eg, SISH), or other metallic particles (eg. eg, gold-facilitated fluorescence in situ hybridization, G ^ol D ^f ISH). Thus, depending on the detection method, the populations of cells obtained from a sample to be tested can be visualized by means of fluorescence microscopy or conventional bright-field light microscopy.

Hybridization assays with cytological and histological samples are important tools for determining the number, size and / or location of specific DNA sequences. For example, in CGH, entire genomes are stained and compared to normal reference genomes for the detection of regions with aberrant copy number. Typically, DNA from subject tissue and normal control tissue is labeled with different stained probes. The DNA pools are mixed and added to a normal chromosome metaphase stretch (or to a microalignment chip, for alignment or array CGH). The color ratios are then compared to identify regions with aberrant copy number.

FISH is commonly used when multi-color imaging is required and / or when the protocol requires quantization of the signals. The technique generally involves preparing a cytological sample, labeling probes, denaturing target chromosomes and the probe, hybridizing the probe to the target sequence, and detecting the signal. Typically, the hybridization reaction fluorescently stains the targeted sequences so that their location, size, or number can be determined using fluorescence microscopy, flow cytometry, or other suitable instrumentation. DNA sequences ranging from entire genomes to several kilobases can be studied using FISH. FISH with metaphase extensions and interphase nuclei can also be used.

FISH has been used successfully to map repetitive and single copy DNA sequences in metaphase chromosomes, interphase nuclei, chromatin fibers, and naked DNA molecules, and for chromosome identification and karyotype analysis across localization. of large repeat families, usually ribosomal DNAs and families of major tandem alignments. One of the most important applications for FISH has been in the detection of single copy DNA sequences, particularly disease-related genes in humans and other eukaryotic model species, and the detection of infectious agents. FISH can be used to detect, for example, chromosomal aneuploidy in prenatal diagnoses, hematologic cancers, and solid tumors; gene abnormalities such as oncogene amplifications, gene deletions or gene fusions; chromosomal structural abnormalities such as translocations, duplications, insertions, or inversions; contiguous gene syndromes such as microdeletion syndrome; the genetic effects of various therapies; viral nucleic acids in somatic cells and sites of viral integration in chromosomes; etc. In multi-color FISH, each chromosome is stained a different color, allowing the normal chromosomes from which the abnormal chromosomes are derived to be determined. Such techniques include multiplex FISH (m-FISH), spectral karyotyping (SKY), combined ratio and binary marking (COBRA), color change karyotyping, interspecies color bands, high resolution multi-color bands, telomeric multiplex FISH (TM- FISH), separate signal FISH (ssFISH) and fused signal FISH.

CISH and SISH can be used for many of the same applications as FISH, and have the additional advantage of allowing analysis of the underlying tissue morphology, for example in histopathology applications. If FISH is performed, the hybridization mix may contain distinct and balanced sets of pairs of probes, as described in US Patent No. 6,730,474. For CIS ^h , the hybridization mix can contain at least one set of probes configured for detection with one or more conventional organic chromogens, and for SISH, the hybridization mix can contain at least one set of probes configured for detection with particles. silver, as described in Powell RD et al., "Metallographic in situ hybridization", Hum. Pathol., 38: 1145-59 (2007).

The compositions disclosed herein can also be used fully or partially in all types of molecular biology techniques involving hybridization, including blotting and probing (eg, Southern, Northern, etc.), alignments, and techniques. of amplification including traditional PCR, RT-PCR, mutational PCR, asymmetric PCR, hot start PCR, reverse PCR, multiplex PCR, nested PCR, quantitative PCR and in situ PCR. In situ PCR is a polymerase chain reaction that takes place within a cell on a slide, eg, the cytology and histology specimens described above. Typically, after adhering the sample to a microscope slide, the cells are rehydrated and permeabilized, and then combined with an appropriate mixture of PCR reagents including polymerase, dNTPs, and primers. PCR can be carried out on a specialized instrument, such as the in situ PCR system. GeneAmp 1000 (Perkin Elmer Biosystems, Foster City, CA), and the amplified product can be detected using labeled probes or by incorporating labeled dNTPs during amplification. The compositions of the invention will improve the efficiency of in situ PCR analysis, for example, by reducing the temperatures and / or the denaturation and hybridization time required in order to run the amplification cycles.

(3). Hybridization conditions

The method of the present invention involves the use of sulfolane as a polar aprotic solvent in in situ hybridization of nucleic acid strands. The compositions of the present invention are particularly useful in such a method and are described in detail in the appended claims. In situ hybridization methods using the compositions of the invention may involve applying the compositions to a sample comprising a target nucleic acid sequence, most likely in a double-stranded form. Typically, in order to ensure access for the probe to hybridize to the target sequence, the sample and composition are heated to denature the target nucleic acids. During denaturation, the polar aprotic solvent interacts with the sequence and facilitates denaturation of the target and re-pairing of the probe with the target. The polar aprotic solvent specified in the present invention accelerates this process considerably and reduces the severity and toxicity of hybridization conditions compared to formamide.

In situ hybridizations using the compositions of the invention can be performed using the same assay methodology as for hybridizations performed with traditional compositions. However, the compositions of the invention allow shorter hybridization times. For example, the heat pretreatment, digestion, denaturation, hybridization, washing, and mounting steps can use the same conditions in terms of volumes, temperatures, reagents, and incubation times as for traditional compositions. There is great variation in traditional hybridization protocols known in the art. For example, some protocols specify an independent denaturation step of possible double-stranded nucleotides with no probe present, prior to the next hybridization step. The compositions of the invention can be used in any of the traditional hybridization protocols known in the art.

Alternatively, assays using the compositions of the invention can be changed and optimized with respect to traditional methodologies, for example, by decreasing hybridization time, increasing or decreasing denaturation and / or hybridization temperatures, and / or increasing or decreasing volumes. hybridization.

For example, in some embodiments, the compositions of the invention will produce strong signals when the denaturation temperature is from 60 to 100 ° C and the hybridization temperature is from 20 to 60 ° C. In other embodiments, the compositions of the invention will produce strong signals when the denaturation temperature is 60 to 70 ° C, 70 to 80 ° C, 80 to 90 ° C, or 90 to 100 ° C, and the temperature Hybridization is from 20 to 30 ° C, from 30 to 40 ° C, from 40 to 50 ° C or from 50 to 60 ° C. In other embodiments, the compositions of the invention will produce strong signals when the denaturation temperature is 72, 82, or 92 ° C, and the annealing temperature is 37, 40, 45, or 50 ° C.

In other embodiments, the compositions of the invention will produce strong signals when the denaturation time is from 0 to 10 minutes and the hybridization time is from 0 minutes to 24 hours. In other embodiments, the compositions of the invention will produce strong signals when the denaturation time is from 0 to 5 minutes and the hybridization time is from 0 minutes to 8 hours. In other embodiments, the compositions of the invention will produce strong signals when the denaturation time is 0, 1, 2, 3, 4, or 5 minutes, and the hybridization time is 0 minutes, 5 minutes, 15 minutes, 30 minutes. , 60 minutes, 180 minutes or 240 minutes. Those skilled in the art will understand that in some cases, eg RNA detection, a denaturation step is not required.

Consequently, hybridizations using the compositions of the invention can be performed in less than 8 hours. In other embodiments, hybridization is done in less than 6 hours. In still other embodiments, hybridization is done within 4 hours. In other embodiments, hybridization is done within 3 hours. In still other embodiments, hybridization is done within 2 hours. In other embodiments, hybridization is done within 1 hour. In still other embodiments, hybridization is done within 30 minutes. In other embodiments, hybridization can take place within 15 minutes. Hybridization can take place even within 10 minutes or in less than 5 minutes. Figures 1 and 2 illustrate a typical time course for hybridizations performed with histological and cytological samples, respectively, using the compositions of the invention compared to hybridizations using traditional compositions.

As the hybridization time changes, the probe concentration can also be varied in order to produce strong signals and / or reduce the background. For example, as the hybridization time decreases, the amount of probe can be increased in order to improve the signal intensity. On the other hand, as the hybridization time decreases, the amount of probe can be decreased in order to improve the background staining.

The compositions of the invention surprisingly eliminate the need for a blocking step during hybridization by improving signal and background intensity by blocking the binding of, for example, sequences repetitive with the target DNA. Thus, there is no need to use total human DNA, blocking PNA, COT-1 DNA, or DNA from any other source as a blocking agent. However, background levels can be further lowered by adding agents that reduce non-specific binding, such as to the cell membrane, such as small amounts of total human DNA or DNA of non-human origin (eg, salmon sperm DNA). to a hybridization reaction using the compositions of the invention.

The aqueous compositions of the invention further provide the possibility of considerably reducing the concentration of nucleic acid sequences included in the composition. Generally, the concentration of probes can be reduced from 2 to 8 times compared to traditional concentrations. For example, if HER2 DNA probes and CEN17 PNA probes are used in compositions of the invention, their concentrations can be reduced by 1/4 and 1/2, respectively, compared to their concentrations in traditional hybridization compositions. . This feature, along with the absence of any blocking DNA requirements, such as blocking PNA or COT1, allows an increase in probe volume in automated instrument systems compared to the traditional 10 µl volume used in compounding systems. traditional, reducing loss due to evaporation, as discussed in more detail below.

Reducing the probe concentration also reduces the background. However, the reduction in probe concentration is inversely related to the hybridization time, that is, the lower the concentration, the longer hybridization time is required. However, even when extremely low concentrations of probe are used with the aqueous compositions of the invention, the hybridization time is still shorter than with traditional compositions.

The compositions of the invention often allow better signal-to-noise ratios than traditional hybridization compositions. For example, with certain probes, a one hour hybridization with compositions of the invention will produce a similar background and more intense signals than overnight hybridization with traditional compositions. No background is seen when no probe is added.

Traditional test methods can also be changed and optimized when using the compositions of the invention depending on whether the system is manual, semi-automated, or automated. For example, a semi-automated or automated system will benefit from the short hybridization times obtained with the compositions of the invention. The short hybridization time can reduce the difficulties encountered when using traditional compositions in such systems. For example, a problem with semi-automated or automated systems is that significant evaporation of the sample can occur during hybridization, since such systems require small sample volumes (eg, 10-150 µl), high temperatures, and holding times. prolonged hybridization (eg, 14 hours). Therefore, the proportions of the components in traditional hybridization compositions are quite unchanged. However, since the compositions of the invention allow faster hybridizations, evaporation is reduced, allowing an increase in flexibility in the proportions of the components in the hybridization compositions used in semi-automated and automated systems.

For example, two automated instruments have been used to perform hybridizations using the disclosed compositions. Compositions comprising 40% (v / v) ethylene carbonate have been used in the apparatus disclosed in PCT application WO2009 / 074154, and compositions comprising 15% (v / v) ethylene carbonate have been used in the HYBRIMASTER HS-300 instrument (Aloka CO. LTD, Japan). When the compositions are used in the HYBRIMASTER HS-300 instrument, the instrument can perform rapid FISH hybridization with water instead of the traditional toxic formamide mix, thereby improving safety and reducing evaporation. Attaching water-moistened strips to the lid on the inside of the Aloka instrument reaction unit (hybridization chamber), for example, as described in US Patent No. 7,901,534, reduces evaporation. even more.

Another problem with automated imaging analysis is the number of images required, the enormous amount of storage space required, and the time required to capture the images. The compositions of the invention address this problem by producing very strong signals compared to traditional compositions. Due to the very strong signals produced by the compositions of the invention, imaging can be performed at lower magnification than required for traditional compositions and can still be detected and analyzed, for example, by algorithms. Since the focal plane becomes wider with lower magnification, the compositions of the invention reduce or eliminate the requirement to capture serial sections of a sample. As a result, overall imaging is much faster, since the compositions of the invention require less or no serial section and each image covers a much larger area. Also, the overall time for analysis is faster, since the total image files are much smaller.

Thus, the compositions and methods of the invention solve many of the problems associated with traditional in situ hybridization methods and compositions.

The disclosure can be more clearly understood with the help of the non-limiting examples that follow, which constitute preferred embodiments of the compositions according to the disclosure. Differently in the examples, or when otherwise indicated, it is to be understood that all numbers expressing amounts of components, reaction conditions, etc., in the specification and claims are in all cases modified by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained herein. At a minimum, each numerical parameter should be interpreted in light of the number of significant figures and common rounding approaches.

Although the numerical ranges and parameters established by the broad scope are approximations, the numerical values set forth in the specific example are reported as precisely as possible. Any numerical value, however, inherently contains certain errors that necessarily result from the standard deviation found in their respective test measurements. The following examples illustrate the present invention.

Examples

The reagents used in the following examples are from Dako's FISH Auxiliary Kit for Histology (K5599) and FISH Auxiliary Kit for Cytology (K5499) (Dako Denmark A / S, Glostrup, Denmark). The kits contain all of the key reagents, except the probe, required to complete a FISH procedure for formalin-fixed, paraffin-embedded tissue section samples. All samples were prepared according to the manufacturer's description. The Dako hybridizer (S2450, Dako) was used for the digestion, denaturation and hybridization steps.

Slide evaluation for FISH was performed within one week after hybridization using a Leica DM6000B fluorescence microscope, equipped with individual DAPI, FITC, Texas Red filters and a FITC / Texas Red double filter under an oil objective. 10x, 20x, 40x and 100x.

CISH slide evaluation was performed using an Olympus BX51 light microscope, under a 4x, 10x, 20x, 40x and 60x objective.

In the examples that follow, "dextran sulfate" refers to the sodium salt of dextran sulfate (D8906, Sigma) having a molecular weight Mw> 500,000. All polar aprotic solvent concentrations are given as percentages v / v. Phosphate buffer refers to a phosphate buffered solution containing NaH2PO4, 2H2O (dihydrate dibasic sodium phosphate) and Na2HPO4, H2O (monohydrate monobasic sodium phosphate). Citrate buffer refers to a citrate buffered solution containing sodium citrate (Na3C6H5O7, 2H2O; 1.06448, Merck) and citric acid monohydrate (C6H8O7, H2O; 1.00244, Merck).

FISH / CISH Procedure for General Histology (Examples 1-20)

Slides with multiple sections of formalin-fixed paraffin embedded (FFPE) tissue arrays cut from humans (tonsils, breast, kidney, and colon carcinoma) were baked at 60 ° C for 30-60 min. , dewaxed in xylene baths, rehydrated in ethanol baths, and then transferred to wash buffer. The samples were then pretreated in pretreatment solution at a minimum of 95 ° C for 10 min and washed 2 x 3 min. The samples were then digested with TUR pepsin at 37 ° C for 3 min, washed 2 x 3 min, dehydrated in a series of ethanol evaporations and air dried. Samples were then incubated with 10 µl of FISH probe as described below in individual experiments. The samples were then washed by stringent washing at 65 ° C 10 min, then washed 2 x 3 min, then dehydrated in a series of ethanol evaporations and air dried. Finally, the slides were mounted with 15 µl of anti-fading mounting medium. When staining was complete, trained observers scored to assess signal intensity, morphology, and background of the stained slides.

FISH Procedure for General Cytology (Examples 21-22)

Slides were fixed with metaphase preparation in 3.7% formaldehyde for 2 min, washed 2 x 5 min, dehydrated in a series of ethanol evaporations, and air dried. Samples were then incubated with 10 µl of FISH probe as described in individual experiments. The samples were then washed by stringent washing at 65 ° C 10 min, then washed 2 x 3 min, then dehydrated in a series of ethanol evaporations and air dried. Finally, the slides were mounted with 15 µl of anti-fading mounting medium. When staining was complete, trained observers scored the signal intensity and background of the stained slides as described in the scoring guidelines sections for tissue sections.

Tissue Section Scoring Guidelines

Signal intensities were evaluated on a scale of 0-3 with 0 meaning no signal and 3 being equal to strong signal. Cellular / tissue structures are evaluated on a scale of 0-3 with 0 meaning absence of structure and no core boundaries and 3 being equal to an intact structure and clear core boundaries. Between 0 and 3 there are additional degrees with a separation of 0.5 from which the observer can evaluate the signal intensity, the tissue structure and the background.

Signal intensity is scored with a graded system on a 0-3 scale.

0 No signal is observed.

1 Signal strength is weak.

2 Signal strength is moderate.

3 Signal strength is strong.

The scoring system allows the use of A grades.

Tissue and nuclear structure are scored with a graded system on a 0-3 scale.

0 Tissue structures and nuclear borders are completely destroyed.

1 Tissue structures and / or nuclear borders are sparse. This grade includes situations in which some areas have empty cores.

2 Tissue structures and / or nuclear borders are observed, but the nuclear borders are not clear. This grade includes situations where a few cores are empty.

3 Tissue structures and nuclear borders are intact and clear.

The scoring system allows the use of A grades.

The fund is scored using a graded system on a 0-3 scale.

0 Little or no background is observed.

1 Some background.

2 Moderate background.

3 High bottom.

The scoring system allows the use of A grades.

Example 1

This example compares the signal intensity and cell morphology of samples treated with the compositions disclosed herein or traditional hybridization solutions as a function of denaturation temperature.

Probe composition for FISH I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide (15515-026, Invitrogen), 5 | iM blocking PNA (see Kirsten Vang Nielsen et al. , PNA Suppression Method Combined with Fluorescence In Situ Hybridization (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006)), Texas-labeled CCND1 gene DNA probe Red 10 ng / | il (RP11-1143E20, size 192 kb).

Probe composition for FISH II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate (03519, Fluka), 5 | iM blocking PNA, labeled CCND1 gene DNA probe with Texas Red 10 ng / | il (RP11-1143E20, size 192 kb).

Different viscosity phases were mixed prior to use, if present. FISH probes were denatured as indicated for 5 min and hybridized at 45 ° C for 60 min.

Results:

Example 2

This example compares the signal intensity and background staining of samples treated with the compositions disclosed herein or traditional hybridization solutions as a function of hybridization time.

Probe composition for FISH I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 | iM blocking PNA, 10 ng / | Texas Red-labeled CCND1 gene DNA probe il.

Probe composition for FISH II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate, 5 | iM blocking PNA, 10 ng Texas Red-labeled CCND1 gene DNA probe / | il.

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 14 hours, 4 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 0 minutes.

Results:

Example 3

This example compares the signal intensity of samples treated with the compositions of the invention and different polar aprotic solvents or traditional hybridization solutions.

Probe composition for FISH I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 | i M blocking PNA, 10 ng / Texas Red-labeled CCND1 gene DNA probe | i l.

Probe composition for FISH II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate (EC), 5 | iM blocking PNA, CCND1 gene DNA probe labeled with Texas Red 10 ng / | i l. Probe composition for FISH III: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% propylene carbonate (PC) (540013, Aldrich), 5 | i M blocking PNA, DNA probe of the CCND1 gene labeled with Texas Red 10 ng / | i l.

Probe composition for IV FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% sulfolane (SL) (T22209, Aldrich), 5 | i M blocking PNA, gene DNA probe CCND1 labeled with Texas Red 10 ng / | i l.

Probe composition for FISH V: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% acetonitrile (NA) (C02CIIX, Lab-Scan), 5 | i M blocking PNA, DNA probe of the CCND1 gene labeled with Texas Red 10 ng / | i l.

Probe composition for FISH VI: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, and 40% butyrolactone (GBL) (B103608, Aldrich), 5 | i M blocking PNA, gene DNA probe CCND1 labeled with Texas Red 7.5 ng / | i l.

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes.

Results:

Example 4

This example compares the signal intensity of treated samples with the compositions disclosed herein having different concentrations of polar aprotic solvent.

Probe compositions for FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10-60% ethylene carbonate (as indicated), 5 | iM blocking PNA, gene / GK DNA probe -Texas Red-labeled constant 7.5 ng / ul ((CTD-3050E15, RP11-1083E8; size 227 kb) and Texas Red-labeled gene / GK-variable DNA probe 7.5 ng / ul (CTD- 2575M21, RP11-122B6, RP11-316G9; size 350 and 429 kb).

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes.

Results:

Example 5

This example compares the signal intensity and background intensity of samples treated with the compositions with and without PNA blocking.

Probe compositions for FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate, 7.5 ng / ul Texas Red labeled CCND1 gene DNA probe.

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes.

Results:

Example 6

This example compares the signal intensity of treated samples with the compositions disclosed herein as a function of probe concentration and hybridization time.

Probe Compositions for FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate, and Texas Red 10, 7.5, 5, or 2.5 labeled CCND1 gene DNA probe ng / ul (as indicated). Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 3 hours, 2 hours, and 1 hour.

Results:

Example 7

This example compares the signal intensity of treated samples with the compositions disclosed herein as a function of salt, phosphate and buffer concentrations.

Probe compositions for FISH: 10% dextran sulfate, ([NaCl], [phosphate buffer], [TRIS buffer] as indicated in the results), 40% ethylene carbonate, labeled CCND1 gene DNA probe with Texas Red 7.5 ng / | il.

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes.

Results:

Example 8

This example compares the signal intensity of treated samples with the compositions disclosed herein as a function of dextran sulfate concentration.

Probe compositions for FISH: 0, 1, 2, 5 or 10% dextran sulfate (as indicated), 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate, DNA probe of the SIL-TAL1 gene labeled with Texas Red 5 ng / | il (RP1-278O13; size 67 kb) and FITC SIL-TAL1 6 ng / | il (ICRFc112-112C1794, RP11-184J23, RP11-8J9, CTD-2007B18, 133B9; size 560 kb).

Different viscosity phases were mixed prior to use, if present. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes. No blocking.

Results:

Example 9

This example compares the signal intensity of samples treated with the compositions disclosed herein as a function of the concentrations of dextran sulfate, salt, phosphate, and polar aprotic solvent.

Probe composition for FISH Ia: 34% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 0% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH Ib: 34% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 0% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH Ic: 34% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 0% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIa: 32% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 5% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIb: 32% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 5% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIc: 32% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 5% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIIa: 30% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 10% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIIb: 30% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IIIc: 30% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 10% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for FISH IVa: 28% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 15% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for IVb FISH: 28% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 15% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe composition for IVc FISH: 28% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 15% ethylene carbonate, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe (size 218 kb) and 50 nM FITC-labeled CEN-7 PNA probe.

Probe Reference for FISH V: Standard retail vial of HER2 PharmDx Probe Mix (K5331, Dako) containing blocking PNA. Hybridization overnight for 20 hours.

All compositions were present as monophase. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 min without blocking, except for the V FISH probe reference, which was PNA blocked and hybridized for 20 hours.

Results:

Example 10

This example compares the signal intensity of treated samples with the compositions disclosed herein as a function of the concentration of polar aprotic solvent and dextran sulfate under conditions of high salt concentration (4x normal).

Probe composition for FISH I: 0% ethylene carbonate, 29% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe and 50 nM FITC-labeled CEN-7 PNA probe. The composition was monophasic.

Probe composition for FISH II: 5% ethylene carbonate, 27% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe, and 50 nM of CEN-7 PNA probe labeled with FITC. The composition was monophasic.

Probe composition for FISH III: 10% ethylene carbonate, 25% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng / | yl Texas Red-labeled HER2 gene DNA probe, and 50 nM of CEN-7 PNA probe labeled with FITC. The composition was monophasic.

Probe composition for IV FISH (not tested): 20% ethylene carbonate, 21% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng / Texas Red labeled HER2 gene DNA probe | il and 50 nM FITC-labeled CEN-7 PNA probe. The composition was biphasic.

Results:

Example 11

This example compares the signal intensity and background of samples treated with different phases of the compositions disclosed herein.

Probe composition for FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene carbonate, 8 ng / | yl Texas Red labeled HER2 gene DNA probe and 8 ng / | yl PNA probe. CEN-17 labeled with 600 nM FITC. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes. No blocking.

Results:

Example 12

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

Probe composition for FISH: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% GBL, 10 ng / | yl Texas Red-labeled CCND1 gene DNA probe and CEN-PNA probe. 17 FITC-labeled 600 nM.

FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes. No blocking.

Results:

Example 13

This example examines the number of phases in the compositions disclosed herein as a function of the concentration of polar aprotic solvent and dextran sulfate.

Probe compositions for FISH: 10% or 20% dextran sulfate; 300 mM NaCl; 5 mM phosphate buffer; EC at 0, 5, 10, 15, 20, 25, 30%; probe 10 ng / ^ l.

Results:

Example 14

This example compares the signal intensity and background of samples treated with different compositions disclosed herein as a function of probe concentration and hybridization time.

Probe composition for FISH I: 10 ng / µl TxRed-labeled HER2 DNA probe (standard concentration) and standard FITC-labeled CEN7 PNA probe concentration (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.

Probe composition for FISH II: TxRed 5 ng / | yl (1/2 conventional concentration) and conventional concentration (50 nM) of FITC-labeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.

Probe composition for FISH III: HER2 DNA probe labeled with TxRed 2.5 ng / | yl (1/4 of the standard concentration) and A of the standard concentration (25 nM) of CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.

Compositions I-III exist as single phase. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 3 hours, 2 hours, and 1 hour.

Results:

Example 15

This example compares the signal intensity and background of samples treated with the compositions disclosed herein as a function of blocking agent.

Probe compositions for FISH: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; TxRed-labeled HER2 DNA probe 2.5 ng / | yl (1/4 of the standard concentration) and A of the standard concentration (300 nM) of FITC-labeled CEN17 PNA probe. Samples were blocked with: (a) nothing; (b) COT1 0.1 | ig / | yl (15279-011, Invitrogen); (c) COT1 0.3 | ig / | yl; or (d) 0.1 µg / µl total human DNA prior to hybridization using the compositions disclosed herein.

All samples were present as single phase. FISH probes were incubated at 82 ° C for 5 min and then at 45 ° C for 60 minutes.

Results:

Example 16

This experiment compares different modes of removal of background staining using the compositions disclosed herein.

All compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, HER22.5 ng / ul DNA probes (1/4 conventional concentration), CEN17300 PNA probe nM (1/2 the conventional concentration), and one of the following background reducing agents:

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

B) COT-1 DNA 0.1 ug / ul

C) Total human DNA (THD) 0.1 ug / ul (sonicated unlabeled THD)

D) 0.1 ug / ul fragmented salmon sperm DNA (AM9680, Ambion)

E) 0.1 ug / ul calf thymus DNA (D8661, Sigma)

F) Herring sperm DNA 0.1 ug / ul (D7290, Sigma)

G) 0.5% formamide

H) 2% formamide

I) 1% ethylene glycol (1.09621, Merck)

J) 1% glycerol (1.04095, Merck)

K) 1,3-propanediol 1% (533734, Aldrich)

L) 1% H2O (control)

All samples were present as single phase. Probes were incubated at 82 ° C for 5 minutes and then at 45 ° C with FFPE tissue sections for 60 and 120 minutes.

Results:

Example 17

This experiment compares the signal intensity of the upper and lower phases using two different polar aprotic solvents.

Probe composition for FISH I: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich), 5 | iM blocking PNA, DNA probe CCND1 gene labeled with Texas Red 10 ng / | yl.

Probe composition for FISH II: 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 | iM blocking PNA, DNA probe of the CCND1 gene labeled with Texas Red 10 ng / | yl.

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

Results:

Example 18.

This experiment examines the ability of various polar aprotic solvents to form a single phase system.

All compositions contained: 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, and either 10, 15, 20, or 25% of one of the following polar aprotic solvents:

sulfolane

y-butyrolactone

ethylene trithiocarbonate

glycol sulfite

propylene carbonate

Results: all polar aprotic solvents at all concentrations examined produced at least one biphasic system in the compositions used. However, this does not exclude that these compounds can produce a monophasic system under the conditions of other compositions.

Example 19

This experiment examines the use of the compositions disclosed herein in chromogenic in situ hybridization (CISH) analysis on multiple FFPE tissue sections.

Probe composition for FISH I: FITC-labeled 4.5 ng / | il (1/4 of the conventional concentration) (RP11-654A2, RP11-246A2, CTP-2355L21, RP11-158G6, RP11-780M2, RP11-481C14; size 1018 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0. Probe composition for FISH II: FITC-labeled TCRAD gene DNA probe 4.5 ng / | yl (1/4 of the conventional concentration) (1018 kb size); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug / ul fragmented salmon sperm DNA.

Probe composition for FISH III: 300 nM of each individual FITC-labeled CEN17 PNA probe (1/2 conventional concentration); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

All samples were analyzed using Dako's DuoCISH protocol (SK108) and split probe compositions with the exception that the stringency wash was performed for 20 minutes instead of 10 minutes, and without the use of the red chromogen step. DuoCISH.

Results:

Example 20

This example compares the signal intensity and background of FFPE tissue sections treated with the compositions disclosed herein with two DNA probes.

Probe composition for FISH I: 9 ng / | yl FITC-labeled IGH gene DNA probe (RP11-151B17, RP11-112H5, RP11-101G24, RP11-12F16, RP11-47P23, CTP-3087C18; size 612 kb); 6.4 ng / | yl Tx Red labeled MYC DNA probe (CTD-2106F24, CTD-2151C21, CTD-2267H22; size 418 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

Probe composition for FISH II: 9 ng / | yl FITC-labeled IGH gene DNA probe; 6.4 ng / µl TxRed-labeled MYC DNA probe; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug / ul fragmented salmon sperm DNA.

Example 21

This experiment examines the use of the compositions disclosed herein with cytological samples.

Probe composition for FISH: 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; HER2 DNA probe labeled with TxRed 5 ng / | yl (1/2 of the conventional concentration) and A of the conventional concentration of CEN7 (25 nM).

FISH probes with metaphase chromosome extensions were incubated at 82 ° C for 5 minutes, then at 45 ° C for 30 minutes, all without blocking.

Results:

Example 22

This example compares the signal intensity and background of DNA probes with cytology samples, metaphase smears, with and without blocking.

Probe composition for FISH I: Texas Red 6 ng / | yl-labeled TCRAD gene DNA probe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb) and FITC-labeled gene DNA probe 4.5 ng / | yl (1/4 of the conventional concentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.

Probe composition for FISH II: Texas Red-labeled TCRAD gene DNA probe 6 ng / | yl (standard concentration) (301 kb size) and FITC-labeled gene DNA probe 4.5 ng / | yl ( 1/4 of the conventional concentration); 15% E ^c , 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug / ul fragmented salmon sperm DNA.

FISH probes with metaphase extensions were incubated at 82 ° C for 5 min, then at 45 ° C for 60 min.

Results:

Claims (15)

i. In situ hybridization composition comprising at least one nucleic acid sequence, a polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences, and a hybridization solution, wherein the polar aprotic solvent is not dimethylsulfoxide (DMSO) , where the polar aprotic solvent is:

Y wherein said composition further comprises at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents, wherein the sulfolane concentration is from 10% to 95% (v / v). 2. In situ hybridization composition according to claim 1, wherein the sulfolane concentration is 10% to 20% (v / v), 20% to 30% (v / v) or 10% to 60% (v / v). 3. In situ hybridization composition according to any one of claims 1 to 2, provided that the composition does not contain formamide. 4. In situ hybridization composition according to any one of claims 1 to 2, provided that the composition contains less than 10% formamide, less than 2% formamide or less than 1% formamide. 5. In situ hybridization composition according to claim 1, wherein the accelerating agent is dextran sulfate and the salts are NaCl and / or phosphate buffer. 6. In situ hybridization composition according to claim 5, wherein the dextran sulfate is present in a concentration of 5% to 40%, the NaCl is present in a concentration of 0mM to 1200mM and / or the phosphate buffer it is present in a concentration of 0 mM to 50 mM. 7. In situ hybridization composition according to claim 6, wherein the dextran sulfate is present in a concentration of 10% to 30%, the NaCl is present in a concentration of 300 mM to 600 mM and / or the phosphate buffer it is present in a concentration of 5 mM to 20 mM. 8. In situ hybridization composition according to claim 1, wherein the accelerating agent is selected from the group consisting of: formamide, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol and 1,3-propanediol, and the buffering agent is citric acid buffer. 9. In situ hybridization composition according to claim 8, in which the formamide is present in a concentration of 0.1-5%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol and 1,3 -propanediol are present at a concentration of 0.1% to 10%, and the citric acid buffer is present at a concentration of 1mM to 50mM. 10. An in situ hybridization composition according to claim 1, wherein the blocking agent is selected from the group consisting of: total human DNA, herring sperm DNA, salmon sperm DNA and calf thymus DNA. 11. In situ hybridization composition according to claim 10, wherein the total human DNA, the herring sperm DNA, the salmon sperm DNA and the calf thymus DNA are present in a concentration of 0.01 to 10 | ig / | il. 12. In situ hybridization composition according to any one of claims 1-11, comprising • 40% sulfolane, 10% dextran sulfate, 300 mM NaCl and 5 mM phosphate buffer, • 15% sulfolane, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer and 0.1 | ig / | yl total human DNA, or • 15% sulfolane, 20% dextran sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2 and 0.1 | ig / | yl DNA from herring sperm, or DNA from salmon sperm, or DNA from calf thymus, or 0.5% formamide or 1% ethylene glycol or 1% 1,3-propanediol. 13. In situ hybridization composition according to any one of claims 1-12, comprising more than one phase at room temperature, preferably comprising two or three phases at room temperature. ambient. 14. In situ nucleic acid sequence hybridization method comprising: - provide a first nucleic acid sequence, and - applying the in situ hybridization composition according to any one of claims 1-13 to said first nucleic acid sequence for at least a period of time sufficient to hybridize the first nucleic acid sequence and the nucleic acid sequence of the composition of in situ hybridization. 15. The method of claim 14, wherein the first nucleic acid sequence is in a cytology or histology sample.