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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6832—Enhancement of hybridisation reaction
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2527/00—Reactions demanding special reaction conditions
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  • C12Q2527/00—Reactions demanding special reaction conditions
  • C12Q2527/137—Concentration of a component of medium

Definitions

• the present invention relates to compositions and methods for performing a stringent wash step in hybridization applications
• the present invention can be used for the in vivo, in vitro, and in situ molecular examination of DNA and RNA.
• the invention can be used for the molecular examination of DNA and RNA in the fields of cytology, histology, and molecular biology.
• the present invention can be used for in situ hybridization (ISH) applications.
• ISH in situ hybridization
• Double stranded nucleic acid molecules ⁇ i.e., DNA (deoxyribonucleic acid), DNA/RNA (ribonucleic acid) and RNA/RNA) associate in a double helical configuration.
• This double helix structure is stabilized by hydrogen bonding between bases on opposite strands when bases are paired in a particular way (A+T/U or G+C) and hydrophobic bonding among the stacked bases.
• Complementary base paring is central to all processes involving nucleic acid.
• nucleic acid probes or primers are designed to bind, or "hybridize,” with a target nucleic acid, for example, DNA or RNA in a sample.
• a target nucleic acid for example, DNA or RNA in a sample.
• in situ hybridization includes hybridization to a target in a specimen wherein the specimen may be in vivo, in situ, or for example, fixed or adhered to a glass slide.
• nucleic acid hybridization assays mostly depend on at least one of three major factors: a) denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions, b) renaturation (i.e., re-annealing of, e.g., two nucleic acid strands) conditions, and c) post-hybridization washing conditions.
• denaturation i.e., separation of, e.g., two nucleic acid strands
• renaturation i.e., re-annealing of, e.g., two nucleic acid strands
• post-hybridization washing conditions e.g., post-hybridization washing conditions.
• Traditional hybridization experiments, such as ISH assays use a formamide-containing solution to denature doubled stranded nucleic acid.
• Formamide disrupts base pairing by displacing loosely and uniformly bound hydrate molecules and by
• a "renaturation” or “reannealing” step allows the primers or probes to bind to the target nucleic acid in the sample. This step is also sometimes referred to as the “hybridization” step.
• the re- annealing step is by far the most time-consuming aspect of traditional hybridization applications. See Figures 1 and 2 (presenting examples of traditional hybridization times).
• the presence of formamide in a hybridization buffer can significantly prolong the renaturation time, as compared to aqueous denaturation solutions without formamide.
• any unbound and mis- paired probe is removed by a series of post-hybridization washes.
• the specificity of the interaction between the probe and the target is largely determined by stringency of these post-hybridization washes.
• Duplexes containing highly complementary sequences are more resistant to high-stringency conditions than duplexes with low complementary. Thus, increased stringency conditions can be used to remove non-specific bonds between the probe and the target nucleic acids.
• Formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
• Time (as the wash time increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
• the present invention provides several potential advantages over prior art hybridization applications, such as increased specificity, lower background, lower wash temperatures, preservation of sample morphology, and less toxic hybridization solvents.
• compositions which result in at least one of the following advantages: increased specificity, lower background, lower evaporation of reagent, preservation of sample morphology, simpler procedure, faster procedure, easier automation, and safer reagents.
• One way in which the present invention achieves those objectives is by providing compositions and methods for performing a stringent wash step in hybridization applications.
• compositions and methods of the invention are applicable to any hybridization technique.
• the compositions and methods of the invention are also applicable to any molecular system that hybridizes or binds using base pairing, such as, for example, DNA, RNA, PNA, LNA, and synthetic and natural analogs thereof.
• the method and compositions of the present invention may be used for the in vivo, in vitro, or in situ analysis of genomic DNA, chromosomes, chromosome fragments, genes, and chromosome aberrations such as translocations, deletions, amplifications, insertions, mutations, or inversions associated with a normal condition or a disease. Further, the methods and compositions are useful for detection of infectious agents as well as changes in levels of expression of RNA, e.g., mRNA and its complementary DNA (cDNA).
• RNA e.g., mRNA and its complementary DNA (cDNA).
• RNA messenger RNA
• viral RNA viral DNA
• small interfering RNA small nuclear RNA
• snRNA non-coding RNA
• ncRNA transfer messenger RNA
• miRNA micro RNA
• piRNA piwi-interacting RNA
• long noncoding RNA small nucleolar RNA
• SNPs single nucleotide polymorphisms
• CNVs copy number variations
• the method and compositions of the present invention are useful for in vivo, in vitro, or in situ analysis of nucleic acids using techniques such as northern blot, Southern blot, flow cytometry, autoradiography, fluorescence microscopy, chemiluminescence, immunohistochemistry, virtual karyotype, gene assay, DNA microarray (e.g., array comparative genomic hybridization (array CGH)), gene expression profiling, Gene ID, Tiling array, gel electrophoresis, capillary electrophoresis, and in situ hybridizations such as FISH, SISH, CISH.
• techniques such as northern blot, Southern blot, flow cytometry, autoradiography, fluorescence microscopy, chemiluminescence, immunohistochemistry, virtual karyotype, gene assay, DNA microarray (e.g., array comparative genomic hybridization (array CGH)), gene expression profiling, Gene ID, Tiling array, gel electrophoresis, capillary electrophores
• the methods and compositions of the invention may be used on in vitro and in vivo samples such as bone marrow smears, blood smears, paraffin embedded tissue preparations, enzymatically dissociated tissue samples, bone marrow, amniocytes, cytospin preparations, imprints, etc.
• the invention provides methods and compositions for performing a stringent wash step in hybridization applications.
• the invention may, for example, eliminate the use of, or reduce the dependence on formamide from, e.g., 50% formamide in traditional stringent wash buffers to 25%, 15%, 10%, 5%, 2%, 1% or 0% formamide in the compositions and methods of the invention.
• the methods and compositions of the invention may also increase the stringency of post- hybridization washes without the use of formamide such that the stringent wash can occur, e.g., at a lower temperature than with traditional non-formamide stringent wash buffers.
• the invention may also allow for stringency washes at lower temperatures (e.g. 20-30° C lower) than traditional hybridization applications. For example, instead of washing single locus probes at 73° C in Ix SSC, 0.3% NP-40, the compositions and methods of the invention may allow such probes to be washed at 45° C, or 40° C, or 35° C, or even at room temperature. The invention may also allow the stringent wash to be performed at the same or at a lower temperature than the hybridization temperature.
• the present invention overcomes major toxicity and temperature problems associated with traditional hybridization assays by allowing for easier automation, by lowering the stringent wash temperature of, e.g,. single locus probes without using the toxic chemical formamide, and by providing stringent wash temperatures that cause less harm to, e.g., biological samples (such as, virus, RNA, DNA, FFPE tissue sections, cryo sections, cytological preparations, etc.) and/or their potential carriers (e.g. nitrocellulose membranes, microarrays, etc).
• biological samples such as, virus, RNA, DNA, FFPE tissue sections, cryo sections, cytological preparations, etc.
• potential carriers e.g. nitrocellulose membranes, microarrays, etc.
• compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non- complementary double-stranded nucleotide sequences.
• One way to test for whether the amount of polar aprotic solvent is effective to denature non-complementary sequences in a hybridization product is to determine whether the polar aprotic solvent, when used in the methods and compositions described herein, yields a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay.
• Non-limiting examples of effective amounts of polar aprotic solvents include, e.g., about 1% to about 95% (v/v). In some embodiments, the concentration of polar aprotic solvent is 5% to 60% (v/v). In other embodiments, the concentration of polar aprotic solvent is 10% to 60% (v/v). In still other embodiments, the concentration of polar aprotic solvent is 30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 60% to 70% (v/v) are also suitable.
• 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).
• aqueous composition comprising a polar aprotic solvent has reduced toxicity compared to traditional formamide-containing stringency wash solutions.
• a less-toxic composition of the invention may comprise a composition with the proviso that the composition does not contain formamide, or with the proviso that the composition contains less than 25%, or less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% formamide.
• a less-toxic composition may also comprise a composition with the proviso that the composition does not contain dimethyl sulfoxide (DMSO), or with the proviso that the composition contains less than 25%, 10%, 5%, 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than 0.01% DMSO.
• DMSO dimethyl sulfoxide
• suitable polar aprotic solvents for use in the invention may be selected based on their Hansen Solubility Parameters.
• suitable polar aprotic solvents may have a dispersion solubility parameter between 17.7 to 22.0 MPa , a polar solubility parameter between 13 to 23 MPa 1 2 , and a hydrogen bonding solubility parameter between 3 to 13 MPa 1/2 .
• suitable polar aprotic solvents for use in the invention are cyclic compounds.
• a cyclic compound has a cyclic base structure. Examples include the cyclic compounds disclosed herein. Li other embodiments, the polar aprotic solvent may be chosen from Formulas 1-4 below:
• suitable polar aprotic solvents for use in the invention may be chosen from Formula 5 below:
• X is optional and if present, is chosen from O or S; where Z is optional and if present, is chosen from O or S; where A and B independently are O or N or S or part of the alkyldiyl or a primary amine; where R is alkyldiyl; and where Y is O or S or C.
• X is non-existing; Z and X are O; X is non-existing; X is non-existing;
• A, B, and Z are O; A and B are part of A is part of the A is part of the
• Y is C; and the alkyldiyl; alkyldiyl; alkyldiyl; alkyldiyl;
• R is Ethane-l,2 diyl; Y is S; and Y is C; Y is C;
• R is Butane- 1,4 diyl; B and Z is O; and B is methylamine;
• R is Propane- 1,3 diyl
• Z is O
• R is Propane- 1,3 diyl
• the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, or carbonate functionality.
• lactone lactone
• sulfone nitrile
• sulfite or carbonate functionality.
• Such compounds are distinguished by their relatively high dielectric constants, high dipole moments, and solubility in water.
• the polar aprotic solvent having lactone functionality is ⁇ -butyrolactone (GBL)
• the polar aprotic solvent having sulfone functionality is sulfolane (SL)
• the polar aprotic solvent having nitrile functionality is acetonitrile (AN)
• the polar aprotic solvent having sulfite functionality is glycol sulfite/ethylene sulfite (GS)
• the polar aprotic solvent having carbonate functionality is ethylene carbonate (EC), propylene carbonate (PC), or ethylene thiocarbonate (ETC)
• the compositions and methods of the invention comprise a polar aprotic solvent, with the proviso that the polar aprotic solvent is not acetonitrile (AN) or sulfolane (SL).
• the invention discloses a method for performing a stringent wash step in a hybridization application comprising: a) providing a hybridization product comprising a first nucleic acid sequence hybridized to a second nucleic acid sequence,
• aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary double-stranded nucleotide sequences
• no additional energy is required to denature any non-complementary binding between the first and second nucleic acid sequences. In other embodiments, a sufficient amount of energy to denature any non-complementary binding between the first and second nucleic acid sequences is provided.
• the energy is provided by heating the aqueous composition and the hybridization product.
• the step of method of the invention may include the steps of heating and cooling the aqueous composition and nucleic acid sequences.
• the energy is provided by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating, or heat lamps.
• the aqueous composition and hybridization product are heated to less than 7O 0 C, such as, for example, 65 0 C, 62 0 C, 6O 0 C, 55 0 C, 52 0 C, 5O 0 C, 45 0 C, 42 0 C, or 4O 0 C.
• the denaturation of any non-complementary binding between the first and second nucleic acid sequences occurs in less than 1 hour, such as, for example, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.
• the invention relates to the use of a composition comprising between 1 and 95% (v/v) of at least one polar aprotic solvent in a stringent wash step in a hybridization application.
• the invention relates to the use of a an aqueous composition as described in this invention for performing a stringent wash step in a hybridization application.
• FIG. 1 depicts a typical time-course for single locus hybridization assay with primary labeled FISH probes on formaldehyde fixed paraffin embedded tissue sections (histological specimens).
• the first bar on the left represents the deparaffmation step; the second bar represents the heat-pretreatment step; the third bar represents the digestion step; the fourth bar represents the denaturation and hybridization steps; the fifth bar represents the stringency wash step; and the sixth bar represents the mounting step.
• FIG. 2 depicts a typical time-course for single locus hybridization assay with primary labeled FISH probes on cytological specimens.
• the first bar on the left represents the fixation step; the second bar represents the denaturation and hybridization steps; the third bar represents the stringency wash step; and the fourth bar represents the mounting step.
• Bio 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 spread, purified nucleic acid sequences, artificially made nucleic acid sequences made by, e.g., a biologic based system or by chemical synthesis, microarray, or other form of nucleic acid chip, hi one embodiment, a sample is a mammalian sample, such as, e.g., a human, murine, rat, feline, or canine sample.
• Nucleic acid means anything that binds or hybridizes using base pairing including, oligomers or polymers having a backbone formed from naturally occurring nucleotides and/or nucleic acid analogs comprising nonstandard nucleobases and/or nonstandard backbones (e.g., a peptide nucleic acid (PNA) or locked nucleic acid (LNA)), or any derivatized form of a nucleic acid.
• PNA peptide nucleic acid
• LNA locked nucleic acid
• 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, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in, e.g., U.S. Pat. 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
• the pendant nucleobase such as, e.g., a purine or pyrimidine base on PNA may be connected to the backbone via a linker such as, e.g., one of the linkers taught in PCT/US02/30573 or any of the references cited therein, hi one embodiment, the PNA has an iV-(2-aminoethyl)-glycine) backbone. PNAs may be synthesized (and optionally labeled) as taught in PCT/US02/30573 or any of the references cited therein.
• PNAs hybridize tightly, and with high sequence specificity, with DNA and RNA, because the PNA backbone is uncharged. Thus, short PNA probes may exhibit comparable specificity to longer DNA or RNA probes. PNA probes may also show greater specificity in binding to complementary DNA or RNA.
• locked nucleic acid or "LNA” means an oligomer or polymer comprising at least one or more LNA subunits.
• LNA subunit means a ribonucleotide containing a methylene bridge that connects the 2'- " oxygen of the ribose with the 4'-carbon. See generally, Kurreck, Eur. J. Biochem., 270:1628-44 (2003).
• nucleic acids and nucleic acid analogs also include polymers of nucleotide monomers, including double and single stranded deoxyribonucleotides (DNA), ribonucleotides (RNA), ⁇ -anomeric forms thereof, synthetic and natural analogs thereof, and the like.
• the nucleic acid chain may be composed entirely of deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids (LNA), synthetic or natural 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 water solubility of at least about 5% (volume) at or near ambient temperature, i.e., about 20°C, and which does not undergo significant hydrogen exchange at approximately neutral pH, i.e., in the range of 5 to 9, or in the range 6 to 8.
• Polar aprotic solvents include those defined according to the Hansen 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 the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene, or alkyne.
• Aqueous solution is to be understood as a solution containing water, even small amounts of water.
• a solution containing 1% water is to be understood as an aqueous solution.
• Hybridization application “hybridization assay,” “hybridization experiment,” “hybridization procedure,” “hybridization technique,” “hybridization method,” etc. are to be understood as referring to any process that involves hybridization of nucleic acids. Such a process may include, for example, a fixation step, a deparaffination step, a heat- pretreatment step, a digestion step, denaturation and hybridization steps, a stringency wash step, and a mounting step. Unless otherwise specified, the terms “hybridization” and “hybridization step” are to be understood as referring to the re-annealing step of the hybridization procedure as well as the denaturation step.
• Hybridization composition refers to an aqueous solution of the invention for performing a hybridization procedure, for example, to bind a probe to a nucleic acid sequence.
• Hybridization compositions may comprise, e.g., 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 triphosphates (dNTPs), for amplifying nucleic acids in a biological sample.
• dNTPs deoxynucleoside triphosphates
• Hybridization solution refers to an aqueous solution for use in a hybridization composition of the invention.
• Hybridization solutions are discussed in detail below and may comprise, e.g., buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents.
• Hansen Solubility Parameters and “HSP” refer to the following cohesion energy (solubility) parameters: (1) the dispersion solubility parameter (6 D , "D parameter”), which measures nonpolar interactions derived from atomic forces; (2) the polar solubility parameter ( ⁇ p, "P parameter”), which measures permanent dipole-permanent dipole interactions; and (3) the hydrogen bonding solubility parameter ( ⁇ , "H parameter”), which measures electron exchange.
• D dispersion solubility parameter
• ⁇ p polar solubility parameter
• H parameter hydrogen bonding solubility parameter
• Rapid reannealing (approximately 25%) and/or intermediately reannealing (approximately 30%) components of mammalian genomes.
• the rapidly reannealing components contain small (a few nucleotides long) highly repetitive sequences usually found in tandem (e.g., satellite DNA), while the intermediately reannealing components contain interspersed repetitive DNA.
• Interspersed repeated sequences are classified as either SINEs (short interspersed repeat sequences) or LINEs (long interspersed repeated sequences), both of which are classified as retrotransposons in primates.
• SINEs and LINEs include, but are not limited to, Alu-repeats, Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotide repeats.
• AIu repeats make up the majority of human SINEs and are characterized by a consensus sequence of approximately 280 to 300 bp that consist of two similar sequences arranged as a head to tail dimer.
• telomere and centromere repeat sequences are localized within a certain 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 labelling of dangerous preparations" (ecb.jrc.it/legislation/1999L0045EC.pdf) ("Directive”). According to the Directive, toxicity is defined using the following classification order: T+ “very toxic”; T “toxic”, C “corrosive”, Xn “harmful”, .Xi “irritant.” Risk Phrases (“R phrases”) describe the risks of the classified toxicity.
• Formamide is listed as T (toxic) and R61 (may cause harm to the unborn child). All of the following chemicals are classified as less toxic than formamide: acetonitrile (Xn, Rl 1, R20, R21, R22, R36); sulfolane (Xn, R22); ⁇ -butyrolactone (Xn, R22, R32); and ethylene carbonate (Xi ,R36, R37, R38). At the time of filing this application, ethylene trithiocarbonate and glycol sulfite are not presently labeled.
• Stringent and “stringency” in the context of post-hybridization washes are to be understood as referring to conditions for denaturing non-complementary base-pairing.
• a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates specific hybridization with little non- complementary base-pairing.
• T m The melting temperature
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• T m can be approximated from the following equation:
• T m 81.5°C.+16.6(log M)+0.41(% GC) -0.61(% form)-500/L
• M is the molarity of monovalent cations
• % GC is the percentage of guanosine and cytosine nucleotides in the DNA
• % form is the percentage of formamide in the hybridization solution
• L is the length of the hybrid in base pairs.
• T m is reduced by about I 0 C for each 1% of mismatching. Accordingly, stringent conditions are generally selected to be about 5 0 C lower than T n , for the specific sequence and its complement at a defined ionic strength and pH.
• room temperature and “RT” refer to about 20 0 C to about 25 0 C, unless otherwise stated.
• Suitable polar aprotic solvents for use in the invention may 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 HSP have been reported for over 1200 chemicals.
• the D parameter may be calculated with reasonable accuracy based on refractive index, or may be derived from charts by comparison with known solvents of similar size, shape, and composition after establishing a critical temperature and molar volume.
• the P parameter may be estimated from known dipole moments (see, e.g., McClellan A.L., Tables of Experimental Dipole Moments (W.H. Freeman 1963)) using Equation 1 :
• HSP characterizations are conveniently visualized using a spherical representation, with the HSP of an experimentally-determined suitable reference solvent at the center of the sphere.
• the radius of the sphere (R) indicates the maximum tolerable variation from the HSP of the reference solvent that still allows for a "good" interaction to take place. Good solvents are within the sphere and bad ones are outside.
• the distance, R a between two solvents based on their respective HSP values can be determined using Equation 2:
• RED numbers less than 1.0 indicate high affinity; RED numbers equal or close to 1.0 indicate boundary conditions; and progressively higher RED numbers indicate progressively lower affinities.
• the D parameters of the polar aprotic solvents of the invention are between 17.7 to 22.0 MPa 1/2 .
• 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 for use in the invention, but may be considered if their P and H parameters are within the ranges discussed below. Since the D parameter is multiplied by 4 in Equation 2, the limits are one-half of R 0 . In addition, it should be noted that D values of around 21 or higher are often characteristic of a solid.
• the P parameters of the polar aprotic solvents of the invention are between 13 to 23 MPa 1/2 .
• Such exceptionally high P parameters are generally associated with solvents having a high dipole moment and presumably also a relatively low molecular volume.
• the dipole moment should be between 4.5 and 3.1.
• the dipole moment should be between 5.6 and 3.9.
• the H parameters of the polar aprotic solvents of the invention are between 3 to 13 MPa 1/2 .
• polar aprotic solvents having an alcohol group are not useful in the compositions and methods of the invention, 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 enters into the evaluation of all three Hansen Solubility Parameters. As molar volume gets smaller, liquids tend to evaporate rapidly. As molar volume gets larger, liquids tend to enter the solid region in the range of D and P parameters recited above. Thus, the polar aprotic solvents of the invention are rather close to the liquid/solid boundary in HSP space.
• the polar aprotic solvents of the invention 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 ⁇ -butyrolactone (GBL)
• an exemplary polar aprotic solvent with sulfone functionality is sulfolane (SL; tetramethylene sulfide-dioxide)
• 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)
• an exemplary polar aprotic solvents with carbonate functionality are ethylene carbonate (EC), propylene carbonate (PC), or ethylene trithiocarbonate (ETC).
• the structures of these exemplary solvents are provided below and their Hansen Solubility Parameters, RED numbers, and molar volumes are given in Table 1.
• Suitable polar aprotic solvents that may be used in the invention are cyclic compounds such as, e.g., ⁇ -caprolactone.
• substituted pyrolidinones and related structures with nitrogen in a 5- or 6-membered ring and cyclic structures with two nitrile groups, or one bromine and one nitrile group, may also be suitable for use in the invention.
• N-methyl pyrrolidinone shown below
• Suitable polar aprotic solvents may contain a ring urethane group (NHCOO-). However, not all such compounds are suitable.
• One of skill in the art may screen for compounds useful in the compositions and methods of the invention as described herein. Exemplary chemicals that may be suitable for use in the invention are set forth in Tables 2 and 3 below.
• Table 2 sets forth an exemplary list of potential chemicals for use in the compositions and methods of the invention based on their Hansen Solubility Parameters. Other compounds, may of course, also meet these requirements such as, for example, those set forth in Table 3.
• the chemicals listed in Tables 2 and 3 have been used in hybridization and/or PCR applications in the prior art (e.g., dimethyl sulfoxide (DMSO) has been used in hybridization and PCR applications, and sulfolane (SL), acetonitrile (AN), 2-pyrrolidone, ⁇ -caprolactam, and ethylene glycol have been used in PCR applications).
• the polar aprotic solvent is not DMSO, sulfolane, acetonitrile, 2- pyrrolidone, ⁇ -caprolactam, or ethylene glycol.
• most polar aprotic solvents have not been used in prior art hybridization applications.
• the prior art did not recognize that they may be advantageously used in the stringent wash step of such hybridization applications, as disclosed in this application.
• Such solutions may comprise, for example, buffering agents, accelerating agents, chelating agents, salts, detergents, and blocking agents.
• the buffering agents may include SSC, HEPES, SSPE, PPES, TMAC, TRIS, SET, citric acid, a phosphate buffer, such as, e.g., potassium phosphate or sodium pyrophosphate, etc.
• the buffering agents may be present at concentrations from 0.0 Ix to 50x, such as, for example, O.Olx, O.lx, 0.5x, Ix, 2x, 5x, 10x, 15x, 2Ox, 25x, 30x, 35x, 4Ox, 45x, or 5Ox.
• the buffering agents are present at concentrations from O.lx to 10x.
• the accelerating agents may 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.
• 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
• organic solvents such as formamide, dimethylformamide, DMSO, etc.
• the accelerating agent may be present at concentrations from 1% to 80% or O.lx to 10x, such as, for example, 0.1% (or O.lx), 0.2% (or 0.2x), 0.5% (or 0.5x), 1% (or Ix), 2% (or 2x), 5% (or 5x), 10% (or 1 Ox), 15% (or 15x), 20% (or 2Ox), 25% (or 25x), 30% (or 30x), 40% (or 4Ox), 50% (or 50x), 60% (or 6Ox), 70% (or 7Ox), or 80% (or 80x).
• formamide is present at concentrations from 25% to 75%, such as 25%, 30%, 40%, 50%, 60%, 70%, or 75%
• DMSO, dextran sulfate, and glycol are present at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%, 9%, or 10%.
• the chelating agents may include EDTA, EGTA, etc.
• the chelating agents may be present at concentrations from 0.1 mM to 10 mM, such as O.lmM, 0.2mM, 0.5mM, ImM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or 1OmM.
• the chelating agents are present at concentrations from 0.5 mM to 5 mM, such as 0.5mM, ImM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, or 5mM.
• the salts may include sodium chloride, sodium phosphate, magnesium phosphate, etc.
• the salts may be present at concentrations from 1 mM to 750 mM, such as ImM, 5mM, 1OmM, 2OmM, 3OmM, 4OmM, 5OmM, 10OmM, 20OmM, 30OmM, 40OmM, 50OmM, 60OmM, 70OmM, or 75OmM.
• the salts are present at concentrations from 10 mM to 500 mM, such as 1OmM, 2OmM, 3OmM, 4OmM, 5OmM, 10OmM, 20OmM, 30OmM, 40OmM, or 50OmM.
• the detergents may include Tween, SDS, Triton, CHAPS, deoxycholic acid, etc.
• the detergent may be present at concentrations from 0.001% to 10%, such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.
• the detergents are present at concentrations from 0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.
• Another example of a typical stringent wash is: first a high stringency wash with 0.4X (or IX) SSC, 0.3% NP- 40, pH 7.0 at 73 0 C for 2 min., followed by a medium stringency wash with 2X SSC, 0.1% NP-40, pH 7.0 at room temperature for 1-10 min.
• a typical stringent wash for nucleic acids which have more than 100 complementary residues is a 0.
• stringent conditions typically involve salt concentrations of less than about 1.5 M, more typically about 0.01 to 1.0 M, at pH 7.0 to 8.3, and the temperature is typically at least about 3O 0 C.
• Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• destabilizing agents such as formamide.
• stringent washes may use 2xSSC and 50% formamide for 3x 5min from 42 to 45° C.
• stringent conditions with formamide may be optimized for different types and lengths of probes (e.g. DNA oligos; LNA; PNA) and different targets (e.g. mRNA, virus).
• compositions of the invention may comprise a wash solution comprising any of the components of traditional components recited above in combination with at least one polar aprotic solvent.
• the traditional components may be present at the same concentrations as used in traditional wash solutions, or may be present at higher or lower concentrations, or may be omitted completely.
• the salts may be present at concentrations of 0-1200 mM NaCl and/or 0-200 mM phosphate buffer.
• the concentrations of salts may be, for example, OmM, 15mM, 3OmM, 45mM, 6OmM, 75mM, 9OmM, 105mM, 12OmM, 135mM, 15OmM, 165mM, 18OmM, 195mM, 21OmM, 225mM, 24OmM 5 255mM, 27OmM, 285mM, or 300 mM NaCl and 5 mM phosphate buffer, or 600 mM NaCl and 10 mM phosphate buffer.
• the concentrations of salts may be, for example, the concentrations present in 0.1X, 0.2X, 0.3X, 0.4X, 0.5X, 0.6X, 0.7X, 0.8X, 0.9X, IX, 2X, 3X, 4X, 5X, 6X, 7X, or 8X SSC.
• compositions of the invention comprise accelerating agents such as dextran sulfate, glycol, or DMSO
• the dextran sulfate may be present at concentrations of from 5% to 40%, the glycol maybe present at concentrations of from 0.1% to 10%, and the DMSO maybe from 0.1% to 10%.
• the concentration of dextran sulfate maybe 10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, or glycerol may be 1% to 10%.
• concentration of DMSO may be 1%.
• the aqueous composition does not comprise DMSO as an accelerating agent
• the aqueous composition does not comprise formamide as an accelerating agent, or comprises formamide with the proviso that the composition contains less than 25%, or less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%, or less than 0.05%, or less than
• compositions of the invention comprise citric acid
• concentrations may range from 1 mM to 100 mM and the pH may range from 5.0 to 8.0.
• concentration of citric acid may be 10 mM and the pH may 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, they may comprise blocking agents to block binding of, e.g., repeat sequences to the target such as larger amounts of total human DNA or repeat enriched DNA or specific blocking agents such as PNA or LNA fragments and sequences. These agents maybe present at concentrations of from 0.01-100 ⁇ g/ ⁇ L or 0.01-100 ⁇ M.
• these agents will be 0.1 ⁇ g/ ⁇ L total human DNA, or 0.1 ⁇ g/ ⁇ L non-human DNA, such as herring sperm, sahnon sperm, or calf thymus DNA, or 5 ⁇ M blocking PNA.
• compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary double-stranded nucleotide sequences.
• One way to test for whether the amount of polar aprotic solvent is effective to denature non-complementary sequences in a hybridization product is to determine whether the polar aprotic solvent, when used in the methods and compositions described herein, yields a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay.
• Non-limiting examples of effective amounts of polar aprotic solvents include, e.g., about 1% to about 95% (v/v). In some embodiments, the concentration of polar aprotic solvent is 5% to 60% (v/v). In other embodiments, the concentration of polar aprotic solvent is 10% to 60% (v/v). hi still other embodiments, the concentration of polar aprotic solvent is 30% to 50% (v/v).
• 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) are also suitable, hi 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).
• 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).
• a composition of the invention comprises a mixture of 20% polar aprotic solvent (v/v) (e.g., ethylene carbonate, "EC") and 2X SSC at pH 7.0.
• v/v polar aprotic solvent
• Another exemplary composition of the present invention comprises a mixture of 50% EC and 2X SSC at pH 7.0.
• polar aprotic solvents may impart different properties on the compositions of the invention.
• the choice of polar aprotic solvent may contribute to the stability of the composition, since certain polar aprotic solvents may degrade over time.
• the polar aprotic solvent ethylene carbonate breaks down 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 of the invention.
• ethylene glycol which is a relatively stable molecule
• carbon dioxide which can interact with water to form carbonic acid, altering the acidity of the compositions of the invention.
• stability can be improved by reducing the pH of the composition, by adding citric acid as a buffer at pH 6.2 instead of the traditional phosphate buffer, which is typically used at about pH 7.4, and/or by adding ethylene glycol at concentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%, etc.
• citric acid as a buffer at pH 6.2 instead of the traditional phosphate buffer, which is typically used at about pH 7.4
• ethylene glycol at concentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%, etc.
• concentrations e.g., between 0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%, etc.
• the compositions of the invention are stable at 2-8 0 C for approximately 8 months. Stability can also be improved if the compositions are stored at low temperatures (e.g
• certain polar aprotic solvents may cause the compositions of the invention to separate into multi-phase systems under certain conditions.
• the conditions under which multi-phase systems are obtained may be different for different polar aprotic solvents.
• compositions comprising low concentrations ethylene carbonate may exist as one phase, while compositions comprising higher concentrations of ethylene carbonate may separate into two, or even three phases.
• compositions comprising 15% ethylene carbonate exist as a single phase at room temperature
• 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.
• compositions comprising greater than 20% EC for example 40% or 50% EC, and 2X SSC may be present as a single phase at room temperature.
• polar aprotic solvents may exist in two phases at room temperature even at low concentrations.
• sulfolane, ⁇ -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.
• compositions of the invention may also be possible to alter the number of phases by adjusting the temperature of the compositions of the invention. Generally, as temperature increases, the number of phases decreases. For example, at 2-8 0 C, compositions comprising 40% ethylene carbonate may separate into a three-phase system.
• Washes may be performed with a one-phase composition of the invention, with individual phases of the multiphase compositions of the invention, or with mixtures of any one or more of the phases in a multiphase composition of the invention.
• compositions of the invention can be varied in order to optimize results for a particular application.
• concentration of polar aprotic solvent, salt, accelerating agent, blocking agent, and/or hydrogen ions may be varied in order to improve results for a particular application.
• concentration of polar aprotic solvent may be varied in order to improve signal intensity and background staining.
• the methods and compositions of the invention may be used fully or partly in all types of hybridization applications in the fields of cytology, histology, or molecular biology.
• the first or the second nucleic acid sequence in the methods of the invention is present in a biological sample.
• samples include, e.g., tissue samples, cell preparations, cell fragment preparations, and isolated or enriched cell component preparations.
• the sample may originate from various tissues such as, e.g., breast, lung, colorectal, prostate, lung, head & neck, stomach, pancreas, esophagus, liver, and bladder, or other relevant tissues and neoplasia thereof, any cell suspension, blood sample, fine needle aspiration, ascites fluid, sputum, peritoneum wash, lung wash, urine, feces, cell scrape, cell smear, cytospin or cytoprep cells.
• tissues such as, e.g., breast, lung, colorectal, prostate, lung, head & neck, stomach, pancreas, esophagus, liver, and bladder, or other relevant tissues and neoplasia thereof, any cell suspension, blood sample, fine needle aspiration, ascites fluid, sputum, peritoneum wash, lung wash, urine, feces, cell scrape, cell smear, cytospin or cytoprep cells.
• the sample may be isolated and processed using standard protocols.
• Cell fragment preparations may, e.g., be obtained by cell homogenizing, freeze-thaw treatment or cell lysing.
• the isolated sample may be treated in many different ways depending of the purpose of obtaining the sample and depending on the routine at the site. Often the sample is treated with various reagents to preserve the tissue for later sample analysis, alternatively the sample may be analyzed directly. Examples of widely used methods for preserving samples are formalin-fixed followed by paraffin-embedding and cryo- preservation.
• cell cultures are generally treated with colcemid, or anther suitable spindle pole disrupting agent, to stop the cell cycle in metaphase.
• the cells are then fixed and spotted onto microscope slides, treated with formaldehyde, washed, and dehydrated in ethanol. Probes are then added and the samples are analyzed by any of the techniques discussed below.
• Cytology involves the examination of individual cells and/or chromosome spreads from a biological sample. Cytological examination of a sample begins with obtaining a specimen of cells, which can typically be done by scraping, swabbing or brushing an area, as in the case of cervical specimens, or by collecting body fluids, such as those obtained from the chest cavity, bladder, or spinal column, or by fine needle aspiration or fine needle biopsy, as in the case of internal tumors, hi a conventional manual cytological preparation, the sample is transferred to a liquid suspending material and the cells in the fluid are then transferred directly or by centrifugation-based processing steps onto a glass microscope slide for viewing.
• 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 placed in contact with a microscope slide.
• the cells are then fixed on the microscope slide before analysis by any of the techniques discussed below.
• slides containing the specimen are immersed in a formaldehyde buffer, washed, and then dehydrated in ethanol.
• the probes are then added and the specimen is covered with a coverslip.
• the slide is optionally incubated at a temperature sufficient to denature any double-stranded nucleic acid in the specimen (e.g., 5 minutes at 82 0 C) and then incubated at a temperature sufficient to allow hybridization (e.g., overnight at 45 0 C).
• the coverslips are removed and the specimens are subjected to a high- stringency wash (e.g., 10 minutes at 65 0 C) followed by a series of low-stringency washes (e.g., 2 x 3 minutes at room temperature). The samples are then dehydrated and mounted for analysis.
• a high- stringency wash e.g., 10 minutes at 65 0 C
• a series of low-stringency washes e.g., 2 x 3 minutes at room temperature.
• RNA hybridization experiments using cytological samples, cells are equilibrated in 40% formamide, Ix SSC, and 10 mM sodium phosphate for 5 min, incubated at 37° C overnight in hybridization reactions containing 20 ng of oligonucleotide probe (e.g mix of labeled 50 bp oligos), IxSSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20 mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. coli tRNA (10 mg/ml), and 10 mM sodium phosphate.
• oligonucleotide probe e.g mix of labeled 50 bp oligos
• IxSSC 40% formamide
• 10% dextran sulfate e.g mix of labeled 50 bp oligos
• BSA 20 mM ribonucleotide vanadyl complex
• Digoxigenin-labeled probes can then e.g. be detected by using a monoclonal antibody to digoxigenin conjugated to Cy3. Biotin-labeled probes can then e.g. be detected by using streptavidin-Cy5. Detection can be by fluorescence or CISH.
• Histology involves the examination of cells in thin slices of tissue.
• a tissue sample for histological examination, pieces of the tissue are fixed in a suitable fixative, typically an aldehyde such as formaldehyde or glutaraldehyde, and then embedded in melted paraffin wax.
• the wax block containing the tissue sample is then cut on a microtome to yield thin slices of paraffin containing the tissue, typically from 2 to 10 microns thick.
• the specimen slice is then applied to a microscope slide, air dried, and heated to cause the specimen to adhere to the glass slide. Residual paraffin is then dissolved with a suitable solvent, typically xylene, toluene, or others.
• a suitable solvent typically xylene, toluene, or others.
• slices may be prepared from frozen specimens, fixed briefly in 10% formalin or other suitable fixative, and then infused with dehydrating reagent prior to analysis of the sample.
• the slides are washed (e.g., 2 x 3 minutes), dehydrated, and probe is applied.
• the specimens are covered with a coverslip and the slide is optionally incubated at a temperature sufficient to denature any double-stranded nucleic acid in the specimen (e.g. 5 minutes at 82 0 C), followed by incubation at a temperature sufficient to allow hybridization (e.g., overnight at 45 0 C).
• the coverslips are removed and the specimens are subjected to a high-stringency wash (e.g., 10 minutes at 65 0 C) followed by a series of low-stringency washes (e.g., 2 x 3 minutes at room temperature).
• the samples are then dehydrated and mounted for analysis.
• slides with FFPE tissue sections are deparaffmized in xylene for 2 x 5 min, immerged in 99% ethanol 2 x 3 min, in 96% ethanol 2 x 3 min, and then in pure water for 3 min.
• Slides are placed in a humidity chamber, Proteinase K is added, and slides are incubated at RT for 5 min- 15 min. Slides are immersed in pure water for 2 x 3 min, immersed in 96% ethanol for 10 sec, and air-dried for 5 min. Probes are added to the tissue section and covered with coverslip. The slides are incubated at 55° C in humidity chamber for 90 min.
• the slides are immersed in a Stringent Wash solution at 55 0 C for 25 min, and then immersed in TBS for 10 sec.
• the slides are incubated in a humidity chamber with antibody for 30 min.
• the slides are immersed in TBS for 2 x 3 min, then in pure water for 2 x 1 min, and then placed in a humidity chamber.
• the slides are then incubated with substrate for 60 min, and immersed in tap water for 5 min.
• RNA target sample is denatured for 10 minutes at 65 0 C in RNA loading buffer and immediately placed on ice.
• the gels are loaded and electrophoresed with Ix MOPS buffer (10X MOPS contains 20OmM morpholinopropansulfonic acid, 5OmM sodium acetate, 1OmM EDTA, pH 7.0) at 25 V overnight.
• the gel is then pre-equilibrated in 2Ox SSC for 10 min and the RNA is transferred to a nylon membrane using sterile 2Ox SSC as transfer buffer.
• the nucleic acids are then fixed on the membrane using, for example, UV-cross linking at 120 mJ or baking for 30 min at 12O 0 C.
• the membrane is then washed in water and air dried.
• the membrane is placed in a sealable plastic bag and prehybridized without probe for 30 min at 68 0 C.
• the probe is denatured for 5 min at 100 0 C and immediately placed on ice.
• Hybridization buffer prewarmed to 68 0 C
• the membrane is then removed from the bag and washed twice for 5 min each with shaking in a low stringency wash buffer (e.g., 2x SSC, 0.1% SDS) at room temperature.
• the membrane is then washed twice for 15 min each in prewarmed high stringency wash buffer (e.g., O.lx SSC, 0.1% SDS) at 68 0 C.
• the membrane may then be stored or immediately developed for detection.
• compositions and methods of the present invention can be used fully or partly in all types of nucleic acid hybridization techniques known in the art for cytological and histological samples.
• Such techniques include, for example, in situ hybridization (ISH), fluorescent in situ hybridization (FISH; including multi-color FISH, Fiber-FISH, etc.), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH), comparative genome hybridization (CGH), chromosome paints, and arrays in situ.
• DNA probes may be present at concentrations of 0.1 to 100 ng/ ⁇ L. For example, in some embodiments, the probes may be present at concentrations of 1 to 10 ng/ ⁇ L.
• PNA probes may be present at concentrations of 0.5 to 5000 nM. For example, in some embodiments, the probes may be present at concentrations of 5 to 1000 nM.
• probes that are suitable for use in the methods of the invention are described, e.g., in U.S. Patent Publication No. 2005/0266459, which is incorporated herein by reference.
• probes may be prepared by chemical synthesis, PCR, or by amplifying a specific DNA sequence by cloning, inserting the DNA into a vector, and amplifying the vector an insert in appropriate host cells.
• Commonly used vectors include bacterial plasmids, cosmids, bacterial artificial chromosomes (BACs), PI diverted artificial chromosomes (PACs), or yeast artificial chromosomes (YACs).
• 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, e.g., as disclosed in PCT/US02/30573.
• the nucleic acid probe may be a double or single stranded nucleic acid fragment or sequence, such as a DNA, RNA, or analogs such as PNA or LNA.
• the probes may be labeled to make identification of the probe-target hybrid possible by use, for example, of a fluorescence or bright field microscope/scanner, hi some embodiments, the probe may be labeled using radioactive labels such as 31 P, 33 P, or 32 S, non-radioactive labels such as digoxigenin and biotin, or fluorescent labels
• the type of probe determines the type of feature one may detect in a hybridization assay.
• total nuclear or genomic DNA probes can be used as a species-specific probe.
• Chromosome paints are collections of DNA sequences derived from a single chromosome type and can identify that specific chromosome type in metaphase and interphase nuclei, count the number of a certain chromosome, show translocations, or identify extra-chromosomal fragments of chromatin.
• Different chromosomal types also have unique repeated sequences that may be targeted for probe hybridization, to detect and count specific chromosomes. Large insert probes may be used to target unique single-copy sequences. With these large probes, the hybridization efficiency is inversely proportional to the probe size.
• Smaller probes can also be used to detect aberrations such as deletions, amplifications, inversions, duplications, and aneuploidy.
• aberrations such as deletions, amplifications, inversions, duplications, and aneuploidy.
• differently-colored locus-specific probes can be used to detect translocations via split-signal in situ hybridization.
• 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 probe length increases. Probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique 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) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences.
• At least one set of the in situ hybridization probes may comprise one or more PNA probes, as defined above and as described in U.S. Patent No. 7,105,294, which is incorporated herein by reference. Methods for synthesizing PNA probes are described in PCT/US02/30573.
• at least one set of the hybridization probes in any of the techniques discussed above may comprise one or more locked nucleic acid (LNA) probes, as described in WO 99/14226, which is incorporated herein by reference. Due to the additional bridging bond between the 2' and 4' carbons, the LNA backbone is pre-organized for hybridization.
• LNA locked nucleic acid
• LNA/DNA and LNA/RNA interactions are stronger than the corresponding DNA/DNA and DNA/RNA interactions, as indicated by a higher melting temperature.
• compositions and methods of the invention which decrease the energy required for hybridization, are particularly useful for hybridizations with LNA probes.
• the probes may comprise a detectable label (a molecule that provides an analytically identifiable signal that allows the detection of the probe-target hybrid), as described in U.S. Patent Publication No. 2005/0266459, which is incorporated herein by reference.
• the detectable label may be directly attached to a probe, or indirectly attached to a probe, e.g., by using a linker. Any labeling method known to those in the art, including enzymatic and chemical processes, can be used for labeling probes used in the methods and compositions of the invention, hi other embodiments, the probes are not labeled.
• in situ hybridization techniques such as FISH, CISH, and SISH for DNA detection
• FISH fluorescence in situ hybridization
• CISH CISH
• SISH single-stranded DNA hybridization
• Traditional methods for decreasing nonspecific probe binding include saturating the binding sites on proteins and tissue by incubating tissue with prehybridization solutions containing ficoll, bovine serum albumin (BSA), polyvinyl pyrrolidone, and nucleic acids.
• BSA bovine serum albumin
• nucleic acids polyvinyl pyrrolidone
• compositions of the invention advantageously reduce and/or eliminate the need for such blocking steps and blocking reagents.
• repetitive sequences may be suppressed according to the methods known in the art, e.g., as disclosed in PCT/US02/30573.
• Bound probes may be detected in cytological and histological samples either directly or indirectly with fluorochromes (e.g., FISH), organic chromogens (e.g., CISH), silver particles (e.g., SISH), or other metallic particles (e.g., gold-facilitated fluorescence in situ hybridization, GOLDFISH).
• fluorochromes e.g., FISH
• CISH organic chromogens
• SISH silver particles
• GOLDFISH gold-facilitated fluorescence in situ hybridization
• Hybridization assays on cytological and histological samples are important tools for determining the number, size, and/or location of specific DNA sequences. For example, in CGH, whole genomes are stained and compared to normal reference genomes for the detection of regions with aberrant copy number. Typically, DNA from subject tissue and from normal control tissue is labeled with different colored probes. The pools of DNA are mixed and added to a metaphase spread of normal chromosomes (or to a microarray chip, for array- or matrix-CGH). The ratios of colors are then compared to identify regions with aberrant copy number.
• FISH is typically used when multiple color imaging is required and/or when the protocol calls for quantification of signals.
• the technique generally entails preparing a cytological sample, labeling probes, denaturing target chromosomes and the probe, hybridizing the probe to the target sequence, and detecting the signal.
• 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 whole genomes down to several kilobases can be studied using FISH. With enhanced fluorescence microscope techniques, such as, for example, deconvolution, even a single mRNA molecule can be detected.
• FISH may also be used on metaphase spreads and interphase nuclei.
• FISH has been used successfully for mapping repetitive and single-copy DNA sequences on metaphase chromosomes, interphase nuclei, chromatin fibers, and naked DNA molecules, and for chromosome identification and karyotype analysis through the localization of large repeated families, typically the ribosomal DNAs and major tandem array families.
• One of the most important applications for FISH has been in detecting single-copy DNA sequences, in particular disease related genes in humans and other eukaryotic model species, and the detection of infections agents.
• FISH may be used to detect, e.g., chromosomal aneuploidy in prenatal diagnoses, hematological 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 viral integration sites in chromosomes; etc.
• each chromosome is stained with a separate color, enabling one to determine the normal chromosomes from which abnormal chromosomes are derived.
• Such techniques include multiplex FISH (m-FISH), spectral karyotyping (SKY), combined binary ration labeling (COBRA), color-changing karyotyping, cross-species color banding, high resolution multicolor banding, telomeric multiplex FISH (TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.
• CISH and SISH maybe used for many of the same applications as FISH, and have the additional advantage of allowing for analysis of the underlying tissue morphology, for example in histopathology applications.
• the hybridization mixture may contain sets of distinct and balanced pairs of probes, as described in U.S. Patent No. 6,730,474, which is incorporated herein by reference.
• the hybridization mixture may contain at least one set of probes configured for detection with one or more conventional organic chromogens
• SISH the hybridization mixture may contain at least one set of probes configured for detection with silver particles, as described in Powell RD et al., "Metallographic in situ hybridization," Hum. Pathol., 38:1145-59 (2007).
• compositions of the invention may also be used fully or partly in all types of molecular biology techniques involving hybridization, including blotting and probing ⁇ e.g., Southern, northern, etc.), and arrays.
• the method of the present invention involves the use of polar aprotic solvents in a stringent wash buffer for hybridization applications.
• the compositions of the present invention are particularly useful in said method.
• the stringent wash compositions of the invention may be used in any of the traditional hybridization protocols known in the art.
• the heat pre-treatment, digestion, denaturation, hybridization, washes, and mounting steps may use the same conditions in terms of volumes, temperatures, reagents (aside from the stringent wash buffer), and incubation times as for traditional hybridization applications.
• the denaturation temperature may vary from 60 to 100 0 C and the hybridization temperature may vary from 20 to 6O 0 C.
• the denaturation temperature may vary from 60 to 7O 0 C, 70 to 8O 0 C, 80 to 9O 0 C or 90 to 100 0 C
• the hybridization temperature may vary from 20 to 3O 0 C, 30 to 4O 0 C, 40 to 50 0 C, or 50 to 6O 0 C.
• the denaturation temperature is 72, 82, or 92 0 C
• the hybridization temperature is 37, 40, 45, or 5O 0 C.
• the denaturation time may vary from 0 to 10 minutes and the hybridization time may vary from 0 minutes to 24 hours, hi other embodiments, the denaturation time may be from 0 to 5 minutes and the hybridization time may be from 0 minute to 8 hours, hi other embodiments, 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. It will be understood by those skilled in the art that in some cases, e.g., RNA detection, a denaturation step is not required.
• assays using the compositions of the invention can be changed and optimized from traditional methodologies, for example, by decreasing the stringent wash time and/or decreasing the stringent wash temperatures.
• the hybridization product will generally comprise complementary base pairing and non-complementary base pairing between the probe and the target nucleic acid. Any non-complementary base pairing is then removed by a series of post- hybridization washes. Four variables are typically adjusted to influence the stringency of the post-hybridization washes:
• Salt conditions as salt concentration decreases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
• Formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).
• Stringent wash methods using the compositions of the invention may involve applying the compositions to a hybridization product comprising a target nucleic acid sequence hybridized to a probe.
• the polar aprotic solvent interacts with the hybridization product and facilitates the denaturation of the mismatched (i.e., non-complementary) sequences.
• the polar aprotic solvents specified in the present invention speed up this process, reduce the temperature required for the stringency wash, and reduce the harshness and toxicity of the stringency wash conditions compared to formamide-containing buffers.
• the concentration of probe may be varied in order to produce strong signals and/or reduce background. For example, reducing probe concentration reduces background. However, reducing the probe concentration is inversely related to the hybridization time, i.e., the lower the concentration, the higher hybridization time required. Background levels can also be reduced by adding agents that reduce non-specific binding, such as to the cell membrane, such as small amounts of total human DNA or non-human-origin
• compositions of the invention often allow for better signal-to- noise ratios than traditional stringent wash compositions.
• compositions and methods of the invention solve many of the problems associated with traditional hybridization compositions and methods.
• the reagents used in the following examples are from Dako's Histology FISH Accessory Kit (K5599) and Cytology FISH Accessory Kit (K5499) (Dako Denmark A/S, Glostrup Denmark).
• the kits contain all the key reagents, except for probe, required to complete a FISH procedure for formalin-fixed, paraffin-embedded tissue section specimens. All samples were prepared according to the manufacturer's description.
• the Dako Hybridizer (S2451, Dako) was used for the digestion, denaturation, and hybridization steps.
• FISH slides were performed within a week after hybridization using a Leica DM6000B fluorescence microscope, equipped with DAPI, FITC, Texas Red single filters and FITC/Texas Red double filter under 1Ox, 2Ox, 4Ox, and 10Ox oil objective.
• extract sulfate refers to the sodium salt of dextran sulfate (D8906, Sigma) having a molecular weight M w > 500,000. All concentrations of polar aprotic solvents are provided as v/v percentages.
• Phosphate buffer refers to a phosphate buffered solution containing NaH 2 PO 4 ,, 2H 2 O (sodium phosphate dibasic dihydrate) and Na 2 HPO 4 , H 2 O (sodium phosphate monobasic monohydrate).
• Citrate buffer refers to a citrate buffered solution containing sodium citrate (Na 3 C 6 H 5 O 7 , 2H 2 O; 1.06448, Merck) and citric acid monohydrate (C 6 H 8 O 7 , H 2 O; 1.00244, Merck).
• FFPE formalin- fixed paraffin embedded
• the samples were then washed by Stringency Wash at 65 0 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 ⁇ L Antifade Mounting Medium. When the staining was completed, observers trained to assess signal intensity, morphology, and background of the stained slides performed the scoring.
• the signal intensities were evaluated on a 0-3 scale with 0 meaning no signal and 3 equating to a strong signal.
• the cell/tissue structures are evaluated on a 0-3 scale with 0 meaning no structure and no nuclei boundaries and 3 equating to intact structure and clear nuclei boundaries. Between 0 and 3 there are additional grades 0.5 apart from which the observer can assess signal intensity, tissue structure, and background.
• the signal intensity is scored after a graded system on a 0-3 scale.
• the scoring system allows the use of 1 A grades.
• tissue and nuclear structure is scored after a graded system on a 0-3 scale.
• tissue structures and/or nuclear borders are poor. This grade includes situations where some areas have empty nuclei.
• the scoring system allows the use of 1 A grades.
• the background is scored after a graded system on a 0-3 scale.
• This example compares the signal intensity and cell morphology from samples treated with the compositions of the invention or traditional hybridization solutions as a function of denaturation temperature.
• FISH Probe composition I 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide (15515-026, Invitrogen), 5 ⁇ M blocking PNAs ⁇ see Kirsten Vang Nielsen et al., PNA Suppression Method Combined with Fluorescence In Situ Hybridisation (FISH) Technique inPRINS and PNA Technologies in Chromosomal Investigation, Chapter 10 (Franck Pellestor ed.) (Nova Science Publishers, Inc. 2006)), 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe (RPl 1-1143E20, size 192 kb).
• FISH Fluorescence In Situ Hybridisation
• FISH Probe composition II 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate (03519, Fluka), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe (RPl 1-1143E20, size 192 kb).
• This example compares the signal intensity and background staining from samples treated with the compositions of the invention or traditional hybridization solutions as a function of hybridization time.
• FISH Probe composition I 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• FISH Probe composition II 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• the 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.
• This example compares the signal intensity from samples treated with the compositions of the invention having different polar aprotic solvents or traditional hybridization solutions.
• FISH Probe composition I 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% formamide, 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• FISH Probe composition II 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate (EC), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• FISH Probe composition III 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Propylene carbonate (PC) (540013, Aldrich), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• PC Propylene carbonate
• FISH Probe composition IV 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Sulfolane (SL) (T22209, Aldrich), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• SL Sulfolane
• FISH Probe composition V 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Aceto nitrile (AN) (C02CIIX, Lab-Scan), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCND 1 gene DNA probe.
• FISH Probe composition VI 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ⁇ -butyrolactone (GBL) (B103608, Aldrich), 5 ⁇ M blocking PNAs, 7,5 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe. Phases of different viscosity, if present, were mixed before use. The FISH probes were incubated at 82°C for 5 min and then at 45°C for 60 minutes.
• GBL ⁇ -butyrolactone
• This example compares the signal intensity from samples treated with the compositions of the invention having different concentrations of polar aprotic solvent.
• FISH Probe Compositions 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10-60% Ethylene carbonate (as indicated), 5 ⁇ M blocking PNAs, 7.5 ng/ ⁇ L Texas Red labeled /GiiT-constant DNA gene probe ((CTD-3050E15, RPl 1-1083E8; size 227 kb) and 7.5 ng/ ⁇ L FITC labeled /G ⁇ -variable gene DNA probe (CTD-2575M21, RPl 1-122B6, RP11-316G9; size 350 and 429 kb).
• the FISH probes were incubated at 82°C for 5 min and then at 45 0 C for 60 minutes.
• This example compares the signal intensity and background intensity from samples treated with the compositions with and without PNA blocking.
• FISH Probe Compositions 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 7.5 ng/ ⁇ L Texas Red labeled CCND 1 gene DNA probe.
• the FISH probes were incubated at 82°C for 5 min and then at 45°C for 60 minutes.
• This example compares the signal intensity from samples treated with the compositions of the invention as a function of probe concentration and hybridization time.
• FISH Probe Compositions 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, and 10, 7.5, 5 or 2.5 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe (as indicated).
• This example compares the signal intensity from samples treated with the compositions of the invention as a function of salt, phosphate, and buffer concentrations.
• FISH Probe Compositions 10% dextran sulfate, ([NaCl], [phosphate buffer], [TRIS buffer] as indicated in Results), 40% Ethylene carbonate, 7.5 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• the FISH probes were incubated at 82°C for 5 min and then at 45 0 C for 60 minutes.
• This example compares the signal intensity from samples treated with the compositions of the invention as a function of dextran sulfate concentration.
• FISH Probe Compositions 0, 1, 2, 5, or 10% dextran sulfate (as indicated), 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 5 ng/ ⁇ L Texas Red labeled SIL- TALl gene DNA probe (RPl -278013; size 67 kb) and 6 ng/ ⁇ L FITC SIL-TALl (ICRFcI 12-112C1794, RP11-184J23, RP11-8J9, CTD-2007B18, 133B9; size 560 kb).
• This example compares the signal intensity from samples treated with the compositions of the invention as a function of dextran sulfate, salt, phosphate, and polar aprotic solvent concentrations.
• FISH Probe Composition Ia 34% dextran sulfate, 0 niM NaCl, 0 mM phosphate buffer, 0% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition Ib 34% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 0% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition Ic 34% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 0% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition Ha 32% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 5% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition lib 32% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 5% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition Hc 32% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 5% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition Ilia 30% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 10% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition HIb 30% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 10% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition IIIc 30% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 10% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition IVa 28% dextran sulfate, 0 mM NaCl, 0 mM phosphate buffer, 15% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition IVb 28% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 15% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Composition IVc 28% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 15% ethylene carbonate, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe (size 218 kb) and 50 nM of FITC-labeled CEN-7 PNA probe.
• FISH Probe Reference V Standard sales vial of HER2 PharmDx probe mix (K5331, Dako) containing blocking PNA. Overnight hybridization for 20 hours.
• FISH probes were incubated at 82°C for 5 min and then at 45°C for 60 minutes with no blocking, except for FISH Probe Reference V, which had PNA blocking and was hybridized for 20 hours.
• composition IVa gave strong DNA signals with no salt. This is not possible with standard FISH compositions, where DNA binding is salt dependent.
• This example compares the signal intensity from samples treated with the compositions of the invention as a function of polar aprotic solvent and dextran sulfate concentration under high salt (4x normal) conditions.
• FISH Probe Composition I 0% ethylene carbonate, 29% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
• FISH Probe Composition II 5% ethylene carbonate, 27% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
• FISH Probe Composition III 10% ethylene carbonate, 25% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition was a single phase.
• FISH Probe Composition IV (not tested): 20% ethylene carbonate, 21% dextran sulfate, 1200 mM NaCl, 20 mM phosphate buffer, 10 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe and 50 nM of FITC-labeled CEN-7 PNA probe. Composition had two phases.
• composition II gave good DNA signals with only 5% EC and strong DNA signals with 10% EC.
• This example compares the signal intensity and background from samples treated with different phases of the compositions of the invention.
• FISH Probe Composition 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% Ethylene carbonate, 8 ng/ ⁇ L Texas Red labeled HER2 gene DNA probe and 600 nM FITC-labeled CEN-17 PNA probe.
• the FISH probes were incubated at 82 0 C for 5 min and then at 45 0 C for 60 minutes. No blocking.
• This example is similar to the previous example, but uses a different DNA probe and GBL instead of EC.
• FISH Probe Composition 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% GBL, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe and 600 nM FITC- labeled CEN- 17 PNA probe.
• the FISH probes were incubated at 82°C for 5 min and then at 45°C for 60 minutes. No blocking.
• This example examines the number of phases in the compositions of the invention as a function of polar aprotic solvent and dextran sulfate concentration.
• FISH Probe Compositions 10 or 20% dextran sulfate; 300 niM NaCl; 5 mM phosphate buffer; 0, 5, 10, 15, 20, 25, 30% EC; 10 ng/ ⁇ L probe.
• This example compares the signal intensity and background from samples treated with different compositions of the invention as a function of probe concentration and hybridization time.
• FISH Probe Composition I 10 ng/ ⁇ L HER2 TxRed labeled DNA probe (standard concentration) and standard concentration of CEN7 FITC labeled PNA probe (50 nM); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.
• FISH Probe Composition II 5 ng/ ⁇ L HER2 TxRed labeled DNA probe (1/2 of standard concentration) and standard concentration (50 nM) of FITC labeled CEN7 PNA probes; 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer.
• FISH Probe Composition III 2.5 ng/ ⁇ L HER2 TxRed labeled DNA probe (1/4 of standard concentration) and 1 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-i ⁇ existed as a single phase.
• the FISH probes were incubated at 82 0 C for 5 min and then at 45°C for 3 hours, 2 hours and 1 hours.
• DNA PNA B.G. DNA PNA B.G.
• DNA PNA B.G. DNA PNA B.G.
• This example compares the signal intensity and background from samples treated with the compositions of the invention as a function of blocking agent.
• FISH Probe Compositions 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; 2.5 ng/ ⁇ L HER2 TxRed labeled DNA probe (1/4 of standard concentration) and 1 A of the standard concentration (300 nM) FITC labeled CEN17 PNA probe. Samples were blocked with: (a) nothing; (b) 0.1 ⁇ g/ ⁇ L COTl (15279-011, Invitrogen); (c) 0.3 ⁇ g/ ⁇ L COTl ; or (d) 0.1 ⁇ g/ ⁇ L total human DNA before hybridization using the compositions of the invention.
• FISH probes were incubated at 82 0 C for 5 min and then at 45°C for 60 minutes.
• AU compositions contained 15% EC, 20% dextran sulfate, 600 mM NaCl, 10 mM phosphate buffer, 2.5 ng/ ⁇ L HER2 DNA probes (1/4 of standard concentration), 300 nM CENl 7 PNA probe (1/2 of standard concentration), and one of the following background- reducing agents:
• THD total human DNA
• This experiment compares the signal intensity from the upper and lower phases using two different polar aprotic solvents.
• FISH Probe Composition I 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% ethylene trithiocarbonate (ET) (E27750, Aldrich), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• E27750 ethylene trithiocarbonate
• E27750 Aldrich
• 5 ⁇ M blocking PNAs 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• FISH Probe Composition II 10% dextran sulfate, 300 mM NaCl, 5 mM phosphate buffer, 40% glycol sulfite (GS) (G7208, Aldrich), 5 ⁇ M blocking PNAs, 10 ng/ ⁇ L Texas Red labeled CCNDl gene DNA probe.
• GS glycol sulfite
• the FISH probes were incubated at 82°C for 5 min and then at 45 0 C for 60 minutes. Results:
• This experiment examines the ability of various polar aprotic solvents to form a one- phase system.
• 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:
• Example 19 This experiment examines the use of the compositions of the invention in chromogenic in situ hybridization (CISH) analysis on multi FFPE tissue sections.
• FISH Probe Composition I 4.5 ng/ ⁇ L TCRAD FITC labelled gene DNA probe (1/4 of standard concentration) (RP 11 -654A2, RP 11 -246 A2, CTP-2355L21 , RP 11 - 158G6, RPl 1-780M2, RPl 1-481C14; size 1018 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
• FISH Probe Composition II 4.5 ng/ ⁇ L TCRAD FITC labelled gene DNA probe (1/4 of standard concentration) (size 1018 kb); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon DNA sperm.
• FISH Probe Composition III 300 nM of each individual FITC labelled PNA CENl 7 probe (1/2 of standard concentration); 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
• This example compares the signal intensity and background from FFPE tissue sections treated with the compositions of the invention with two DNA probes.
• FISH Probe Composition I 9 ng/ ⁇ L IGH FITC labelled gene DNA probe (RPl 1- 151B17, RPl 1-112H5, RPl 1-101G24, RPl 1-12F16, RPl 1-47P23, CTP-3087C18; size 612 kb); 6.4 ng/ ⁇ L MYC Tx Red labeled 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.
• FISH Probe Composition II 9 ng/ ⁇ L IGH FITC labelled gene DNA probe; 6.4 ng MYC TxRed labeled DNA probe; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.
• Example 21 This experiment examines the use of the compositions of the invention on cytological samples.
• FISH Probe Composition 15% EC; 20% dextran sulfate; 600 mM NaCl; 10 mM phosphate buffer; 5 ng/ ⁇ L HER2 TxRed labeled DNA probe (1/2 of standard concentration) and Vi of the standard concentration of CEN7 (25 nM).
• the FISH probes were incubated on metaphase chromosome spreads at 82 0 C for 5 minutes, then at 45 0 C for 30 minutes, all without blocking. Results:
• This example compares the signal intensity and background from DNA probes on cytology samples, metaphase spreads, with and without blocking.
• FISH Probe Composition I 6 ng/ ⁇ L TCRAD Texas Red labelled gene DNA probe (standard concentration) (CTP-31666K20, CTP-2373N7; size 301 kb) and 4.5 ng/ ⁇ L FITC labelled gene DNA probe (1/4 of standard concentration); 15% EC, 20% dextran sulfate; 600 niM NaCl; 10 mM citrate buffer, pH 6.0.
• FISH Probe Composition II 6 ng/ ⁇ L TCRAD Texas Red labelled gene DNA probe (standard concentration) (size 301 kb) and 4.5 ng/ ⁇ L FITC labelled gene DNA probe (1/4 of standard concentration); 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0; 0.1 ug/uL sheared salmon sperm DNA.
• the FISH probes were incubated on metaphase spreads at 82 0 C for 5 min, then at 45 0 C for 60 min. Results:
• This example compares the background and signal intensity as a function of the stringency wash conditions.
• Stringency wash composition II 2XSSC (pH 7.0), 50% EC (E2,625-8, Sigma- Aldrich)
• Stringency wash composition III 2XSSC (pH 7.0), 50% formamide (15515-026, Invitrogen)
• FISH Probe Composition A 2.5 ng/ ⁇ L HER2 TxRed labeled DNA probe (1/4 of standard concentration) and 1 A of the standard concentration (300 nM) of CENl 7 PNA probes; 15% EC, 20% dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.0.
• FISH Probe Composition B Standard sales vial of HER2 PharmDx probe mix (K5331, Dako) containing blocking PNA and formamide.
• This example used a standard sales vial of HER2 PharaiDx probe mix (K5331, Dako) containing 10 ng/ ⁇ L HER2 TxRed labeled DNA probe and (600 nM) of CENl 7 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mM Phosphate buffer, 5 ⁇ M unlabelled blocking PNAs.
• Stringency wash composition I 2X SSC (pH 7.0), 20% EC (E2,625-8, Sigma- Aldrich)
• Stringency wash composition II 2X SSC (pH 7.0), 50% EC (E2,625-8, Sigma- Aldrich)
• Stringency wash composition EI IX Stringency Buffer (vial 4, K5599, Dako) The samples were denatured at 82° C for 5 min. and hybridized overnight (20 hours). The stringency wash for compositions I and II was performed at 45 °C for 15 min. followed by a wash in 2X SCC at 45 0 C for 3 x 5 min. The stringent wash for composition III was performed at 65°C for 10 min. followed by a wash for 2 x 3 min. in Wash buffer (Vial 3, K5599). Results:
• This example compares the background and signal intensity as a function of the stringency wash conditions.
• This example used a standard sales vial of HER2 PharmDx probe mix (K5331, Dako) containing 10 ng/ ⁇ L HER2 TxRed labeled DNA probe and (600 nM) of CENl 7 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mM Phosphate buffer, 5 ⁇ M unlabelled blocking PNAs.
• Stringency wash composition I 2xSSC (pH 7.0), 20% SL
• the samples were denatured at 82° C for 5 min. and hybridized overnight (20 hours).
• the stringency wash for compositions I and II was performed at 45 0 C for 15 min. followed by a wash in 2X SCC at 45 °C for 3 x 5 min.
• the stringency wash for composition III was performed at 65 0 C for 10 min. followed by a wash for 2 x 3 min. in Wash buffer (Vial 3, K5599). Results:
• This example compares the background and signal intensity as a function of the stringency wash conditions.
• This example used a standard sales vial of HER2 PharmDx probe mix (K5331, Dako) containing 10 ng/ ⁇ L HER2 TxRed labeled DNA probe and (600 nM) of CENl 7 PNA probes, 45% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mM Phosphate buffer, 5 ⁇ M unlabelled blocking PNAs.
• Stringency wash composition II 0.5xSSC (pH 7.0), 20% EC
• Stringency wash composition IV Ix Stringency Buffer vial 4, K5599 (Dako)
• the samples were denatured at 82° C for 5 min. and hybridized overnight (20 hours).
• the stringency wash for compositions I-ffl was performed at 45 °C for 15 min. followed by a wash in 2X SCC at 45 0 C for 3 x 5 min, then washed Ix 3 min. in Wash buffer (Vial 3, K5599).
• the stringency wash for composition IV was performed at 65 0 C for 10 min. followed by a wash for 2 x 3 min. in Wash buffer (Vial 3, K5599).
• the buffers of the invention produce similar levels of background as traditional stringent wash buffers at 65 0 C.
• the buffers of the invention can be used to produce low background under low temperature stringency wash conditions with different salt concentrations.
• a kit for performing a hybridization application comprising:
• composition for performing a stringent wash, said composition comprising at least one polar aprotic solvent in an amount effective to denature non- complementary sequences in a hybridization product, and a hybridization solution,
• polar aprotic solvent is not dimethyl sulfoxide (DMSO).
• Embodiment 2 The kit according to embodiment 1, wherein the concentration of polar aprotic solvent in the aqueous composition is about 1% to 95% (v/v)
• Embodiment 3 The kit according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent in the aqueous composition is 5% to 10% (v/v).
• Embodiment 4 The kit according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent in the aqueous composition is 10% to 20% (v/v).
• Embodiment 5 The kit according to embodiment 1 or 2, wherein the concentration of polar aprotic solvent in the aqueous composition is 30% to 95% (v/v).
• Embodiment 6. The kit according to any one of embodiments 1 to 5, wherein the aqueous composition is non-toxic.
• Embodiment 7 The kit according to any one of embodiments 1 to 6, with the proviso that the aqueous composition does not contain formamide.
• Embodiment 8 The kit according to embodiment 6, with the proviso that the aqueous composition contains less than 25% formamide.
• Embodiment 9 The kit according to embodiment 8, with the proviso that the aqueous composition contains less than 10% formamide.
• Embodiment 10 The kit according to embodiment 9, with the proviso that the aqueous composition contains less than 2% formamide.
• Embodiment 11 The kit according to embodiment 10, with the proviso that the aqueous composition contains less than 1% formamide.
• Embodiment 12 The kit according to any of embodiments 1 to 11, wherein the polar aprotic solvent has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.
• Embodiment 13 The kit according to any one of embodiments 1 to 12, wherein the polar aprotic solvent has a dispersion solubility parameter between 17.7 to 22.0 MPa 1/2 , a polar solubility parameter between 13 to 23 MPa 1/2 , and a hydrogen bonding solubility parameter between 3 to 13 MPa 1/2 .
• Embodiment 14 The kit according to any one of embodiments 1 to 13, wherein the polar aprotic solvent has a cyclic base structure.
• Embodiment 15 The kit according to any one of embodiments 1 to 13, wherein the polar aprotic solvent is selected from the group consisting of:
• X is optional and if present, is chosen from O or S
• Z is optional and if present, is chosen from O or S
• a and B independently are O or N or S or part of the alkyldiyl or a primary amine, where R is alkyldiyl
• Y is O or S or C.
• Embodiment 16 The kit according to any one of embodiments 1 to 15, wherein the polar aprotic solvent is selected from the group consisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate, ⁇ -butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, l,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2- dinitrobenzene, 2,4-dinitro
• Embodiment 17 The kit according to any one of embodiments 1 to 15, wherein the polar aprotic solvent is selected from the group consisting of:
• Embodiment 18 The kit according to any one of embodiments 1 to 15, wherein the polar aprotic solvent is:
• Embodiment 19 The kit according to any one of embodiments 1 to 18, wherein the aqueous composition further comprises at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, and detergents.
• Embodiment 20 The kit according to embodiment 19, wherein the salts are NaCl and/or phosphate buffer and/or citrate buffer.
• Embodiment 21 The kit according to embodiment 19, wherein the salts are NaCl and/or phosphate buffer and/or SSC.
• Embodiment 22 The kit according to embodiment 20, wherein the NaCl is present at a concentration of 0 mM to 1200 mM and/or the citrate buffer is present at a concentration of O mM to lOO mM.
• Embodiment 23 The kit according to embodiment 22, wherein the NaCl is present at a concentration of 50 mM to 600 mM and/or the citrate buffer is present at a concentration of 5 mM to 50 niM.
• Embodiment 24 The kit according to embodiment 19, wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, 1,2 propanediol, and 1,3 propanediol.
• Embodiment 25 The kit according to embodiment 24, wherein the formamide is present at a concentration of 0.1-5%, the DMSO is present at a concentration of 0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, 1,2 propanediol, 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 1 mM to 100 mM.
• Embodiment 26 The kit according to any one of embodiments 1-25, wherein the aqueous composition comprises 50% of at least one polar aprotic solvent and 0.05X to 4X SSC.
• Embodiment 27 The kit according to any one of embodiments 1-25, wherein the aqueous composition comprises 20% of at least one polar aprotic solvent and 2X SSC.
• Embodiment 28 The kit according to any one of embodiments 1-25, wherein the aqueous composition comprises 15% of at least one polar aprotic solvent and 2X SSC.
• Embodiment 29 The kit according to any one of embodiments 1-28, wherein the aqueous composition comprises more than one phase at room temperature.
• Embodiment 30 The kit according to embodiment 29, wherein the aqueous composition comprises two phases at room temperature.
• Embodiment 31 The kit according to embodiment 29, wherein the aqueous composition comprises three phases at room temperature.
• Embodiment 32 A method for performing a stringent wash step in a hybridization application comprising:
• a hybridization product comprising a first nucleic acid sequence hybridized to a second nucleic acid sequence
• aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary double-stranded nucleotide sequences
• polar aprotic solvent is not dimethyl sulfoxide (DMSO).
• Embodiment 33 A method for performing a stringent wash step in a hybridization application comprising:
• a hybridization product comprising a first nucleic acid sequence hybridized to a second nucleic acid sequence
• Embodiment 34 The method according to embodiment 32, wherein the polar aprotic solvent is defined according to any of embodiments 2-5 or 12-18.
• Embodiment 35 The method according to embodiment 32 or 33, wherein a sufficient amount of energy to denature any non-complementary binding between the first and second nucleic acid sequences is provided.
• Embodiment 36 The method according to embodiment 35, wherein the energy is provided by heating the combination produced in (c).
• Embodiment 37 The method according to embodiment 36, wherein the energy is provided by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating, or heat lamps.
• Embodiment 38 The method according to embodiment 36, wherein the combination produced in (c) is heated to less than 7O 0 C, such as, for example, 65 0 C, 62°C, 6O 0 C, 55 0 C, 52 0 C, 5O 0 C, 45 0 C, 42 0 C, 4O 0 C, 35 0 C, 3O 0 C, or room temperature.
• 7O 0 C such as, for example, 65 0 C, 62°C, 6O 0 C, 55 0 C, 52 0 C, 5O 0 C, 45 0 C, 42 0 C, 4O 0 C, 35 0 C, 3O 0 C, or room temperature.
• Embodiment 39 The method according to any of embodiments 32 to 38, wherein the denaturation of any non-complementary binding between the first and second nucleic acid sequences occurs in less than 1 hour, such as, for example, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.
• Embodiment 40 The method according to any one of embodiments 32-39, wherein the first nucleic acid sequence is in a biological sample.
• Embodiment 41 The method according to embodiment 40, wherein the biological sample is a cytology or histology sample.
• Embodiment 43 The method according to any one of embodiments 32-41, wherein the aqueous composition comprises one phase at room temperature.
• Embodiment 44 The method according to any one of embodiments 32-43, wherein the aqueous composition comprises multiple phases at room temperature.
• Embodiment 45 The method according to embodiment 44, wherein the aqueous composition comprises two phases at room temperature.
• Embodiment 46 The method according to embodiment 44 or 45, wherein the phases of the aqueous composition are mixed.
• Embodiment 47 An aqueous composition for performing a stringent wash in a hybridization application, said composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary sequences in a hybridization product, and a hybridization solution, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).
• DMSO dimethyl sulfoxide
• Embodiment 48 The aqueous composition of embodiment 47, wherein the concentration of polar aprotic solvent is defined as in any one of embodiments 2 to 5.
• Embodiment 49 The aqueous composition of embodiment 47 or 48, wherein the polar aprotic solvent is defined as in any one of embodiments 12 to 18.
• Embodiment 50 The aqueous composition of any one of embodiments 47 to 49, wherein the aqueous composition is further defined as in any one of embodiments 6 to 11 or 19 to 31.
• Embodiment 51 Use of an aqueous composition comprising between 1 and 95% (v/v) of at least one polar aprotic solvent for a stringent wash step in a hybridization application.
• Embodiment 52 Use of a composition according to embodiment 51, wherein the aqueous composition is defined as in any one of embodiments 1 to 31.

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