Hybridization compositions and methods for making and using compositions

Abstract

To provide hybridization buffers for use in hybridization, for example, for use in in situ hybridization (ISH).SOLUTION: Disclosed are hybridization buffers and compositions comprising a sulfone solvent and polyvinylsulfonic acid or a salt thereof, as well as methods for making and using the same.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS No. 60 / 339,993, filed on Oct. 23, 2019, both of which are incorporated herein by reference. U.S. Provisional Application No. 62 / 838,813, filed April 25, 2019 and May 16, 2019 The filing date and benefit of priority to U.S. Provisional Application No. 62 / 848,964, filed on Assert.

[0002] The present disclosure provides methods for use in hybridization methods, e.g., in si A hybridization kit for use in the ISH method. More generally, the present disclosure relates to methods for the preparation of cytological, histological, and molecular biology buffers. Related to the field. [Background technology]

[0003] Hybridization is an important and well-known property of nucleic acids. A sequence structure is one in which, when bases are paired in a particular way (A+T or G+C), the opposite sequence It is stabilized by hydrogen bonds with the bases of the nucleotides. Thus, a nucleic acid fragment or sequence binds to a complementary nucleic acid fragment or sequence. Hybridization assays and other methods involve the use of a target nucleic acid, e.g., DNA or RNA. A nucleic acid probe designed to specifically bind to A is used. Hybridization assay, in situ hybridization (ISH), is a method The hybridization of a nucleic acid probe to a target nucleic acid in a sample, where the sample is The cells may be in vivo or may be, for example, fixed or placed on a glass slide. The probe may be labeled to provide fluorescent or bright field imaging. Use of a microscope / scanner allows for identification of probe-target hybrids. For nucleic acid targets, the probes are typically double-stranded or single-stranded nucleic acids, e.g. For example, DNA, RNA, or analogs. In some embodiments, the probe is radiolabeled. Knowledge, for example, 31 P, 33 P, 32 S, non-radioactive labels, e.g., digoxigenin and biphenylsulfonyl Such labeled probes can be labeled with thiol, ... to detect genetic abnormalities in target sequences, e.g., prenatal disorders, cancer, and Valuable information about other genetic or infectious diseases can be obtained.

[0004] The efficiency and accuracy of nucleic acid hybridization assays are primarily determined by three major factors: a) denaturation (i.e., the denaturation of, for example, two nucleic acid strands) b) renaturation (i.e., reannealing of, for example, two nucleic acid strands) conditions. and c) post-hybridization washing conditions.

[0005] Traditional hybridization methods, such as ISH assays, involve altering a double-stranded nucleic acid target. Formamide-containing solutions have been used to activate the denaturing agent. Although highly effective, formamide is a toxic and hazardous material and must be handled with care in its use and disposal. Furthermore, the use of formamide is subject to strict regulations regarding the conditions and nuclei used. Depending on the acid fragment or sequence, the amount of The time required for the denaturation step increases. For example, after the denaturation step, This is followed by a hybridization step, which can be performed, for example, by conventional fluorescent in situ hybridization. For redimination (FISH), the protocol takes 14-24 hours, up to 72 hours. It may even take.

[0006] The process by which two complementary strands of a nucleic acid are reannealed (i.e., hybridized). This step is the time consuming aspect of an assay that uses hybridization. Hinders the Watson-Crick binding sites of acids and bases, thus forming hydrogen bonds between complementary nucleobases Chaotropic agents, such as formamide, guanidinium hydrogen, and urea, which disrupt the Chaotropes have been used to lower the melting temperature (Tm) of complementary strands. The use of chaotropic agents reduces Tm, but these agents are not effective in aqueous solutions that do not contain chaotropic agents. Prolonged hybridization times compared to hybridization in solution do.

[0007] Certain existing hybridization buffers are suitable for formalin fixation and paraffin fixation. Required for performing in situ hybridization on embedded (FFPE) tissue There is a wide variability in the time required. While some of the more common precipitation buffers require overnight incubation, certain improved The compositions require only 1-2 hours of incubation (e.g., these entire US20110229975 and US20110 (See US Pat. No. 236997.) Short incubation periods are preferred, and such benefits Hybridization buffers that yield high resolution results are preferred. The bridization buffer is significantly more effective than previous teratogenic formamide compositions. A new improvement was announced by Agilent Technologies, Inc. This is a safety concern for workers using in situ hybridization techniques. The improved buffer also provides the necessary buffer for hybridization. The time required was significantly reduced to 1-2 hours.

[0008] However, faster incubation times for certain existing hybridization buffers are possible. Although the hybridization time is highly desirable and a significant improvement, The hybridization buffer has been further improved to provide a more stable and easier to use hybridization solution. There are certain characteristics that can be achieved by the buffer. Improved application, e.g. lower viscosity, higher stability, higher homogeneity, and more flexibility It exhibits reasonable storage and mixing requirements while maintaining a short 1-2 hour incubation period. There remains a need in the art for hybridization buffers that maintain .

[0009] Certain existing hybridization buffers, for example, are formed between glass slides. This indicates slow, non-strong diffusion in the capillary field created by the hybridization. This is undesirable as it can leave air spaces where the functionality of the distillation buffer is not present.

[0010] Certain existing ISH hybridization buffers are typically stored at temperatures below -18°C. At such storage temperatures, such buffers will not solidify. tend to form large precipitates and / or exhibit phase separation. Most hybridization buffers are thawed and equilibrated to 20-25°C before use. Then, extensive mixing must be performed to solubilize the precipitate and homogenize the liquid phase. Immediately after use, these hybridization buffers should be stored at temperatures below -18°C. Therefore, such hybridization buffers must be frozen. It is susceptible to damage from freezing and thawing and can only withstand about 10 freeze-thaw cycles. I can't.

[0011] Certain existing hybridization buffers also exhibit heterogeneity at low temperatures, In some cases, special attention to the temperature equilibration and mixing process is required. In some cases, a separate device, e.g., the Dako Omnis Mixing Device Prior to use, the probe product is diluted in a certain existing hybridization buffer. Must be used for melting and homogenization.

[0012] In addition, certain existing hybridization buffers exhibit instability. For example, Buffer solutions that use ethylene carbonate as a solvent are unstable due to hydrolysis of the ethylene carbonate. Ethylene carbonate hydrolysis is temperature dependent and therefore, Buffers are less desirable for use at temperatures above 66°C.

[0013] Matthieu, the entire disclosure of which is incorporated herein by reference. Sen et al., WO2009 / 144581, discloses a method for hybridizing at least one molecule to a target. The present invention provides methods and compositions for detecting hyaluronan in a human body. Eliminate the use of formamide in hybridization or reduce reliance on formamide. These compositions contain at least one polar aprotic The present invention includes an aqueous composition comprising a solvent in an amount effective to denature double-stranded nucleotide sequences.

[0014] Matthieu, the entire disclosure of which is incorporated herein by reference. WO2010 / 097707 to Sen describes probes for hybridization applications. and methods and compositions for differentially denaturing targets. The invention, for example, eliminates the use of formamide in hybridization applications, These compositions can reduce dependency on glycerol or formamide. A polar aprotic solvent is contained in an amount effective to denature the double-stranded nucleotide sequence. The present invention includes an aqueous composition comprising:

[0015] Battersby et al., U.S. Patent Application Publication No. 20110236997, discloses a hybrid The present application describes methods and reagents for the isoformization assay. The polymer and hybridization conditions have been modified to allow for shorter incubation times. It is said to be a ras. Summary of the Invention

[0016] In one embodiment, the present disclosure provides a method for preparing a soluble ... and formula (I), (Ia), (II), (IIa) or (III): [ka] [In the formula, R 1 is hydrogen or C 1~6 aliphatic, and L, if present, is a covalent bond or C 1~6 Aliphatic, R 2 is a covalent bond, if present, C 1~6 Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms, R 2 is one or two substituted with a SO3H group or its salt, except that R 2 If is a covalent bond, one Only SO3H groups or their salts are present, and R 2 -R 3 , halogen, -CN , -NO2, -OR 3 , -N(R 3 )2, and -SR 3 1 to 2 independently selected from Optionally substituted with one group, each R 3 are independently hydrogen or C 1~6 Alki n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I) and any one of the salts of sulfo The hybridization method includes the steps of: In some embodiments, a lysis buffer is provided. 1 is hydrogen. So, R 1 is C 1~6 In certain embodiments, L is a covalent bond. In one embodiment, L is C 1~6 In further embodiments, L is methylene. .

[0017] In some embodiments, the hybridization buffer of the present technology further comprises water. In some embodiments, the sulfonic acid polymer or salt thereof is polyvinyl sulfonic acid or In another embodiment, the concentration of the sulfone solvent is about 10% to about 20% (v / v). In additional embodiments, the sulfone solvent is dimethyl sulfone, diphenyl sulfone , methyl phenyl sulfone, tetramethylene sulfone (sulfolane), and mixtures thereof In another embodiment, the sulfone solvent is sulfolane.

[0018] In some embodiments, the hybridization buffer of the present technology comprises about 20% to about In another embodiment, the composition comprises a sulfonic acid polymer or a salt thereof at a concentration of 40% (w / v). The hybridization buffer of the present technology contains 20% (w / v) dextran sulfate. or less, or less than 15% (w / v), or less than 10% (w / v) nothing.

[0019] In additional embodiments, the hybridization buffer is At 25°C, the viscosity is about 60 mPa.s or less, or about 50 mPa.s or less, or In certain embodiments, the high molecular weight polymer of the present invention has a viscosity of about 40 mPa.s or less. The hybridization buffer can be stored at 2-8°C for at least 2 months or for at least 4 In another embodiment, the hybridoma is stable for 6, 8, 12, 16, 18, or 24 months. The redox buffer can be stored at 2-8°C for at least 2 months or for at least 4 It is homogenous after storage for 6, 8, 12, 16, 18 or 24 months. The hybridization buffer should be stored at 2-8°C for at least 2 months, or at least All products maintained homogeneity after storage for 4, 6, 8, 12, 16, 18 or 24 months. However, after centrifugation of the hybridization buffer containing erioglaucine, the hybridization The transparency of the lysis buffer is determined by visual assessment.

[0020] In some embodiments, the hybridization buffer of the present technology comprises a buffering agent, salt, At least one selected from the group consisting of enhancers, chelating agents, detergents, screening agents, and combinations thereof. In certain embodiments, the hybridization product further comprises at least one additional component. The ionization buffer contains NaCl and 3-(N-morpholino)propanesulfonic acid (MOP S).

[0021] In other embodiments, the hybridization buffer contains less than about 20% (w / v). The composition further comprises enhancers, salts, and buffers at a concentration of about 10% (w / v) or less, and the composition further comprises a sulfone. The solvent is sulfolane at a concentration of about 10% (v / v), and the sulfonic acid polymer or its salt is In some embodiments, the hybridization technique of the present invention is carried out at a concentration of about 30% (w / v). The elution buffer further comprises at least one nucleic acid probe.

[0022] In another aspect, the present disclosure provides a method for hybridizing nucleic acid sequences, comprising: At least for a period of time sufficient to allow the nucleic acid probes to hybridize to the target nucleic acid sequence. applying at least one nucleic acid probe and a hybridization buffer to the target nucleic acid sequence; The hybridization buffer is at a concentration of about 5 to about 30% (v / v). with a sulfone solvent of formula (I), (Ia), (II), (IIa) or (III): [ka] [In the formula, R 1 is hydrogen or C 1~6 aliphatic, and L, if present, is a covalent bond or C 1~6 Aliphatic, R 2 is a covalent bond, if present, C 1~6 Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms, R 2 is one or two substituted with a SO3H group or its salt, except that R 2 If is a covalent bond, one Only SO3H groups or their salts are present, and R 2 -R 3 , halogen, -CN , -NO2, -OR 3 , -N(R 3 )2, and -SR 3 1 to 2 independently selected from Optionally substituted with one group, each R 3 are independently hydrogen or C 1~6 Alki n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I), and the sulfonic acid polymer or its salt is about 5 to about The method further comprises the steps of:

[0023] In some embodiments of the present technology, the target nucleic acid sequence is present in an in situ biological sample. In another embodiment, the current art method comprises subjecting the target nucleic acid sequence to a temperature of about 65° C. or higher. In another embodiment, the current art method comprises denaturing the target nucleic acid sequence for about 8 minutes. In another embodiment, the method of the current art comprises denaturing the target The nucleic acid sequence is incubated at a temperature of about 60° C. to about 95° C., for example, at about 60° C. to about 70° C., or at about 70° C. ℃ to about 80 ℃, or about 80 ℃ to about 90 ℃, or about 90 ℃ to about 95 ℃, or these endpoints In another embodiment, the denaturation of the hybridoma of the present invention is carried out at any temperature between 100° C. and 150° C. The hybridization buffer has a denaturation temperature of about 72°C, about 82°C, or about 92°C. In certain embodiments, at least one nucleic acid probe The period of time sufficient for the hybridization of the nucleic acid to the target nucleic acid sequence is 4 hours or less. In the method, hybridization of at least one nucleic acid probe to a target nucleic acid is performed at 50° C. It is carried out at the following temperatures:

[0024] In some embodiments, current art methods include hybridization from storage at temperatures below 8° C. obtaining a hybridization buffer, and combining the probe with the hybridization buffer. Hybridization was performed without mixing the hybridization buffer before The method further comprises combining the buffer with the at least one nucleic acid probe.

[0025] In another embodiment, the method further comprises the step of using dimethyl sulfone, diphenyl sulfone, methylphenyl The group consisting of tetramethylene sulfone (sulfolane), tetramethylene sulfone (sulfolane), and mixtures thereof In some embodiments, the sulfone solvent is selected from the group consisting of sulfones. It's an orchid.

[0026] In an additional aspect, the current technology is a method for preparing a hybridization composition. The hybridization buffer is then stored at a certain temperature for at least 2 months, or The method further comprises storing the mixture for 4, 6, 8, 12, 16, 18 or 24 months, followed by high The hybridization buffer is combined with at least one nucleic acid probe to perform hybridization. and forming a hydration composition. The hybridization buffer is stored at a temperature above 0°C.

[0027] In some embodiments, the salt of polyvinylsulfonic acid is a sodium salt suitable for the intended purpose. The salt may be a salt, a potassium salt, a lithium salt or any other salt. Any salt of polyvinylsulfonic acid suitable for use in ion buffers may be used. In some embodiments, the hybridization buffer may contain sulfuric acid. Further containing acid dextran or another enhancer, but not more than 20% (w / v) or less than 15% (w / v), or less than 10% (w / v), and the present disclosure 60 mPa.s at 25°C when measured with an ovis 2000 M microviscometer A hybrid having a viscosity of 50 mPa.s or less, or 40 mPa.s or less. Stability studies, including accelerated stability studies, are provided in accordance with the present disclosure. Hybridization buffer should be stored at 2-8°C for at least 2 months or at least Stable and / or homogeneous after storage for 4, 6, 8, 12, 16, 18 or 24 months This proves that.

[0028] In another aspect, the disclosure provides a method for hybridizing nucleic acid sequences. providing a target nucleic acid sequence; and at least one type of hybridization composition. The nucleic acid probe of the present invention is allowed to hybridize to the target nucleic acid sequence for a period of time at least sufficient to applying a hybridization composition disclosed therein to the target nucleic acid sequence. The target nucleic acid sequence can be present in a biological sample in situ, and the method includes denaturing the target nucleic acid sequence at a temperature of about 66° C. to about 80° C. or higher; and / or A hybridization step may be further included, and at least one nucleic acid probe may be used. The enzyme and the target nucleic acid sequence are at a temperature of about 55° C. to about 50° C. for a period of 4 hours or less. In an embodiment, the method further comprises: removing the hybridization buffer from storage at a temperature of 2 to 8° C. and performing hybridization without premixing the hybridization buffer. The hybridization buffer is combined with at least one nucleic acid probe to perform hybridization. The method further comprises forming a hybridization composition with a probe. Once the buffers are combined to form the hybridization composition, the hybridization sequence is The hybridization compositions are mixed prior to use in a hybridization method such as FISH.

[0029] In another aspect, the disclosure provides a method for preparing a hybridization composition. The method comprises combining a hybridization buffer with at least one nucleic acid probe. forming a hybridization composition; and The composition is stored at a temperature for at least 2 months, or for at least 4, 6, 8, 12, or 1 The method includes storing the product at a temperature above 0° C. for 6, 18 or 24 months. Some embodiments of the hybridization buffer are homogenous and do not require mixing prior to use. Although not required, hybridization compositions containing probes may be used in certain embodiments. They should then be mixed prior to use in the hybridization method.

[0030] The present disclosure provides, for example, hybridization that is homogenous and stable over a wide variety of storage conditions. For example, certain embodiments of the present disclosure provide a method for freezing Less sensitive to freezing and melting and more stable when stored at higher temperatures than prior art compositions The increased stability of the hybridization buffer of the present invention is also due to the fact that the hybridization temperature is increased from about 66° C. The hybridization enzyme of the present disclosure can support denaturation at a temperature of, for example, about 80° C. The hybridization buffer also has a lower pH than certain previously existing hybridization buffers. Viscosity and therefore capillary fields, e.g., between the slide and the slide cover This facilitates mixing, pipetting and spreading in the space between the electrodes.

[0031] In an additional aspect, the present disclosure provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising at least one nucleic acid sequence, sulfolane, polyvinylsulfone, or a combination thereof. A hybridization assay comprising a phosphonic acid or a salt thereof and one or more additional components. The additional components include buffers, salts, enhancers, chelating agents, detergents, screening agents, etc. a blocking agent, a blocking agent, or a combination thereof.

[0032] The present disclosure also provides increased homogeneity and increased stability, making it easier to store and use. A hybridization buffer is provided. In some embodiments, the hybridization buffer of the present invention The hybridization buffer is designed to be used at higher temperatures than certain existing hybridization buffers, e.g. For example, it is stable when stored at temperatures above 0° C. Increased stability of the denaturing buffer also contributes to hybridization procedures that involve steps such as high temperature denaturation. In some embodiments, the hybridization buffer also comprises a specific These features of the present disclosure may exhibit lower viscosities than existing hybridization buffers. The features are recognized by those skilled in the art when compared to certain existing hybridization buffers. Among other advantages that will be recognized are mixing in capillary fields, pipetting It offers advantages in hybridization methods including manipulation and diffusion.

[0033] In certain preferred embodiments, the present disclosure provides at least one nucleic acid, about 13% (v / v v) sulfolane at a concentration of about 30% (w / v), polyvinyl sulfonic acid or its salt, dextran sulfate at a concentration of about 10% (w / v), NaCl at a concentration of about 600 mM, and and MOPS at a concentration of about 40 mM.

[0034] According to yet another aspect, the present disclosure provides hybridization protocols and assays. , as described herein, for use in, for example, those described below. The use of a hybridization buffer containing sulfolane and PVSA or a salt thereof is Regarding use. [Brief description of the drawings]

[0035] [Figure 1] 1 is a graphical representation showing that the FISH hybridization buffer of the present disclosure is more stable than certain existing FISH buffers. [Diagram 2] 1 is a graphical representation showing that the FISH hybridization buffers of the present disclosure are less viscous than certain existing FISH buffers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure relates to a hybridization enzyme comprising a sulfone solvent and polyvinyl sulfonic acid or a salt thereof. In some embodiments, the hybridization buffer comprises water. The hybridization buffer is used in accordance with the hybridization method, e.g. Suitable for use in in situ hybridization (ISH). , a method used for visualization of genetic structures or genetic events, which is partly In the embodiment, formalin-fixed paraffin-embedded tissue (FF) is placed on a glass slide. PE), metaphase spreads, cytospins, histological smears or other biological material. The base pairing probes are capable of detecting target material, e.g., DNA, during the ISH staining process. For example, DNA or RNA can be labeled, thereby identifying the relevant genetic information in the target material. The stained slides may, in some embodiments, be fluorescently stained, allowing for subsequent identification and quantification of the stained slides. Examination using a microscope or brightfield microscope and interpretation of the information gathered may be diagnostic, prognostic or Can be used for research purposes.

[0037] Thus, ISH allows the identification of genetic and biological structures in the context of undisrupted morphological structures. Provides rapid and accurate description of mutations, such as amplifications, deletions, translocations, and chromosomal copy number changes. In some cases, there is a correlation between genetic abnormalities, their frequency, and the specific cells that harbor these changes. The link can be searched by doctors.

[0038] In some embodiments, the nucleic acid probe is dissolved in a hybridization buffer at a relevant concentration. and form a hybridization composition. The dyes are mixed together and then used in the staining process, which allows the detection of the dyes in the biological material of interest. The environment is suitable for denaturation and hybridization of the probe and target nucleotides. One important function of the hybridization composition is to regulate the DNA melting temperature. This reduces the degree of hybridization, thereby allowing hybridization at lower temperatures, The aim of this study is to reduce tissue damage by

[0039] The hybridization buffer of the present invention is a hybridization buffer that is compatible with certain existing hybridization It offers several advantages over buffer solutions. For example, the hybridization buffer solutions of the present disclosure The buffer may, in certain embodiments, be a hybridization buffer that is more suitable than certain existing hybridization buffers. The lowest viscosity (for example, less than 1000 mPa·s or less than 100 mPa·s at 25°C) The present disclosure provides an ISH hybridization buffer having a viscosity of less than 1.5 μm. The lower viscosity associated with the hybridization buffer reduces the Promotes rapid and strong diffusion of the hybridization buffer. This is the most common method used in the past. This has the advantage over hybridization buffers, as it prevents the formation of cytoplasmic ions between glass slides. Slow, non-strong diffusion in the capillary field can lead to, for example, hybridization This is because the buffer may leave air spaces where there is no functionality. The lower viscosity exhibited by the solution buffer also reduces the difficulty of pipetting with the more viscous buffers. This makes it easier for users to create and mix.

[0040] In addition, the hybridization buffer of the present disclosure is compatible with certain existing hybridization buffers. It offers improved homogeneity and long-term storage when compared to lysis buffers. In embodiments, the hybridization buffer of the present disclosure is stored long term at 2-8° C. This is because certain products must generally be stored at temperatures below -18°C. The present invention provides significant advantages over existing ISH hybridization buffers. Hybridization buffers are stable to the extent found in many buffers at these storage temperatures. The present invention does not coagulate, form large precipitates, and / or exhibit phase separation. Hybridization buffers do not need to be stored at temperatures below -18°C, so these The solution was thawed and allowed to equilibrate to 20-25°C, then mixed extensively to remove any precipitate. There is no need to solubilize and homogenize the liquid phase, and after use, the solution can be refrozen at temperatures below -18°C. As a result, the hybridization buffer of the present invention is free from freeze-thaw damage. The hybridization buffer of the present disclosure does not need to be frozen for long-term storage. Instead, it can be stored for a long time at temperatures between 2 and 8°C, for example. It offers the advantage of being easy to use.

[0041] Certain existing hybridization buffers also exhibit heterogeneity at low temperatures, In some cases, special attention to the temperature equilibration and mixing process is required. In some cases, a separate device, e.g., the Dako Omnis Mixing Device Prior to use, the probe product is dissolved in a certain existing hybridization buffer. However, it is necessary to use a fusion and homogenization method for the hybridization of the present invention. The buffer maintains homogeneity during long-term storage and does not require extra mixing steps or devices. do not.

[0042] In addition, certain existing hybridization buffers exhibit instability. For example, Buffer solutions that use ethylene carbonate as a solvent are unstable due to hydrolysis of the ethylene carbonate. The hydrolysis of ethylene carbonate is temperature dependent, and therefore Buffers containing 1,2-dichlorophenyl 2,4-dichlorophenyl 3,5-dichlorophenyl 2,6-dichlorophenyl 3,7-dichlorophenyl 2,7-dichlorophenyl 3,8-dichlorophenyl 2,7-dichlorophenyl 3,8-dichlorophenyl 2,7-dichlorophenyl 3,7 ... The hybridization buffer provided by For example, the hybridization relaxation provided by the present disclosure is more stable at high temperatures. Certain embodiments of the buffer solution can be used at a temperature of 80°C.

[0043] In some embodiments, the present disclosure does not require hardware-dependent mixing and is automated and A novel hybridization technique that can be used at low viscosity to facilitate easy manual handling. The hybridization buffer of the present invention also provides improved solubility and The stability of the hybridization buffer of the present disclosure is acceptable at 2 to 8°C. These buffers provide long-term storage without phase separation or precipitation. Therefore, the hybridization buffer of the present invention is in a homogeneous state before use. Thorough mixing is not required. Therefore, the hybridization buffer according to the present disclosure may be transferred directly from storage at 2-8°C to in-process conditions.

[0044] The hybridization buffers and compositions of the present invention also incorporate certain existing ISH hybridization techniques. It exhibits lower viscosity compared to buffer solutions. Without wishing to be bound by theory, high molecular weight The reduction in the concentration of components such as dextran sulfate (MW 500.000) is lower This is likely the main reason for the viscosity. The compositions and buffers of the present disclosure are It provides up to 39% viscosity reduction (at 25°C) compared to the liquid. Without wishing to be bound by the above, synthetic sulfonate polymers, e.g., polyvinyl sulfonate, The addition of sodium salt MW 3,000 to 10,000 (PVSA) to the composition of the present invention is It is predicted to functionally counteract the reduction of dextran sulfate (MW 500,000), This allows the compositions of the present invention to continue to provide high quality dye results even at lower viscosities. Similarly, without wishing to be bound by theory, the superior properties of the compositions of the present disclosure are The ability and functionality of the polymer is improved by the addition of a synthetic sulfonate polymer, such as PVSA or its salt. It is believed that this will be maintained better.

[0045] In some embodiments, the hybridization buffers and compositions of the present disclosure include certain A replacement for ethylene carbonate and other solvents present in certain existing hybridization buffers. In addition, sulfone solvents, such as sulfolane, are included. Without wishing to be bound by theory, Sulfolane has extremely high chemical and thermal stability compared to ethylene carbonate. It is believed that the hybridization buffers and compositions of the present disclosure therefore can be stored at higher temperatures (2-8°C) and used at higher denaturation temperatures. Utilizing higher denaturation temperatures in hybridization protocols The ability to perform more efficient denaturation of target nucleic acids and hybridization compositions. and / or performing the entire hybridization protocol in a shorter time. This makes it possible to do so.

[0046] The hybridization buffers and compositions of the present disclosure also contain commercially available hybridization buffers. The results provide high quality signal staining quality similar to that obtained with the solution buffer. Without wishing to impose restrictions on the use of hybridization buffers and compositions of the present invention, the thermal stability of the hybridization buffers and compositions of the present invention is not intended to be limiting. The higher the yield, the better in line with recommendations for ISH probes, e.g., SureFISH probes. It is believed that this allows for their use at lower denaturation temperatures.

[0047] Table 1 shows the hybridization buffers of the present invention compared to certain existing hybridization buffers. A comparison of the characteristics of various embodiments of the redox buffer is provided. This characteristic provides significant advantages over certain existing hybridization buffers. It is not necessary for an embodiment to have all of these features, as they can still provide advantages. It should be recognized that

[0048] JPEG2025066727000004.jpg91159

[0049] In the context of the present disclosure, the following terms shall be understood as follows:

[0050] "Hybridization" refers to the act of hybridizing a nucleic acid molecule or sequence to a complementary nucleic acid molecule or sequence. "Hybridization buffer" refers to, for example, the binding of a target nucleic acid probe to a target nucleic acid. Suitable for carrying out hybridization protocols to promote binding to nucleic acid sequences. A hybridization buffer refers to a composition that contains, for example, at least one solvent. The hybridization composition may include a solvent, at least one promoter, and water. "Composition" refers to a composition comprising a hybridization buffer and at least one nucleic acid probe. Hybridization buffers and compositions in the current technology have various concentrations It is to be understood that the present invention includes additional components that may be present in the composition. The hybridization buffer and solvent concentrations of the compositions are on a volume to volume (v / v) basis. Whereas the concentrations of additional solid components are provided herein in volumetric terms, the concentrations are expressed as weight by volume (w / v) basis.

[0051] The "hybridization process" generally refers to a process that occurs in a larger way. refers to the period, or portion, of conditions that are present for one nucleic acid to hybridize to another. The hybridization process is provided by the method of The denaturation procedure can be understood to incorporate both denaturation and reannealing ( (e.g., if the procedure does not include a separate denaturation step). "A" means a hybridization process and one or more other processes, e.g. For example, this refers to a process that includes a separation or rinsing process.

[0052] "Biological sample" means any in vivo, i.e., sample of one or more cells or cell fragments. In vitro or in situ samples are to be understood as samples that are, for example, Single or multicellular organisms, tissue sections, cytological samples, chromosome spreads, purified nuclei nucleic acid sequences, artificially prepared nucleic acid sequences, e.g., by biologically based systems or synthetic The nucleic acid chip may be a microarray or other form of nucleic acid chip, such as one prepared by chemical synthesis. In one embodiment, the sample is a mammalian sample, e.g., a human, mouse, rat, or mouse. The sample may be from a dog or cat.

[0053] "Nucleic acid," "nucleic acid strand," and "nucleic acid sequence" refer to nucleic acid sequences that contain non-standard nucleobases and / or non-standard nucleic acid bases. A synthetic backbone (e.g., peptide nucleic acid (PNA) or locked nucleic acid (LNA)), or any from naturally occurring nucleotides and / or nucleic acid analogs, including any derivatized form of nucleic acid. including oligomers or polymers having a structured backbone that are linked or bonded using base pairs. means anything that hybridizes.

[0054] As used herein, the term "peptide nucleic acid" or "PNA" refers to a peptide nucleic acid. It has a polyamide backbone with nucleotide nucleobases (naturally occurring and modified). It refers to a synthetic polymer. The bases may be linked to the backbone via a linker. -(2-aminoethyl)-glycine) backbone. PNAs are the same as those mentioned in the entire disclosure of the present application. No. 6,393,311, filed on Oct. 13, 2002, which is hereby incorporated by reference in its entirety; or Synthesize as taught by any of the references cited in Since the PNA backbone is uncharged, PNAs can be firmly It hybridizes with DNA and RNA with high sequence specificity. A probes may exhibit equal specificity to longer DNA or RNA probes. The probes may also exhibit greater specificity in binding to complementary DNA or RNA. As used herein, the term "locked nucleic acid" or "LNA" refers to at least Also, oligomers or polymers containing one or more LNA subunits are As used herein, the term "LNA subunit" refers to a ribose means a ribonucleotide containing a methylene bridge connecting the 2'-oxygen to the 4'-carbon of Generally, the entire disclosures of which are incorporated herein by reference. Kurreck, Eur. J. Biochem., 270:1628-44 (200 Please refer to the 3rd year.

[0055] Examples of nucleic acids and nucleic acid analogs include polymers of nucleotide monomers, which The double-stranded and single-stranded deoxyribonucleotides (DNA), ribonucleotides (R Nucleic acid chains include the deoxyribonucleic acid (NA), its anomeric forms, and its synthetic and natural analogues. Xyribonucleotides, ribonucleotides, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) NA), its synthetic or natural analogues, or mixtures thereof. DNA, RNA, or other nucleic acids as defined herein may be used in the methods and methods of the present disclosure. and compositions.

[0056] "Polar aprotic solvent" means a solvent with a dipole moment of about 2 Debye units or more, Solubility in water of at least about 5% (by volume) at or near a temperature of about 20°C. and is particularly effective at approximately neutral pH, i.e., in the range of 5 to 9, or in the range of 6 to 8. This refers to an organic solvent that does not easily undergo hydrogen exchange.

[0057] By "aqueous solution" it is meant to be understood a solution that contains water, even if only in small amounts. For example, a solution containing 1% water shall be understood to be an aqueous solution.

[0058] "Repetitive sequences" refer to sequences that reanneal rapidly (approximately 25%) and / or moderately This refers to the components of mammalian genomes that reanneal together (approximately 30%). It is understood that

[0059] "Non-toxic" and "less toxic" are defined with respect to the toxicity of formamide. Formamide is toxic and may harm an unborn baby. In addition, the hybridization buffers and compositions of the present technology do not contain formamide. and is more toxic than certain existing hybridization buffers that contain formamide. is low.

[0060] The terms "aliphatic" or "aliphatic group," as used herein, refer to a fully saturated or containing one or more units of unsaturation. or branched, substituted or unsubstituted hydrocarbon chains, or fully saturated or or monocyclic or bicyclic hydrocarbons containing multiple unsaturated units, but which are not aromatic It can be fully saturated or contain one or more units of unsaturation. Non-aromatic monocyclic or bicyclic hydrocarbons such as Aliphatic groups are generally referred to as "cycloaliphatic" or "cycloalkyl". It may have one point of attachment to the linker, provided, for example, that it is an intramolecular linker. In some embodiments, the lipid may have more than one attachment point. The aliphatic groups contain 1 to 6 aliphatic carbon atoms, provided that the aliphatic groups do not contain additional carbon atoms. The aryl group may have one or more substituents.

[0061] Sulfones for use in the hybridization buffers, compositions and methods of the invention Solvents include sulfolane and other solvents with sulfone functionality. , sulfone solvents include dimethyl sulfone, diphenyl sulfone, methyl phenyl sulfone, tetramethylene sulfone (sulfolane), and mixtures thereof. In some embodiments, the sulfone solvent may comprise a cyclic sulfone moiety, e.g. One embodiment of the present disclosure is a hybridization kit for use in hybridization. In some embodiments, the hybridization buffer is a hybridization buffer or a hybridization composition. In some embodiments, the hybridization buffer may comprise an aqueous composition. A hybridization solution comprising a hybridization buffer and at least one nucleic acid probe. In some embodiments, the hybridization buffer comprises a target At least an amount effective to promote hybridization of a nucleic acid probe to a target nucleic acid sequence. Each solvent contains one sulfone solvent. An amount effective to promote hybridization is an amount that provides a usable level for the intended purpose. For example, the amount of sulfone solvent is sufficient to facilitate hybridization. One method for testing whether a compound is effective in enabling a When used in hybridization methods and compositions, this sulfone solvent is detectable. The key to success is to determine whether a given signal is generated.

[0062] Non-limiting examples of effective amounts of sulfone solvents include, for example, about 1% to about 95% (v / v). In some embodiments, the concentration of the sulfone solvent is 5% to 60% (v / v), or In yet another embodiment, the concentration of the sulfone solvent is 10% to 60% (v / v). %~50%, 5%~30%, 10%, 10%~20%, 10%~30%, or 1 0% to 25% (v / v) is also suitable. In some embodiments, the sulfone solvent is at least At most about 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, In other embodiments, it is present in a concentration of 10%, 11%, 12% or 13% (v / v). Sulfone solvents are available in concentrations up to 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 1 0.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 1 4.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 1 Present in concentrations of 8.5%, 19%, 19.5%, or 20% (v / v). It is expressly contemplated that any of the values ​​and maximum values ​​may be combined to form a range. It is also explicit that any concentration between the minimum and maximum values ​​in the range may be used. It is expected that.

[0063] The hybridization buffer of the present invention contains a sulfonic acid polymer or a salt thereof. In some embodiments, the sulfonic acid polymer has the general formula (I): [ka] [In the formula, R 1 is hydrogen or C 1~6Aliphatic, L is a covalent bond or C 1~6 In aliphatic where n is an integer greater than 10. or a salt thereof. In some embodiments, R 1 is hydrogen In some embodiments, R 1 is C 1~6 In some embodiments, L is aliphatic. In some embodiments, L is a bond. 1~6 In some embodiments, L is methylene. In some embodiments, the sulfonic acid polymer or salt thereof has the general formula ( Ia): [ka] [In the formula, R 1 is hydrogen or C 1~6 aliphatic] or a salt thereof. In some embodiments, the sulfonic acid polymer In some embodiments, the sulfonic acid or salt thereof is polyvinylsulfonic acid or a salt thereof. The acid polymer or its salt is polyanetholesulfonic acid. onic), the general formula (II): [ka] [In the formula, R 1 is hydrogen or C 1~6 Aliphatic, R 3 is hydrogen or C 1~6 With alkyl where n is an integer greater than 10. or a salt thereof.

[0064] In some embodiments, the sulfonic acid polymer or salt thereof has the general formula (IIa): [ka] or a salt thereof. In some embodiments, the sulfonic acid polymer or a salt thereof is polyanetholesulfonic acid or a salt thereof.

[0065] In any one of the above embodiments, n is 10 to 200, 10 to 150, 10 to 10 0, 10~90, 10~80, 10~70, 20~150, 30~150, 40~150 , 50~150, 60~150, 70~150, 80~150, 90~150, 100~ 150, 50~120, 60~120, 70~120, 80~120, 90~120, etc. may be an integer.

[0066] In any one of the above embodiments, the sulfonic acid polymer or salt thereof is about 500 to 1000 μg / ml. Approximately 1,000 Da, approximately 1,000 to approximately 2,000 Da, approximately 2,000 to approximately 4,000 Da , about 3,000 to about 5,000 Da, about 4,000 to about 6,000 Da, about 5,000 to Approximately 7,000 Da, approximately 6,000 to approximately 8,000 Da, approximately 7,000 to approximately 9,000 Da , about 8,000 to about 10,000 Da, about 2,000 to about 20,000 Da, about 3,00 0 to about 20,000 Da, about 4,000 to about 20,000 Da, about 5,000 to about 20, 000Da, about 6,000 to about 20,000Da, about 7,000 to about 20,000Da, About 2,000 to about 9,000 Da, about 3,000 to about 9,000 Da, about 4,000 to about 9,000 Da, about 5,000 to about 9,000 Da, about 6,000 to about 9,000 Da, Average range of about 7,000 to about 9,000 Da, or about 8,000 to about 9,000 Da In some embodiments, the average molecular weight is less than about 1,000 Da, about Less than 2,000 Da, less than about 3,000 Da, less than about 4,000 Da, about 5,000 Da less than about 6,000 Da, less than about 7,000 Da, less than about 8,000 Da, less than about 9,0 00 Da, less than about 10,000 Da, or less than about 20,000 Da. It is explicitly contemplated that any of the minimum and maximum values ​​may be combined to form a range. It is also understood that any average molecular weight between the minimum and maximum values ​​in the range can be used. It is also explicitly assumed.

[0067] As demonstrated in the examples, the lower molecular weight sulfonic acid polymers are more stable than other polymeric anions. Thus, in some embodiments, the sulfonic acid polymer had better performance than the carboxylic acid polymer. The polymer or a salt thereof has the general formula (III): [ka] [In the formula, R 1 is hydrogen or C 1~6 Aliphatic, R 2 is a covalent bond, C 1~6 aliphatic, phenyl, or a 6-membered heteroaryl ring having 1-3 nitrogen atoms; R 2 If or two SO3H groups or their salts, except that R 2 is a covalent bond In the case of R, only one SO3H group or salt thereof shall be present, 2 -R 3 , Haloge N, -CN, -NO2, -OR 3 , -N(R 3 )2, and -SR 3 Selected independently from and each R 3 are independently hydrogen or C1 ~6alkyl group, n being an integer greater than 10, preferably at most a sulfonic acid is an integer such that the polymer or salt thereof has an average molecular weight within the ranges described herein. ] is a sulfonic acid polymer.

[0068] In some embodiments, the sulfonic acid polymer of formula (III) or salt thereof is from about 500 to about 500 Approximately 1,000 Da, approximately 1,000 to approximately 2,000 Da, approximately 2,000 to approximately 4,000 Da , about 3,000 to about 5,000 Da, about 4,000 to about 6,000 Da, about 5,000 to Approximately 7,000 Da, approximately 6,000 to approximately 8,000 Da, approximately 7,000 to approximately 9,000 Da , about 8,000 to about 20,000 Da, about 2,000 to about 20,000 Da, about 3,00 0 to about 20,000 Da, about 4,000 to about 20,000 Da, about 5,000 to about 20, 000Da, about 6,000 to about 20,000Da, about 7,000 to about 20,000Da, About 2,000 to about 9,000 Da, about 3,000 to about 9,000 Da, about 4,000 to about 9,000 Da, about 5,000 to about 9,000 Da, about 6,000 to about 9,000 Da, Average range of about 7,000 to about 9,000 Da, or about 8,000 to about 9,000 Da In some embodiments, the average molecular weight is less than about 1,000 Da, about 2,000 Da, or less than about 1,000 Da. Less than 00 Da, less than about 3,000 Da, less than about 4,000 Da, less than about 5,000 Da, Less than about 6,000 Da, less than about 7,000 Da, less than about 8,000 Da, less than about 9,000 Da a, less than about 10,000 Da, or less than about 20,000 Da. and maximum values ​​may be combined to form a range. It is also understood that any average molecular weight between the minimum and maximum values ​​in the range can be used. Explicitly assumed.

[0069] In some embodiments, n in general formula (III) is 10 to 200, 10 to 150, or 10 to 100, 10~90, 10~80, 10~70, 20~150, 30~150, 40~1 50, 50~150, 60~150, 70~150, 80~150, 90~150, 10 0~150, 50~120, 60~120, 70~120, 80~120, 90~120 It may be an integer such as

[0070] The concentration of sulfonic acid polymer in the hybridization buffer determines the amount of nuclei present in the assay. In some embodiments, the hybridization rate of the acid may be affected. The concentration of the polymer in the solution buffer is between about 0.1 and about 40%, by weight to volume, about 0.2 % to about 15%, about 0.2% to about 10%, about 0.2% to about 9%, about 0.2% to Between about 8%, Between about 0.2% and about 7%, Between about 0.2% and about 6%, Between about 0.2% and about 6% Between about 0.5% and about 5%, Between about 1% and about 5%, Between about 1% and about 4%, Between about 0.5% and Between about 4%, Between about 0.5% and about 3%, Between about 1% and about 2.5%, Between about 5% and about 40% , between about 10% and about 35%, between about 15% and about 30%, or between about 25% and about 35% Any of the preceding minimum and maximum values ​​may be combined to form a range. Explicitly assumed. Any cardinality between the minimum and maximum values ​​in the range may be used. It is also explicitly envisaged.

[0071] The hybridization buffer of the present invention may be any of the hybridization buffers known in the art or later developed. For example, the hybridization buffer may contain a buffer containing One or more of the following components: buffers, enhancers, chelating agents, salts, detergents, and blocking agents may also contain

[0072] In some embodiments, the hybridization buffer contains NaCl as a salt. Cl is present in the hybridization buffer at a concentration of about 1 mM to about 1200 mM. In some embodiments, the hybridization buffer contains N In some embodiments, the hybridization buffer contains 3-(N-mono It contains 2,4-difluoromethylaminopropanesulfonic acid (MOPS) as a buffer. in the dilution buffer at a concentration of about 1 mM to about 50 mM, and at a concentration of about 6.0 to about 8.0 In certain embodiments, the hybridization buffer may be present at about 40 mM and at a pH of about 7.2.

[0073] For example, the hybridization buffer of the present invention may include one or more buffering agents. In some embodiments, the buffering agent is 3-morpholinopropane-1-sulfonic acid (MO PS), SSC, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane Sulfonic acid (HEPES), SSPE, PIPES, TMAC, TRIS, SET, Citric acid Acids, phosphate buffers, such as potassium phosphate, sodium phosphate, or sodium pyrophosphate The buffer is present at a concentration of 0.5x to 50x. Typically, the buffering agent may be present at a concentration of about 2x to about 10x. The concentration of the buffering agent may be If necessary, the hybridization buffers and compositions of the present technology are adjusted to about neutral pH. In order to stabilize the concentration, the concentration can be adjusted within the range of about 1 mM to about 500 mM.

[0074] The hybridization buffer of the present invention may contain one or more enhancing agents. In some embodiments, the enhancer is a polymer, e.g., FICOLL, PVP, heparin, de dextran, and dextran sulfate; proteins, e.g., BSA; glycols, e.g. For example, ethylene glycol, glycerol, 1,3 propanediol, propylene glycol ethylene glycol, or diethylene glycol, and combinations thereof. For example, the hybridization buffer may be selected from Denhardt's solution and BLOTTO, as well as organic solvents such as formamide, dimethylformamide, DM The accelerator may be present in a concentration of about 1% to about 80%. In particular embodiments, formamide is present at about 1% to about 75% volume to volume. or at a concentration of about 25% to about 75%. DMSO, dextran sulfate, and glycosyltransferase are The dextran sulfate is present at a concentration of about 5% to about 20%. Other concentrations are possible depending on the molecular weight of the dextran sulfate. It should be appreciated that dextran sulfate of the formula (I) may be used in certain embodiments. The hybridization buffer contains about 5% to about 20% (w / v) dextran sulfate ( In some embodiments, the hybridization buffer contains about 1 In another embodiment, the hybridization step comprises a step of hybridizing the hybridization medium with a pH of 10 or less than 0.01. The reaction buffer does not contain formamide.

[0075] In some embodiments, the hybridization buffer of the present disclosure contains dextran sulfate ( In some embodiments, the hybridization buffer contains a lower concentration of 1,2-dihydropyridine (DS) than conventional hybridization buffers. The concentration of dextran sulfate is about 20% (w / v) or less, or about 15% (w / v) or less, or about 10% (w / v) or less. The concentration of tolan is about 1% to about 20%, or about 5% to about 15% (w / v). In embodiments, dextran sulfate is about 0.1%, about 0.25%, about 0.5%, about 1% , about 2%, about 3%, about 4%, or about 5% (w / v). In embodiments, dextran sulfate is about 7%, about 7.5%, about 8%, about 8.5%, about 9% , about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12. 5%, approx. 13%, approx. 13.5%, approx. 14%, approx. 14.5%, approx. 15%, approx. 15.5%, approx. 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, It will be present at a concentration of about 19.5%, or about 20% (w / v). It is expressly contemplated that any of the above may be combined to form a range. In one embodiment, the hybridization buffer of the present disclosure contains about 10% (w / v) dextran sulfate. ) at a concentration of

[0076] The hybridization buffer of the present invention may contain one or more chelating agents. In some embodiments, the chelating agent is selected from EDTA, EGTA, or others. The chelating agent is present at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. It can exist in multiples of 100 degrees.

[0077] The hybridization buffer of the present invention may contain one or more salts. In this embodiment, the salt is selected from the group consisting of sodium chloride, sodium phosphate, magnesium phosphate, and the like. The salt may be at a concentration of about 1 mM to about 1200 mM, or any concentration between these endpoints. Typically, the salt is present at a concentration of about 10 mM to about 500 mM or less. It exists in any concentration between the endpoints.

[0078] The hybridization buffer of the present invention may contain one or more detergents. Examples of such antibacterial agents include Tween, SDS, Triton, CHAPS, and deoxycholic acid. The detergent may be present in an amount of about 0.01% to about 10%, or about 0.1% to about 1% (w / w). v) or any concentration between these endpoints.

[0079] The hybridization buffer of the present invention may contain one or more nucleic acid blocking agents, e.g., enzymes. Maternal tRNA, homopolymeric DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA A, COT1 DNA, etc. The blocking nucleic acid may be about 0.05 mg / mL to about It may be present at a concentration of 100 mg / mL or any concentration between these two endpoints.

[0080] The hybridization buffer of the present invention does not contain any enhancer, e.g., dextran sulfate, glycerol, etc. When it contains cholor, or DMSO, dextran sulfate is about 20% (w / v) or less (in some or about 15% or about 10% or less), and glycol may be present at about 0.1%. may be present at a concentration of about 10% or any concentration between these two endpoints, DMSO can be about 0.1% to about 10%, or any concentration between these two endpoints. In this embodiment, the hybridization buffer does not contain DMSO as a promoter. In some embodiments, the hybridization buffer does not contain formamide, or less than about 10%, or less than about 5%, or less than about 2% hybridization buffer; or less than about 1%, or less than about 0.5%, or less than about 0.1%, or less than about 0.05% Formamide, provided that the product contains less than, or about 0.01% formamide. Includes do.

[0081] When the hybridization buffer of the present invention contains citric acid, its concentration is about 1 M to may be in the range of about 50 mM and the pH may be in the range of about 5.0 to about 8.0; or Any concentration between these endpoints may be used. In some embodiments, the concentration of citric acid is about It may be 10 mM and the pH may be about 6.2.

[0082] The hybridization buffer of the present invention is, for example, a hybridization buffer for a cell membrane, such as salmon sperm or It may also contain agents that reduce non-specific binding to small amounts of total human DNA, or, e.g. For example, the hybridization buffer of the present invention can be used to detect, for example, a target, e.g., a large amount of whole human Blocking agents that block the binding of repetitive sequences to DNA or repeat-rich DNA, or These agents may include certain blocking agents, such as PNA or LNA fragments and sequences. The agent may be from about 0.01 μg / μL to about 100 μg / μL or from about 0.01 μM to about 100 μM or any concentration between these endpoints. For example, in some embodiments These drugs contain approximately 0.1 μg / μL of total human DNA, or approximately 0.1 μg / μL non-human DNA, such as herring sperm, salmon sperm, or calf thymus DNA, or about 5 μM blocking PNA.

[0083] The hybridization buffer of the present invention is used in a hybridization assay. If present, they may be used as one or more nucleic acid fragments to form a hybridization composition. The probe may further comprise a detectable compound, e.g., an enzyme, a chromophore, DNA probes may be directly or indirectly labeled with fluorochromes, haptens, and the like. The lobe may be present at a concentration of about 0.1 ng / μL to about 100 ng / μL. In this embodiment, the probe may be present at a concentration of about 1 ng / μL to about 10 ng / μL. The NA probe may be present at a concentration of about 0.5 nM to about 5000 nM. For example, in some embodiments In the present embodiment, the probe may be present at a concentration of from about 5 nM to about 1000 nM.

[0084] In one embodiment, the hybridization buffer contains 13% sulfone solvent (v / v). (e.g., sulfolane), 10% dextran sulfate (w / v), 30% polyvinyl chloride sulfonic acid or its salt (w / v), 600 mM NaCl, 40 mM MOPS buffer solution Another exemplary hybridization buffer of the present disclosure comprises a mixture of 14% H7.2. (v / v) sulfone solvent (e.g., sulfolane), 10% dextran sulfate (w / v ), 30% polyvinylsulfonic acid (w / v), 600 mM NaCl, 40 mM MO It contains a mixture of PS buffer pH 7.2, and 0.1 μg / μl total human DNA. An exemplary hybridization buffer is 15% polar aprotic solvent (v / v) (e.g., sulfolane), 10% dextran sulfate (w / v), 30% polyvinyl sulfate Phosponic acid or its salt (w / v), 600 mM NaCl, 40 mM MOPS buffer, H7.2, and 0.1 μg / μL of non-human DNA (e.g., herring sperm, salmon sperm, , or calf thymus) or 0.5% formamide or 1% glycol (e.g., ethyl glycol, 1,3 propanediol, or glycerol).

[0085] In some embodiments, the sulfone solvent is the only solvent present in the hybridization buffer. In another embodiment, the hybridization The buffer contains one or more other or second polar aprotic solvents. In some embodiments, the hybridization buffer of the present invention may contain one or more additional solvents (solvents). For example, the hybridization buffer may contain one or more Polar aprotic solvents, such as lactones, nitriles, sulfites, and / or carbohydrates. Such solvents may include those having ester functional groups due to their relatively high dielectric constant. They are distinguished by their high dipole moment and solubility in water. They contain lactone functional groups. An exemplary polar aprotic solvent is y-butyrolactone (GBL), which contains a nitrile functional group. An exemplary polar aprotic solvent having a sulfite functional group is acetonitrile (AN). An exemplary polar aprotic solvent having and an exemplary polar aprotic solvent having a carbonate functionality is ethylene carbonate. (EC), propylene carbonate (PC), or ethylene trithiocarbonate (ETC) Different polar aprotic solvents impart different properties to the hybridization buffer. For example, the choice of polar aprotic solvent can be influenced by the The stability of the hybridization buffer must be considered, as carboxylic acid solvents may degrade over time. For example, the polar aprotic solvent ethylene carbonate can ethanol (which is a relatively stable molecule), and carbon dioxide (which can interact with water to form carbonic acid). Without being bound by theory, the acidity of the hybridization buffer is altered. The pH change due to the decomposition of ethylene carbonate has no effect on hybridization. It is believed to reduce the effectiveness of the hybridization buffer. In some embodiments, the hybridization buffer of the present invention contains substantially no ethylene carbonate. Free or substantially free of polar aprotic solvents with carbonate functionality .

[0086] However, for example, sulfolane, dextran sulfate, and PVSA or a salt thereof may be used. The hybridization buffer of the present disclosure has been stored at 2-8° C. for approximately 12 months. For example, sulfolane at a concentration of about 13% (v / v) is mixed with polyvinyl chloride. Dextran sulfate was added at a concentration of about 10% (w / v) and dextran sulfonic acid was added at a concentration of about 30% (w / v). NaCl at a concentration of about 600 mM and MOPS buffer pH 7.2 at a concentration of about 40 mM Hybridization buffer containing 100 μM of ... It results in a homogenous solution that can be used with minimal mixing.

[0087] According to another aspect of the disclosure, certain embodiments of the hybridization buffer of the present invention is less toxic than conventional hybridization buffers. Hybridization buffers that are less toxic than the hybridization solutions are described herein. As stated, the hybridization buffer does not contain formamide. or the hybridization buffer is less than about 10%, or about 5% Less than, or less than about 2%, or less than about 1%, or less than about 0.5%, or Formamide less than 0.1%, or less than about 0.05%, or less than about 0.01% The hybridization buffer may include a hybridization buffer that contains The hybridization buffer also contains dimethyl sulfoxide (DMSO). or hybridization buffer is less than about 10%, about 5% less than about 2%, or less than about 1%, or less than about 0.5%, or about 0.1% Contains less than, or about 0.05%, or about 0.01% DMSO The hybridization buffer may comprise a hybridization buffer that satisfies the following conditions:

[0088] In some embodiments, the hybridization methods and hybridization systems of the present invention The reaction buffer is suitable for the separation of genomic DNA, chromosomes, chromosome fragments, genes, and chromosomal aberrations, e.g. , translocations, deletions, amplifications, insertions, mutations, or inversions associated with normal conditions or disease Further, the present method and the method of the present invention are useful for in vivo or in vitro analysis of The hybridization buffer and the hybridization buffer are useful for the detection of infectious agents as well as for the detection of RNA, e.g., mRNA. and its complementary DNA (cDNA) for altering the expression levels.

[0089] Other uses include messenger RNA (mRNA), RNA viruses, DNA viruses, Small interfering RNA (siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g., tRNA and rRNA), transfer messengers -RNA (tmRNA), microRNA (miRNA), piwi-binding RNA (piRNP A), long non-coding RNA, small nucleolar RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA), methylation and other base modifications, single nucleotide polymorphisms (S NP), copy number variation (CNV), and, for example, radioisotopes, fluorescent molecules, biotin , digoxigenin (DIG), or antigen-labeled nucleic acids alone or unlabeled. This includes in vivo or in vitro analysis in combination with nucleic acids.

[0090] The hybridization method and hybridization buffer of the present invention include the following techniques: For example, Northern blot, Southern blot, flow cytometry, autoradiography Physiology, Fluorescence microscopy, Chemiluminescence, Immunohistochemistry, Virtual karyotype, Genetic assay, DNA Microarrays (e.g., array comparative genomic hybridization (array CGH), Gene expression profiling, gene ID, tiling array, gel electrophoresis, capillary Electrophoresis, and in situ hybridization, e.g., FISH, SI Useful for in vivo or in vitro analysis of nucleic acids using SH, CISH, etc. The methods and hybridization buffers of the present invention are useful in in vitro and in vivo hybridization. in vivo samples, e.g. bone marrow smears, blood smears, paraffin-embedded tissue preparations, for chemically dissociated tissue samples, bone marrow, amniotic cells, cytospin preparations, imprinting, and other techniques. It can be used.

[0091] The hybridization buffer of the present invention can be varied for a particular application. For example, sulfone solvents, salts, facilitators, blocking agents, and / or hydrogen ions (i.e. The concentration of the solvent (pH) may be varied to improve results for a particular application. For example, The concentration of sulfone solvent was varied to improve signal intensity and background staining. Generally, as the concentration of the solvent increases, the signal intensity increases and the Ground staining is reduced.

[0092] In addition, the type of probe used in the hybridization compositions of the present invention can affect the results. For example, in some embodiments of the present disclosure, DNA / DNA profiling may be used. The probe combinations are the DNA / PNA probes of the hybridization compositions of the present invention. On the other hand, the PNA probe can exhibit a lower background than the combination at low salt concentrations. and / or show stronger signals than DNA probes at low polar aprotic solvent concentrations In fact, PNA probes tend to give no signal even in the absence of sulfolane. whereas the DNA probe showed weak signals or no signal without sulfolane. or no signal.

[0093] The methods and hybridization buffers of the present invention are useful in the study of cytology, histology, or molecular biology. Complete or partial hybridization in all kinds of biological applications According to one embodiment, the first or second nucleic acid sequence is detected in a biological sample. Examples of such samples include, for example, tissue samples, cell preparations, cell fragment preparations, etc. Samples include preparations of isolated or enriched cellular components. Samples can be prepared from a variety of tissues, e.g. For example, breast, lung, colorectal, prostate, lung, head and neck, stomach, pancreas, esophagus, liver, and bladder. Bladder, or other associated tissues and neoplasms thereof; any cell suspension, blood sample, fine needle aspirate specimen, ascites, sputum, peritoneal washing, lung washing, urine, feces, cell scrape, cell smear, cytology It may be derived from a spin or cytological preparation.

[0094] Samples can be isolated and processed using standard protocols. Cell Fragment Preparation The product can also be obtained, for example, by cell homogenization, freeze-thaw treatment or cell lysis. The isolated samples may be of many types depending on the purpose for which the sample is obtained and on local practice. The samples can be processed in different ways. In most cases, the samples are prepared by isolating the tissue for subsequent sample analysis. The samples may be treated with various reagents for storage or may be analyzed directly. An example of a widely used method for preserving tissue is formalin fixation followed by paraffin embedding. Buried and frozen.

[0095] For metaphase spreads, cell cultures are typically arrested in metaphase To achieve this, cells are treated with colcemid, or another suitable spindle pole disrupting agent. The cells are then fixed. , spotted onto a microscope slide, treated with formaldehyde, washed, and dissolved in ethanol. The probe is then added and the sample is analyzed by one of the techniques discussed below. do.

[0096] Cytology involves the examination of individual cells and / or chromosomal spreads from a biological sample. Cytology of a sample begins with obtaining a sample of cells, which typically consists of a cervical sample. by scraping, swabbing or brushing the area, as in By collecting bodily fluids, such as fluids obtained from the thoracic cavity, bladder, or spinal column, Or, as in the case of internal tumors, it can be performed by fine needle aspiration or fine needle biopsy. In traditional manual cytological preparation, the sample is transferred to a liquid suspending material and then the cells in the fluid are separated. The cells were then cultured under a glass microscope for observation, either directly or via a centrifugation-based processing step. In a typical automated cytological preparation, the filter assembly is The cells are placed in a liquid suspension and the filter assembly disperses the cells and transfers them onto the filter. The filter is then removed and placed in contact with a microscope slide. The cells are then fixed onto a microscope slide and then cultured using one of the techniques discussed below. Analyze by:

[0097] Conventional hybridization protocols using cytological samples contain samples The slides are immersed in buffered formaldehyde, washed, and then dehydrated in ethanol. The probe is then added and the sample is covered with a coverslip. The slide is then used to identify any nucleic acids in the sample. Incubate at a temperature sufficient to denature (e.g., 82°C for 5 minutes) and then hyaluronan. Incubate at a temperature sufficient to allow hybridization (e.g., 45°C overnight). After hybridization, the coverslip is removed and the sample is incubated at high stringency. The specimens are then subjected to a series of low-strength washes (e.g., 65°C for 10 min) followed by Subject the specimen to a rapid wash (e.g., 2 x 3 min at room temperature). Then, dehydrate the specimen. , and fixed for analysis.

[0098] Histology involves the examination of cells in thin sections of tissue. To prepare tissue samples for histological examination To do this, small pieces of tissue are typically soaked in an aldehyde such as formaldehyde or glutaraldehyde. The tissue is then fixed in a suitable fixative, typically ethanol, and then embedded in molten paraffin wax. The wax block containing the tissue sample is cut on a microtome, typically at 2-30° C. A 10 micron thick section of paraffin containing the tissue is then prepared. The slide was applied to a microscope slide, air dried, and heated to adhere the sample to the glass slide. The remaining paraffin is then dissolved in a suitable solvent, typically xylene, toluene, or Cl. These so-called The deparaffinization solvent is removed with a cleaning and dehydrating type reagent, and then the gel is subjected to one of the techniques discussed below. Alternatively, slices can be prepared from frozen samples and rapidly The specimens were fixed in 0.1% formalin or other suitable fixative, then infused with a dehydration reagent before A fee analysis may be performed.

[0099] Traditional hybridization experiments using histological samples use formalin-fixed paraffin. The polyvinyl chloride embedded tissue samples are cut into 2-6 µm sections and collected onto slides. Melt the paraffin (e.g., at 60 °C for 30-60 min) and then add xylene (or xylene). The tissue is then deparaffinized, for example by washing with toluene (a paraffin substitute) for 2 x 5 min. The material is rehydrated, washed, and then pretreated (e.g., at 95-100°C for 10 minutes). The idos are washed and then incubated with pepsin or another suitable permeabilizing agent, for example, at 37°C for 3-15 min. The slides are washed (e.g., 2 x 3 min), dehydrated, and the probe is applied. Cover the sample with a coverslip and place the slide in a microwave oven at a temperature sufficient to denature any nucleic acids in the sample. (e.g., 82°C for 5 min) followed by hybridization. Incubation is performed at a temperature sufficient to allow for the precipitation of the chromium (e.g., 45°C overnight). After hybridization, the coverslip was removed and the sample was washed with high stringency wash ( For example, 65°C for 10 minutes) followed by a series of low stringency Place under wash (e.g., 2 x 3 min at room temperature). Samples are then dehydrated and prepared for analysis. Fix.

[0100] The compositions and methods of the present disclosure are directed to methods known in the art for cytological and histological samples. For use completely or partially in all types of nucleic acid hybridization techniques Such techniques include, for example, in situ hybridization. ISH, Fluorescence in situ Hybridization (FISH; multicolor FISH, Fiber-FISH, etc.), chromogenic in situ hybridization Computed In situ Hybridization (CISH), Silver In situ Hybridization (SISH), Comparative Genome Computational chromosome hybridization (CGH), chromosome painting, and array in situ Some examples include:

[0101] Molecular probes suitable for use in the hybridization methods and compositions of the present invention Lobe, for example, is incorporated herein by reference in its entirety. A probe is described in U.S. Patent Publication No. 2005 / 0266459. Generally, the probe is By chemical synthesis or by cloning, inserting the DNA into a vector and transforming it into a suitable host Amplification of specific DNA sequences by intracellular amplification of vectors and inserts Commonly used vectors include bacterial plasmids, smid, bacterial artificial chromosome (BAC), PI-derived artificial chromosome (PAC), or yeast artificial chromosome The amplified DNA is then extracted, purified, and used as a probe. The probes are labeled for use in a method for the preparation and / or synthesis of the probes. No. 6,393,996, the disclosures of which are incorporated herein by reference in their entirety. This is disclosed in PCT / US02 / 30573.

[0102] Generally, the type of probe determines the characteristics that can be detected in a hybridization assay. For example, total nuclear or total genomic DNA probes can be used as species-specific probes. Chromosome painting is a method for mapping DNA sequences derived from a single chromosome type. This is a collection of chromosomes that identify specific chromosome types in metaphase and interphase nuclei and Count the number of specific chromosomes, indicate translocations, or identify extrachromosomal fragments of chromatin Different chromosomal types can also be used to detect and count specific chromosomes. To achieve this, the nucleic acid has a unique repeat sequence that can be targeted by probe hybridization. Large insert probes can be used to target unique single copy sequences For these large probes, hybridization efficiency is inversely proportional to probe size. Smaller probes can also be used to identify abnormalities, such as deletions, amplifications, inversions, duplications, and aneuploidies. For example, differently colored locus-specific promoters can be used to detect The probe was used to detect translocations via split signal in situ hybridization. It can be used for this purpose.

[0103] In general, the difference in thermal stability between wild-type and mutant complexes increases with increasing probe length. The ability to discriminate between closely related sequences decreases with increasing hybridization time. The power dissipation is inversely proportional to the length of the probe. Generally, probes longer than 10 hp are more suitable for the purpose. required to obtain the sequence diversity necessary to correctly identify a unique organism or disease state. On the other hand, single base (point) mutations in very short oligomers (<10 base pairs) ) can result in greater hybridization to a complementary nucleic acid target sequence compared to a non-target sequence. may be sufficient to allow discrimination of the quantification.

[0104] In one embodiment, at least one set of in situ hybridization probes The term "substrate" refers to a substance that is a component of a pharmaceutical composition, as defined above, and the entire disclosure of which is incorporated herein by reference. One or more of the above-described compounds may be used as described in U.S. Pat. No. 7,105,294. The method for synthesizing the PNA probe may include any number of PNA probes. PCT / US02 / 3057, which is incorporated herein by reference in its entirety. Alternatively, or in addition, at least one of the techniques discussed above may be used. Both sets of hybridization probes are incorporated herein by reference in their entirety. One or more of the compounds described in WO 99 / 14226, which is incorporated herein by reference. may contain multiple locked nucleic acid (LNA) probes. The additional cross-linking ensures that the LNA backbone is pre-organized for hybridization. LNA / DNA and LNA / RNA interactions are indicated by higher melting temperatures. As a result, these interactions are stronger than the corresponding DNA / DNA and DNA / RNA interactions. The compositions and methods of the present invention reduce the energy required for hybridization. , are particularly useful for hybridization with LNA probes.

[0105] In one embodiment, the probe is a nucleotide sequence similar to that described herein, the entire disclosure of which is incorporated herein by reference. Such a detection device as described in U.S. Patent Publication No. 2005 / 0266459 shall be a label (an analytically distinguishable signal that allows for detection of the probe-target hybrid) The detectable label may be directly attached to the probe or may include a molecule that provides a Alternatively, it may be indirectly attached to the probe, for example by using a linker. Any labeling method known in the art can be used in the present invention, including enzymatic and chemical processes. It can be used to label the probes used in the methods and compositions of the present invention. In an embodiment, the probe is unlabeled.

[0106] Generally, in situ hybridization techniques, such as CGH, FISH, CISH and SISH are methods that use different strings of DNA to target genes or gene fragments to chromosomes of cells. Utilizing large, largely unspecific nucleic acid probes that hybridize at high sequence By using large probes, in situ hybridization techniques However, the sensitivity of the hybridization assay is very high. The successful use of large genomic probes has been demonstrated, for example, by the ability to target repetitive sequences present throughout the genome. This relies on blocking of unwanted background staining resulting from such blocking. The king step is time consuming and expensive. As discussed below, the method and The hybridization buffer eliminates the need for such a blocking step. However, in one embodiment, the repetitive sequences are advantageously reduced and / or eliminated, e.g. No. 6,399,943, the entire disclosure of which is incorporated herein by reference. The inhibitors can be inhibited according to methods known in the art, such as those disclosed in US Pat. No. 5,733,573.

[0107] The bound probes are then coupled to fluorescent dyes (e.g., FI) in cytological and histological samples. SH), organic chromogens (e.g., CISH), silver particles (e.g., SISH), or other of metal particles (e.g., gold-enhanced fluorescence in situ hybridization, GOLDFI These can be detected directly or indirectly using the serotype (SH). Therefore, depending on the detection method, Populations of cells from samples can be visualized via fluorescence microscopy or conventional brightfield light microscopy. It can be visualized.

[0108] Hybridization assays for cytological and histological samples have been used to identify specific D It is an important tool for determining the number, size, and / or location of NA sequences. For example, in CGH, the whole genome is stained and normal genomes are used to detect regions with abnormal copy numbers. Compare with a reference genome. Typically, DNA from the tissue of interest and from a normal control tissue is The DNA pools are mixed and labeled with different colored probes to identify normal chromosomes. Add to metaphase spreads (or myc for array- or matrix-CGH) The color ratios are then compared to identify regions with abnormal copy numbers. Identify the area.

[0109] FISH is typically used when multicolor imaging is required and / or the protocol is simple. This technique is generally used when quantification of signal is desired by preparing a cytological sample and The probe is labeled, the target chromosome and the probe are denatured, and the probe is hybridized to the target sequence. Typically, hybridization is performed by detecting the signal. The targeting reaction can be used to detect the location, size, or number of targeted sequences using techniques such as fluorescence microscopy, flow cytometry, or immunofluorescence. These are then fluorescently stained so that they can be determined using chromatometry, or other appropriate instrumentation. Use FISH to study DNA sequences ranging from whole genomes to several kilobases in length. FISH can also be used on metaphase spreads and interphase nuclei. It is also possible.

[0110] FISH is a method for detecting antisense oligonucleotides on metaphase chromosomes, interphase nuclei, chromatin fibers, and naked DNA molecules. For mapping repeat and single copy DNA sequences, as well as large repeated families - typically stained through localization of ribosomal DNA and the major tandem array families It has been successfully used for chromosome identification and karyotyping. The most important applications of FISH One of these is the identification of single-copy DNA sequences in humans and other eukaryotic model species, particularly those involved in disease FISH is used in, for example, prenatal testing, detection of disease-related genes, and detection of infectious agents. Chromosomal aneuploidies in hematological cancers and solid tumors; genetic abnormalities, e.g., cancer genes amplifications, gene deletions, or gene fusions; chromosomal structural abnormalities, e.g., translocations, duplications, insertions, or or inversions; contiguous gene syndromes, e.g., microdeletion syndromes; genetic effects of various treatments; Use to detect viral nucleic acids in cells and viral integration sites in chromosomes In multicolor FISH, each chromosome is stained a different color, allowing the identification of different This technique allows the determination of the normal chromosome from which the normal chromosome was derived. In addition, multiplex FISH (m-FISH), spectral karyotyping (SKY), and combined dual ratio labeling (COBRA), Color Change Karyotyping, Cross-Species Color Banding, High Resolution Multicolor Banding Chromosome binding, telomere multiplex FISH (TM-FISH), split signal FISH ( ssFISH), and fusion signal FISH.

[0111] CISH and SISH can be used for many of the same applications as FISH, e.g. In histopathology applications, this has the added advantage of allowing analysis of the underlying tissue morphology. When FISH is performed, the hybridization mixture is No. 6,730,474, which is incorporated herein by reference. Contains a set of distinct and balanced probe pairs as described For CISH, the hybridization mixture may comprise one or more Contains at least one set of probes configured for detection using conventional organic chromogens For SISH, the hybridization mixture may be a silver particle probe. The present invention may include at least one set of probes configured for detection, which is described in detail throughout the disclosure. Powell RD et al. al., “Metallographic in situ hybridization n”, as described in Hum. Pathol., 38:1145-59 (2007) It is

[0112] The hybridization buffer of the present invention is suitable for blotting and probing (e.g., Hybridization, including, for example, Northern blotting, as well as in all types of molecular biology techniques involving arrays, either completely or partially It can also be used for.

[0113] The method of the present disclosure includes a hybridization reaction comprising a sulfone solvent and PVSA or a salt thereof. The hybridization buffer may be used in the hybridization of nucleic acid molecules or sequences. The hybridization buffers of the present disclosure are particularly useful in such methods.

[0114] The hybridization method using the hybridization buffer of the present invention is A hybridization buffer and at least one nucleic acid probe in a double-stranded form are added. This may involve applying the probe to a sample containing the target nucleic acid sequence most likely to be present. To ensure that the nucleotide sequence has access to hybridize with the target sequence, the sample as well as the hybridization A hybridization buffer comprising at least one nucleic acid probe and The application composition is mixed and heated to denature the target nucleic acid. During denaturation, the sulfone solvent interacts with the target sequence, denaturing the target sequence and reannealing the probe to the target strand. The sulfone solvents identified in this disclosure significantly speed up this process. This reduces the stringency and toxicity of hybridization.

[0115] Hybridization using the hybridization buffer of the present invention is performed in a manner similar to that of conventional hybridization. The same assay as hybridization carried out using hybridization buffer. However, the hybridization relaxation method of the present invention can be carried out using the The buffer allows for shorter hybridization times. For example, pre-heat treatment, digestion, The denaturation, hybridization, washing, and mounting steps were performed with varying volumes, temperatures, and reagents. The same conditions as for the conventional composition in terms of the concentration and incubation time are used. Conventional hybridization protocols known in the art have significant limitations. For example, some protocols require the following hybridization steps: A separate denaturation step of potential duplex nucleotides in the absence of probe was performed prior to the duplication. The hybridization buffer of the present invention is similar to that of conventional hybridization buffers known in the art. The present invention can be used in any of the hybridization protocols.

[0116] Alternatively, hybridization using the hybridization buffer of the present invention The assay may be modified, for example, by reducing hybridization time, denaturing and / or or increasing or decreasing the hybridization temperature and / or By increasing or decreasing the hybridization volume, the conventional methodology can be modified and and can be optimized.

[0117] According to another aspect of the present disclosure, the hybridization energy is A hybridization composition comprising a hybridization buffer and at least one nucleic acid probe. The hybridization step is thus performed by heating the hybridization product. This may include the steps of heating and cooling the dyeing composition.

[0118] In some embodiments, the denaturation and hybridization steps may occur separately. For example, the sample is denatured with a solution without the probe and then hybridized with the probe. may be omitted.

[0119] A further aspect of the present disclosure is a method for providing a sufficient amount of energy to hybridize nucleic acids. The process used is microwave, high temperature bath, hot plate, heating wire, Peltier element, induction This includes methods that include a heating process carried out by heating or the use of heat lamps.

[0120] According to another aspect, the present disclosure provides a method comprising a hybridization protocol, The hybridization protocol may be performed for less than about 4 hours, alternatively for less than about 3 hours, or is performed in less than about 2 hours, alternatively in less than about 1 hour.

[0121] For example, in some embodiments, the hybridization buffer of the present invention has a denaturation temperature of about 60°C to about 95°C or any temperature between these endpoints, A strong signal is obtained when the temperature is between about 20° C. and about 60° C. or any temperature between these endpoints. In another embodiment, the hybridization buffer of the present invention produces a denaturing temperature about 60°C to about 70°C, or about 70°C to about 80°C, or about 80°C to about 90°C, or The hybridization temperature is about 90° C. to about 95° C. or any temperature between these endpoints. The irradiated temperature is about 20°C to about 30°C, or about 30°C to about 40°C, or about 40°C to about About 50°C, or about 50°C to about 55°C, or about 55°C to about 60°C, or any of their endpoints Any temperature between these will produce a strong signal. The hybridization buffer of the invention has a denaturing temperature of about 72° C., about 82° C., or about 92° C. °C, and the hybridization temperature is about 37°C, about 40°C, about 45°C, about 50°C or In another embodiment, the hybridization method of the present invention produces a strong signal when the temperature is about 55° C. The denaturation buffer should be chosen so that the denaturation time is between about 0 minutes and about 10 minutes or between these two endpoints. The hybridization time is about 0 minutes to about 24 hours or any of these. Any time between the two endpoints produces a strong signal. The compositions of the present invention have a denaturation time of about 0 minutes to about 5 minutes, or any time between these two endpoints. hours, and the hybridization time is from about 0 minutes to about 8 hours, or between these two endpoints. In another embodiment, the hybrid of the present invention generates a strong signal when the The hybridization buffer was used with denaturation times of about 0, 1, 2, and 3 minutes. The hybridization time is about 0 minutes, about 5 minutes, or about 4 minutes. If the time is about 15 minutes, about 30 minutes, about 60 minutes, about 180 minutes, or about 240 minutes, Generates a strong signal. In some cases, e.g., RNA detection, a denaturing process is required. It is understood by those skilled in the art that this is not possible.

[0122] Thus, the hybridization process using the compositions of the present invention can be completed in less than 8 hours. In some embodiments, the hybridization process may be carried out for less than 6 hours. In other embodiments, the hybridization process is carried out for less than about 4 hours. , or within about 3 hours, or within about 2 hours, or within about 1 hour, or within about 3 hours Within 0 minutes, or within about 15 minutes, or within about 10 minutes, or within about 5 minutes Hybridization times are varied to produce a strong signal. Vary the concentration of the probe to achieve high resolution and / or reduce background. For example, if hybridization time is decreased, the amount of probe may be increased. On the other hand, the hybridization time is reduced. The amount of probe can then be reduced to improve background staining.

[0123] In some embodiments, the methods and hybridization buffers of the present invention comprise During the chromatographic process, there is essentially no blocking process or blocking agent. In hybridization protocols, the blocking process is used to block, for example, Blocking the binding of repeat sequences reduces signal and background intensity In the method and hybridization buffer of the present invention, As blocking agents, total human DNA, blocking-PNA, COT-1 DNA, or any There may be no need to use DNA from other sources. However, background The levels can be increased by, for example, adding a substance that reduces non-specific binding to cell membranes, such as a small amount of whole human D NA or DNA of non-human origin (e.g., salmon sperm DNA) can be used in the hybridization of the present invention. Further, a hybridization buffer may be added to the hybridization reaction. It can be further reduced.

[0124] The hybridization buffer of the present invention is used in the hybridization process. It further provides the possibility to significantly reduce the concentration of probe nucleic acid sequences that are present in the probe. The concentration of the lobe can be reduced by 1 / 2 to 1 / 8 compared to the conventional concentration. For example, a HER2 DNA probe and a CEN17 PNA probe are used in the hybridization assay of the present invention. When used with a hybridization buffer, these concentrations are comparable to those used in conventional hybridization. These concentrations can be reduced compared to those in the blotting buffer. There is no requirement for blocking DNA, e.g., blocking-PNA or COT1. Combined with the absence of conventional 10μL or less liquids used in conventional manual systems, This allows for an increase in probe volume in automated instrument systems compared to the volume of the , which reduces evaporative losses, as discussed in more detail below.

[0125] Reducing the probe concentration also reduces background. The decrease is inversely proportional to the hybridization time, i.e., the lower the concentration, the longer the hybridization time. Hybridization time is required. Nevertheless, very low concentrations of probe When used with the hybridization buffer of the present invention, The hybridization time is still shorter than with conventional hybridization buffers.

[0126] The hybridization buffer of the present invention is often used in conventional hybridization. This allows for a better signal to noise ratio than the ethanol buffer. Hybridization for 1 hour using the hybridization buffer of the present invention This is similar to overnight hybridization in conventional hybridization buffer. It produces a signal that is stronger than the background.

[0127] Conventional hybridization assay methods also include the hybridization buffer of the present invention. When buffer solutions are used, the system may be manual, semi-automated, or automated. The method can be modified and optimized depending on how the system is being used, for example in a semi-automated or automated manner. The hybridization system of the present invention has short hybridization times and The short hybridization time benefits from the reduced need for mixing with ELISA buffer. However, problems encountered when conventional hybridization buffers are used in such systems include For example, one problem with semi-automated and automated systems is The problem is that such systems are limited by the small sample volumes (e.g., 10–150 μL), high temperatures, and and longer hybridization times (e.g., 14 hours). Significant evaporation of the sample can occur during hybridization. The proportions of the components in the hybridization buffer are fairly constant. However, The hybridization buffer of the present invention allows for faster hybridization. This reduces evaporation and allows for hybridization in semi-automated and automated systems. This allows for more flexibility in the ratios of components in the application buffer.

[0128] The present disclosure may be more clearly understood with the aid of the following non-limiting examples, which These examples constitute preferred embodiments of the compositions according to the present disclosure. Except as otherwise noted, the amounts of ingredients, reaction conditions, and other terms used in the specification and claims are intended to be illustrative and not restrictive. All numbers expressing quantities, amounts, etc. are, in all cases, modified by the term "about." Accordingly, unless specifically indicated to the contrary, the following specifications The numerical parameters set forth in the specification and appended claims are intended to be within the scope of the present invention. These are approximations that may vary depending on the desired properties as determined. Not as an attempt to limit the application of the principle of equivalence of ranges, should be interpreted taking into account the number of significant digits and ordinary rounding practices.

[0129] Although the numerical ranges and parameters indicating broad ranges are approximations, specific The numerical values ​​set forth in the examples are reported as precisely as possible. However, any numerical values ​​are subject to change without notice. Each test measurement inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. .

[0130] Any ranges set forth herein are intended to include all numbers between the endpoints of the range. It is being done.

[0131] All references cited herein are hereby incorporated by reference in their entirety. is incorporated into.

[0132] The examples which follow are illustrative of the disclosure and are not to be construed as limiting the invention in any way. This should not be allowed. EXAMPLES

[0133] [Example 1] The hybridization buffer according to the present disclosure was tested in a FISH staining procedure. The components of the dilution buffer are shown in Table 2.

[0134] JPEG2025066727000010.jpg50159

[0135] [Example 2] In this example, the hybridization buffer of Example 1 was combined with the probe and the hybridization The hybridization buffer was CY3 / F ITC-tagged DNA-based sureFISH RET break apart (BA) Probe or DNA / PNA base with Texas Red / FITC label A dilution of the HER2 / CEN-17 counting probe from Agilent Technologies In conjunction with probes available from Genome Technologies, a hybridization composition is formed. These hybridization compositions were used to generate hybridization assays for FFPE human tissue samples. FISH staining was performed on slides. The slides were tonsillar, breast cancer, kidney and thyroid tissues. The samples contained IgG and colon tissue. The standard method was used except that a higher denaturation temperature (80°C versus 66°C) was used to The tissue was treated with the hybridization composition according to the histology protocol. The protocol was as follows: 1. Deparaffinization in Xylene and Alcohol 2. Rehydration in Alcohol 3. Pretreatment in microwave for 10 min in pretreatment buffer 4. Pepsinization with pepsin by immersion at 37°C 5. Dehydration in Alcohol 6. Application of Probe-Containing Hybridization Buffer to Tissue 7. Denaturation at 80°C for 10 minutes 8. Hybridization at 45°C for 2 hours 9. Stringent wash in stringent wash buffer at 63°C for 10 minutes 10. Dehydration in Alcohol 11. Mounting in Fluorescent Mounting Medium 12. Fluorescence Microscopy and Assessment of Staining Quality

[0136] After completion of the FISH staining procedure, the samples were analyzed for the detection of Texas Red, CY3 and FI Use fluorescence microscopy with TC-associated filters to visualize the hybridization assay. The staining quality was determined by the composition. The staining result (signal quality) was given a score of 0 to 3. A score of 0 indicates no signal and a score of 3 indicates maximum signal. A score of 2 or greater is acceptable. The staining results are shown. Scores may be given in halves and quarters. The staining scores for FFPE tissues using hybridization buffer are shown in Table 3 below. Probe concentrations are given relative to the commercially relevant 1× concentration.

[0137] JPEG2025066727000011.jpg70159

[0138] Hybridization assays were performed at low probe concentrations compared to commercially relevant probe concentrations. The dye composition, when stained as described above, provides high quality fluorescence of FFPE tissue samples. These results demonstrate that ISH staining provides

[0139] [Example 3] Compared to existing hybridization buffers containing ethylene carbonate, the hybridization buffer of the present disclosure The stability of the hybridization buffer was analyzed in an accelerated stability study. The hybridization buffer (test) and the hybridization buffer included as a control were It had the following components:

[0140] JPEG2025066727000012.jpg53159

[0141] To facilitate stability testing, probes based on the hybridization buffers in Table 4 The mixes were stored at 2-8°C, 22°C and 37°C. The temperatures 2-8°C reflected the desired storage. where temperatures 22°C and 37°C reflect accelerated storage. This is based on the Q rule and the Arrhenius equation. Using a model based on the 1.8 model, the 1.8 at higher temperatures is calculated using a conservative Q10 value. The days are to reflect the greater number of days of desired storage. Probe and control hybridization buffers mixed with the disclosed test hybridization buffer. A hybridization composition containing a hybridization buffer was prepared. A hybridization composition containing the probe mixed with a hybridization buffer is prepared. The following day, the samples were transferred to the storage conditions listed below at time point zero (T0). Generate FISH performance data. 250 μL screw-top PE tubes were placed in this stable Used in the experiment to reduce the volume of air above the probe mix. They were named according to the table below and stored in the designated locations. Prior to any testing, they were mixed with the existing buffer. was performed by pipette mixing to provide worst-case conditions for homogeneity and stability. In addition, the buffer of the present disclosure was not mixed. In addition, samples were drawn for FISH performance testing. In this case, pipetting from these test samples was performed from the upper part of the liquid volume.

[0142] Details of the calculated accelerated storage times are as follows (Tables 5 and 6):

[0143] JPEG2025066727000013.jpg60159

[0144] JPEG2025066727000014.jpg61159

[0145] FFPE tissue blocks were used in this study. Red and green signaling on FFPE tissue The null intensity is calculated at the following times (T0, T1, T2 and T3) as shown below: The following FISH tests were performed (Table 7):

[0146] JPEG2025066727000015.jpg72159

[0147] The FISH protocol consisted of a pepsin soak at 37°C for 30 min, denaturation at 80°C for 10 min, and and hybridization at 45°C for 120 minutes.

[0148] Texas Red and FITC signals from the HER2 / CEN-17 probe or for CY3 and FITC signals from sureFISH RET BA probes. The tissues were then scored according to a standard scoring procedure on a scale of 0 to 3. To monitor possible heterogeneity effects, a hybridization buffer containing the disclosed The test vials were not mixed prior to testing. Control buffer was included as a control. The samples were mixed according to the manufacturer's instructions. Scoring was performed by two independent observers. The study was conducted in a blinded manner, and the results were unblinded after scoring was completed.

[0149] The first fish test was performed at T0 (Table 8) for each batch of probe mix before stable storage. The results at T0 were as follows:

[0150] JPEG2025066727000016.jpg111162

[0151] For each batch of probe mix, a second FISH test was performed at T1 (Table 9). The results for T1 were as follows:

[0152] JPEG2025066727000017.jpg103159

[0153] All vials were equilibrated to room temperature before use. #A and B vials were all probe-compatible. The vials were mixed before use (tip mixing), but the remaining vials were not mixed. This is done to include worst-case scenarios of instability and heterogeneity for C and D. The signal scores are indicated, with green indicating a green signal score.

[0154] A third FISH test was performed at T2 for each batch of probe mix prior to stable storage. The results at T2 were as follows (Table 10):

[0155] JPEG2025066727000018.jpg117159

[0156] The fourth FISH test was performed at T3 for each batch of probe mix before stable storage. The results at T3 were as follows:

[0157] JPEG2025066727000019.jpg99170

[0158] All vials were equilibrated to room temperature before use. All #A and #B vials were The vials were mixed before application of the lobe, but the remaining vials were not mixed. and D to include worst-case scenarios of instability and heterogeneity.

[0159] An analysis of the data collected from the accelerated stability study is presented in FIG. The hybridization buffer was tested at temperatures up to 37°C for comparative hybridization. The results demonstrate that the elution buffer can withstand storage much better than the elution buffer. The x-axis defines the number of equivalent days from the start of acceleration at 5°C, and the y-axis defines the function for different labels. These data provide a useful FISH staining intensity. The slope of the sodium salt buffer is smaller, so the slope of the buffer containing sulfolane and PVSA sodium salt is The hybridization buffer is shown to have better stability than the control buffer.

[0160] Table 12 shows the effect of the disclosed (test) buffers compared to the control buffer under different storage conditions. Transposed mean data (average of observer 1 / observer 2) were provided to demonstrate changes in the condition is doing.

[0161] JPEG2025066727000020.jpg103159

[0162] In conclusion, the accelerated stability data demonstrates that the hybridization buffers of the present disclosure, e.g. SureFI prepared using a buffer containing sulfolane and PVSA sodium salt Very good stability of SH RET BA or HER2 / CEN-17 probe mix The hybridization buffer of the present invention exhibits the following properties: It is more stable than buffers and more convenient to use since no mixing is required.

[0163] Also, samples from a probe mix containing the hybridization buffer of the present disclosure The samples were taken from the top of the tube without mixing at any time during the test. Once the mix is ​​mixed to homogeneity, they are stored at storage temperatures (5°C, 22°C and 37°C). These data also show that the solubility of the solubility-remaining solubility-remaining homogeneous regardless of the solubility of the solubility-remaining ... For hybridization buffer, use a pipette to mix the tip with the sample before use. Thus, the data generated was consistent with the hybridization buffer of the present disclosure. This corresponds to the worst case scenario for a liquid, and even in this scenario, the buffer of the present invention Finally, the SAS-JMP software-based degradation / stability analysis The stability model was shown to be stable for 34 months at 5°C for the hybridization buffer of the present disclosure. It predicts a qualitative minimum.

[0164] [Example 3] The hybridization buffer of the present disclosure is used in conjunction with evaporation and filtration in the capillary field. The antibodies were tested for their robustness and for performance in FISH assays. reported that the hybridization buffer of the present disclosure is less than 13%, 14.3% and 14.9%. % (v / v) sulfolane to determine the solvent concentration that influences water loss / phase separation and FISH performance We compare these to understand how they affect the robustness to

[0165] The different hybridization buffers of the present disclosure that were tested were The solvent concentrations were designated as Tests 1, 2, and 3 (Table 13). Prepare hybridization buffer in a volume of 5 mL using The lysis buffer contained the following components:

[0166] JPEG2025066727000021.jpg42159

[0167] Use of the blue dye erioglaucine to facilitate visual monitoring The homogeneity of the hybridization buffer was determined by the following method. Dispense 1 mL of the dilution buffer into a test tube with a screw cap (2 mL Sarste dt test tubes) plus 4 μL of 5% (w / v) erioglaucine solution added to each tube The sample with erioglaucine was then mixed with Ole Dic The mixture was centrifuged at 5000×g for 1 hour at 2°C or 15°C in a centrifuge. After separation, the hybridization buffer samples were visually assessed for homogeneity and phase separation. The results, provided in Table 14 below, show that the hybridization buffer of the present disclosure is homogeneous and has been demonstrated to maintain homogeneity under the conditions tested.

[0168] JPEG2025066727000022.jpg44159

[0169] The prepared hybridization buffer is then used to generate the capillary field. Hybridizer configuration with spacers at the corners of the slide / coverslip for In a simulated denaturation and hybridization study, The robustness of these was tested. Phase separation during denaturation and hybridization was The distribution of blue dye was changed to a dark blue phase and a more transparent phase, and homogeneous regions were quantified from the images. It is possible.

[0170] To perform these tests, a Flex slide was used as the bottom slide and The Flex slide cut to the staining area (25 × 45 mm) was used as the upper slide. The Kaplon tape was then attached to the four corners of a 25 x 45 mm flex glass. In addition, the capillary gap size was specified. The calculated volume of the capillary field is 25mm x 45mm x 0.1mm = 112.5 mm 3 or μL. Use 125 μL of hybridization buffer as specified. This capillary field is filled using

[0171] Simulated denaturation at 80°C for 10 min and hybridization at 45°C for 120 min. The aliquots were then placed on two moistened hyaluronic acid filters, as used during standard manual FISH staining procedures. The simulation was performed using a hybridizer with a medium strip. Images of the diffusing reagent were taken before and after the simulation to determine the percentage of homogenous buffer area. The calculation was carried out.

[0172] The results from the hybridization simulations summarized in the table below are During the denaturation and hybridization steps of the H procedure, the hybridization sequence of the present disclosure The application buffer is homogenous and maintains acceptable homogeneity in the capillary field. This proves that (Table 15).

[0173] JPEG2025066727000023.jpg35159

[0174] [Example 4] Hybridization buffers of the present disclosure having various NaCl concentrations were then loaded onto capillary tubes. These exemplary hybridization assays of the present disclosure were tested for performance in field experiments. The ionization buffer contains a relatively high NaCl concentration (e.g., 13.1% (v / v) sulfolane, 30% (w / v) PVSA sodium salt, 10% (v / v) DS500, 40 mM MO PS pH 7.2, and 900 mM NaCl). Without any doubt, it is believed that high NaCl concentrations reduce evaporation in the capillary field. In this example, the magnitude of effect and the effect on functional performance are examined. To assess the effect of NaCl on the cellular metabolism, four different NaCl concentrations were tested.

[0175] Add 2.5 mL of the following four exemplary hybridization buffers of the present disclosure (Table 16): 1 mL of each homogenized hybridization buffer was added to blue The color dye was added to a screw-top test tube (1 mL Sarstedt test tube). Add 4 µL of the blue dye erioglaucine (5% per mL of hybridization buffer) Erioglaucine (w / v) was used to facilitate visual monitoring of phase separation. After homogeneity testing by centrifugation as described above, all four buffers were homogeneous, permeable. The solution was clear and no phase separation was observed at room temperature or at 15°C and 2°C.

[0176] JPEG2025066727000024.jpg52163

[0177] Four exemplary hybridization plates of the present disclosure containing various NaCl concentrations were then used. The ethanol buffer was tested for robustness in capillary fields.

[0178] Hybridization buffer is added to the slide to generate the capillary field. In a hybridizer configuration with spacers at the corners of the cover glass, These robustness against heterogeneity during accelerated denaturation and hybridization studies The activity of the denatured and hybridized proteins was tested as described below. Phase separation was characterized by a change in the distribution of the erioglaucine dye to a dark blue phase and a more transparent phase. The homogeneous areas were then quantified from the stained buffer images.

[0179] To perform these tests, a Flex slide was used as the bottom slide and A Flex slide cut to the staining area (25 × 45 mm) was used as the top layer. Kaplon tape was applied to the four corners of a 25 x 45 mm Flex glass. The capillary gap size was defined by the double Kaplon tape layer. The calculated volume of the capillary field is 25mm x 45mm x 0.1mm = 112. 5mm 3 or μL. Add 125 μL of hybridization buffer as specified. Use to fill this capillary field.

[0180] Denaturation at 80°C for 10 min and hybridization at 45°C for 120 min. The simulation was performed using two moistened tissue slides, as used during standard FISH staining procedures. The simulation was performed using a hybridizer with a humidity strip. Images of the diffusing reagent were taken before and after the simulation to determine the homogeneous buffer region. Cents were calculated.

[0181] The calculated areas were based on standard determination of homogeneous areas, and the results are summarized as follows (Table 17):

[0182] JPEG2025066727000025.jpg38159

[0183] Hybridization buffers of the present disclosure containing various NaCl concentrations were then used for FI The performance of FISH staining procedures was analyzed (Table 18). Commercially relevant probe concentrations in hybridization buffer: 0.125x and 0 HER2 / CEN-17 probe was applied to FFPE tissue at a relative concentration of .25× The experiment was carried out using 15% (v / v) ethylene carbonate, 20% (w / v) dextran sulfate, A control buffer containing 600 mM NaCl and 10 mM citrate buffer pH 6.2 Buffer was also included for reference and as a control.

[0184] The FISH staining procedure consisted of pepsin immersion at 37°C for 30 min, denaturation at 80°C for 10 min, and The incubation included 10 min at 45° C. and 120 min at 45° C.

[0185] Use a fluorescent microscope with appropriate filters for Texas Red and FITC. The buffer used was scored blindly by an observer on a 0 to 3 scale. The tissues were scored according to standard scoring procedures, with 0 being signal A positive result of 0.05% was determined by the FISH staining. The results from the colors are as follows:

[0186] JPEG2025066727000026.jpg119159

[0187] In conclusion, the four hybridizations tested with decreasing NaCl concentrations The buffer is homogeneous when prepared and after homogeneity testing at 0 and 15°C. The hybridization buffer of the present disclosure also provides a method for preparing hybridization buffers for use in non-protected capillary fields. The robustness against evaporation was demonstrated in the capillary leaf. Without wishing to be bound by theory, It is believed that modifying the NaCl concentration is likely to cause changes in vapor pressure. The average homogenously stained area decreased by approximately 1% when NaCl was reduced from 900 mM to 300 mM. It was reduced by 8% (nominal 11.5%).

[0188] Regarding FISH staining performance, reducing NaCl from 900 mM to 300 mM The data showed that the red signal score increased slightly by 0.25–0.5 depending on the dye concentration. The data shows.

[0189] [Example 5] To determine whether the source of sulfolane affects the performance of the buffer, The homogeneity and performance of hybridization buffers were obtained from two different manufacturers. In addition, the hybridization buffer of the present disclosure was tested using sulfolane. The inclusion of low concentrations of sodium phosphate was investigated. The following hybridization buffer was used: Tested (Table 19):

[0190] JPEG2025066727000027.jpg73170

[0191] To test for homogeneity, add blue dye (100 ml per mL of hybridization buffer). Add 4 μL of 5% (w / v) erythroglaucine (Erioglaucine) to a tube with a screw cap. 1 mL of each homogenized hybrid in a test tube (2 mL Sarstedt tube) The addition of distillation buffer allowed for easy visual monitoring of homogeneity. The samples with erioglaucine were then centrifuged at 5000 x g in an Ole Dich centrifuge. The samples were centrifuged for 1 h at 0° C., 2° C. and 15° C. After each centrifugation, the samples were analyzed for homogeneity and phase separation. The separation was evaluated visually, with the results being as follows (Table 20):

[0192] JPEG2025066727000028.jpg62159

[0193] The results of the homogeneity test show that the performance of the hybridization buffer of the present invention is consistent with that of the manufacturer. The results do not show any differences between the sulfolane obtained from subjects 1 or 2.

[0194] The hybridization assays of the present disclosure were then performed using sulfolane from different manufacturers. The immunization buffer was subjected to FISH staining procedure performance testing. The commercially relevant probe concentrations in different hybridization buffers were 0.125 Tests were performed using HER2 / CEN-17 probes at relative concentrations of 0.25× and 0.30×. The experiment was carried out using 15% (v / v) ethylene carbonate, 20% (w / v) dextran sulfate, 600 The existing buffer solution containing 10 mM NaCl and 10 mM citrate buffer pH 6.2 is also were included for reference and as controls.

[0195] The standard FISH staining procedure involves pepsin immersion at 37°C for 30 minutes, followed by incubation at 80°C for 10 minutes. Denaturation and hybridization at 45°C for 120 minutes were included.

[0196] Scoring is performed for each slide on a fluorescent microscope with the relevant filters. The study was conducted blinded to temperature. Scoring was done on a standard scale of 0 to 3. A scale was used, with 0 indicating no signal, 3 indicating maximum signal, and 2 indicating the maximum acceptable positive signal. The results are presented in the following table (Table 21):

[0197] JPEG2025066727000029.jpg89159

[0198] The results of the FISH performance test are consistent with the homogeneity data and the sulfolane manufacturer is committed to the use of the mitigation methods disclosed herein. It has been demonstrated that it has no effect on the buffer.

[0199] [Example 6] The hybridization buffers of the present disclosure were tested at various hybridization temperatures. The hybridization buffer of the present invention is compatible with certain existing FISH hybridization enzymes. They are more stable than ionization buffers and therefore have a broader range of hybridization This example is expected to be able to support temperature. Hybridization buffer composition with SureFISH RET BA probe Hybridization buffers of the present disclosure when used in Evaluate the reaction temperature.

[0200] The hybridization buffer of the present disclosure used in this example was 13.1% (v / v v) Sulfolane, 30% (v / v) PVSA sodium salt, 10% (v / v) DS500 , 40 mM MOPS pH 7.2, and 900 mM NaCl. For FFPE breast tissue, commercial relevant probe concentrations of 0.125× and 0.2 HER2 / CEN-17 in hybridization buffer at a relative concentration of 5x The RET probe mix was used in the experiment. A standard FISH staining protocol was used, including denaturation at 80°C for 10 min and incubation at 4°C for 10 min. Redization was carried out at test temperatures ranging from 35 to 50°C for 120 minutes as follows: (Table 22):

[0201] JPEG2025066727000030.jpg156159

[0202] Scoring was performed using a fluorescent microscope with the relevant filters to identify hybridization sites. The FFPE tissues were scored according to standard scoring procedures. The samples were scored on a scale of 0 to 3, with 0 indicating no signal and 3 indicating maximum signal. 1 indicates positive signal, and 2 indicates acceptable positive signal. The results from FISH staining are as follows: be:

[0203] JPEG2025066727000031.jpg174159

[0204] Using the hybridization buffer of the present disclosure, the data in Table 23 were obtained at 38°C to A wide hybridization temperature range of 50°C was achieved with low concentrations of HER2 / CEN-17 and and RET BA probe sets resulting in acceptable signal strength. This clearly shows:

[0205] [Example 7] Certain existing hybridization buffers often have capillary fillers. In the field, they exhibit high viscosities that present challenges in terms of handling, pipetting, and spreading. However, the hybridization buffer of the present disclosure still produces high quality FISH staining results. In this experiment, the hybridization method of the present disclosure was The viscosity of the buffer solution is determined at the relevant handling temperatures, e.g., 15, 20, 25, 32 and 37°C. The viscosity of the hybridization buffer is compared to that of a control hybridization buffer at .

[0206] The hybridization buffer of the present disclosure tested (test) was 13% (v / v) sulfo. Ran, 10% (w / v) dextran sulfate, 30% (w / v) sodium polyvinylsulfonate sodium salts, 200 mM NaCl and 50 mM phosphate buffer, pH 7.4. The control hybridization buffer was 15% (v / v) ethylene carbonate, 20% (w / v) dextran sulfate, 600 mM NaCl and 10 mM citrate buffer, It contained a pH of 6.2.

[0207] A microviscometer working by the rolling ball principle was calibrated at 25 °C and according to the manufacturer's instructions. The test hybridization buffer or control hybridization buffer of the present disclosure were used according to the method described above. The device was filled with lysis buffer and the viscosity and density were recorded at different temperatures. The results from the viscosity and density measurements are presented in the following table (Table 24):

[0208] JPEG2025066727000032.jpg43159

[0209] The data from this experiment are summarized in Figure 2, where viscosity is plotted on the y-axis. The x-axis is plotted against temperature. The lysis buffer had a pH of 62.9 mPa.s at 25°C relative to the control buffer. The results demonstrate that the viscosity of the cellulose acetate ester is reduced from 1.0 to 38.3 mPa.s. This corresponds to a roughly 40% decrease at higher temperatures. At higher temperatures, the decrease is slightly more than 40%. At lower temperatures, the reduction is slightly less than 40% (see table above). The results of the study show that the disclosed buffers, when compared to a control hybridization buffer, , it has been determined that the solubility of the solubility-containing polymer exhibits a lower viscosity.

[0210] Exemplary embodiments Exemplary embodiments provided in accordance with the presently disclosed subject matter are not limited to But includes: 1. A sulfone solvent having a concentration of about 5 to about 30% (v / v) and a compound represented by formula (I), (Ia), or (II) , (IIa) or (III): [ka] [In the formula, R 1 is hydrogen or C 1~6 aliphatic, and L, if present, is a covalent bond or C 1~6 Aliphatic, R 2 is a covalent bond, if present, C 1~6 Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms, R 2 is one or two substituted with a SO3H group or its salt, except that R 2 If is a covalent bond, one Only SO3H groups or their salts are present, and R 2 -R 3 , halogen, -CN , -NO2, -OR 3 , -N(R 3 )2, and -SR 3 1 to 2 independently selected from Optionally substituted with one group, each R 3 are independently hydrogen or C 1~6 Alki n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I) and any one of the salts of sulfo The hybridization method includes the steps of: Isolation buffer. 2.R 1 2. The hybridization buffer of embodiment 1, wherein 3.R 1 C 1~6 2. The hybridization buffer of embodiment 1, which is aliphatic. 4. The hybridization method according to any one of embodiments 1 to 3, wherein L is a covalent bond. Buffer solution. 5. L is C 1~6 The hybridization enzyme according to any one of embodiments 1 to 3, which is aliphatic. Isolation buffer. 6. The hybridization method according to any one of embodiments 1 to 3, wherein L is methylene. Buffer solution. 7. The hybridization buffer according to any one of embodiments 1 to 6, further comprising water. liquid solution. 8. The sulfonic acid polymer or its salt is polyvinyl sulfonic acid or its salt. 8. The hybridization buffer according to any one of aspects 1 to 7. 9. Any of embodiments 1 to 8, wherein the concentration of the sulfone solvent is about 10% to about 20% (v / v). 2. A hybridization buffer according to claim 1 . 10. The sulfone solvent is dimethyl sulfone, diphenyl sulfone, methyl phenyl sulfone. tetramethylene sulfone (sulfolane), and mixtures thereof. 10. The hybridization buffer according to any one of embodiments 1 to 9, 11. The hybrid according to any one of the preceding embodiments 1 to 10, wherein the sulfone solvent is sulfolane. Hybridization buffer. 12. The concentration of the sulfonic acid polymer or its salt is about 20% to about 40% (w / v); 12. The hybridization buffer according to any one of embodiments 1 to 11. 13. Dextran sulfate is not more than 20% (w / v), or not more than 15% (w / v), or or at a concentration of 10% (w / v) or less. of hybridization buffer. 14. When measured by microviscosity measurement, the viscosity is approximately 60 mPa.s or less at 25°C. or has a viscosity of about 50 mPa.s or less, or about 40 mPa.s or less. 14. A hybridization buffer according to any one of claims 1 to 13. 15.2-8°C for at least 2 months or at least 4, 6, 8, 12, 16, 15. The hybridization method according to any one of embodiments 1 to 14, which is stable for 18 or 24 months. Dimerization buffer. 16. At least 2 months at 2-8°C or at least 4, 6, 8, 12, 16, 16. The method according to any one of claims 1 to 15, which is homogenous after storage for 18 or 24 months. Hybridization buffer. 17. Hybridization buffer should be stored at 2-8°C for at least 2 months or Maintains homogeneity and uniformity after storage for at least 4, 6, 8, 12, 16, 18 or 24 months. The quality of the hybridization buffer with erythroglaucine was determined by centrifugation. The clarity of the redox buffer is determined by visual assessment. 17. The hybridization buffer according to any one of 1 to 16. 18. Consists of buffers, salts, enhancers, chelating agents, detergents, blocking agents, and combinations thereof Any of embodiments 1 to 17, further comprising at least one additional component selected from the group: A hybridization buffer according to any one of claims 1 to 4. 19. NaCl and 3-(N-morpholino)propanesulfonic acid (MOPS) 19. The hybridization buffer according to any one of embodiments 1 to 18, comprising 20. Enhancers, salts, at a concentration of about 20% (w / v) or less, or about 10% (w / v) or less and a buffer, the sulfone solvent being sulfolane at a concentration of about 10% (v / v). and the sulfonic acid polymer or its salt is at a concentration of about 30% (w / v). 19. The hybridization buffer according to any one of claims 19 to 20. 21. A hybridization buffer according to any one of embodiments 1 to 20, and at least A hybridization composition comprising at least one nucleic acid probe. 22. A method for hybridizing nucleic acid sequences, comprising: At least one nucleic acid probe is allowed to hybridize to a target nucleic acid sequence for at least a sufficient period of time. applying a hybridization buffer to the target nucleic acid sequence; The hybridization buffer contains a sulfone solvent at a concentration of about 5 to about 30% (v / v) and (I), (Ia), (II), (IIa) or (III): [ka] [In the formula, R 1 is hydrogen or C 1~6aliphatic, and L, if present, is a covalent bond or C 1~6 Aliphatic, R 2 is a covalent bond, if present, C 1~6 Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms, R 2 is one or two substituted with a SO3H group or its salt, except that R 2 If is a covalent bond, one Only SO3H groups or their salts are present, and R 2 -R 3 , halogen, -CN , -NO2, -OR 3 , -N(R 3 )2, and -SR 3 1 to 2 independently selected from Optionally substituted with one group, each R 3 are independently hydrogen or C 1~6 Alki n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I), and the sulfonic acid polymer or its salt is about 5 to about The method is present at a concentration of 50% (w / v). 23. The method of embodiment 22, wherein the target nucleic acid sequence is present in an in situ biological sample. . 24. The embodiment further comprising the step of denaturing the target nucleic acid sequence at a temperature of about 65° C. or higher. 24. A method according to any one of 22 to 23. 25. A method for hybridizing at least one nucleic acid probe to a target nucleic acid sequence, comprising: 25. The method of any one of embodiments 22-24, wherein the time period is 4 hours or less. 26. Hybridization of at least one nucleic acid probe to a target nucleic acid is performed at 50°C or higher. 26. The method of any one of embodiments 22 to 25, wherein the method is carried out at a temperature below 100° C. Obtaining the hybridization buffer from storage at a temperature of 27.8° C. or less; Mix the probe with the hybridization buffer before combining it with the hybridization buffer. The hybridization buffer is mixed with at least one nucleic acid probe without mixing the hybridization buffer. 27. The method according to any one of embodiments 22 to 26, further comprising the step of combining. 28. The sulfone solvent is dimethyl sulfone, diphenyl sulfone, methylphenyl sulfone. tetramethylene sulfone (sulfolane), and mixtures thereof. The method according to any one of embodiments 22 to 27, 29. The method of any one of embodiments 22-28, wherein the sulfone solvent is sulfolane. 30. A method for preparing a hybridization composition, comprising: The buffer solution is stored at a certain temperature for at least 2 months, or for at least 4, 6, 8, 12, or 16 months. 18 or 24 months, followed by the step of storing the hybridization buffer. The nucleic acid sequence is combined with at least one nucleic acid probe to form a hybridization composition. and 31. The method according to embodiment 30, wherein the hybridization buffer is stored at a temperature above 0° C. The method described.

[0211] The foregoing description of exemplary or preferred embodiments does not limit the invention as defined by the embodiments. As will be readily appreciated, the present invention should be considered illustrative rather than defining. Many variations and combinations of the features described above may be made without departing from the invention as described in the embodiments. Such variations are not to be regarded as a departure from the scope of the invention. and all such variations are intended to be included within the scope of the following embodiments. All references cited herein are incorporated by reference in their entirety. do.

Claims

1. a sulfone solvent at a concentration of about 5 to about 30% (v / v); Formula (I), (Ia), (II), (IIa) or (III): 【Chemistry 1】 [In the formula, R １ is hydrogen or C １～６ aliphatic, and L, if present, is a covalent bond or C １～６ is aliphatic, R ２ is a covalent bond, if present, C １～６ Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms; R ２ is one or two SO ３ H group or its salt, with the proviso that R ２ If is a covalent bond, one Only SO ３ H group or its salt is present, R ２ is -R ３ , halogen, -CN , -NO ２ , -OR ３ , -N(R ３ ) ２ , and -SR ３ 1 to 2 independently selected from R ３ are independently hydrogen or C １～６ Alki and n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I) any one of the salts wherein the sulfonic acid polymer or salt thereof is , a hybridization buffer present at a concentration of about 5 to about 50% (w / v).

2. R １ The hybridization buffer of claim 1 , wherein is hydrogen.

3. R １ C １～６ 2. The hybridization buffer of claim 1 which is aliphatic.

4. The hybridization method according to any one of claims 1 to 3, wherein L is a covalent bond. buffer solution.

5. L is C １～６ The hybridization enzyme according to any one of claims 1 to 3, which is aliphatic. ion buffer.

6. 4. The hybridization method according to claim 1, wherein L is methylene. buffer solution.

7. The hybridization buffer according to any one of claims 1 to 6, further comprising water. liquid.

8. The sulfonic acid polymer or its salt is polyvinyl sulfonic acid or its salt.

8. The hybridization buffer according to any one of claims 1 to 7.

9. 9. Any of claims 1 to 8, wherein the concentration of the sulfone solvent is about 10% to about 20% (v / v). A hybridization buffer according to any one of claims 1 to 4.

10. The sulfone solvent is dimethyl sulfone, diphenyl sulfone, methylphenyl sulfone, tetramethylene sulfone (sulfolane), tetramethylene sulfone (sulfolane), and mixtures thereof 10. The hybridization buffer according to claim 1 ,

11. 10. The hybrid of claim 1, wherein the sulfone solvent is sulfolane. Redox buffer.

12. The concentration of the sulfonic acid polymer or its salt is about 20% to about 40% (w / v); A hybridization buffer according to any one of claims 1 to 11.

13. 13. Any of claims 1 to 12, further comprising dextran sulfate at a concentration of 20% (w / v) or less. A hybridization buffer according to any one of claims 1 to 4.

14. It has a viscosity of about 60 mPa.s or less at 25°C as measured by microviscometry. The hybridization buffer according to any one of claims 1 to 13,

15. The composition according to any one of claims 1 to 14, which is stable at 2 to 8°C for at least 2 months. of hybridization buffer.

16. 16. Any of claims 1 to 15, which is homogeneous after storage at 2-8°C for at least 2 months.

2. The hybridization buffer according to claim 1.

17. The hybridization buffer is equilibrated after storage at 2-8° C. for at least two months. and said homogeneity is maintained by said hybridization relaxation with erioglaucine. After centrifugation of the buffer, the transparency of the hybridization buffer is evaluated visually. The hybridization according to any one of claims 1 to 16, buffer solution.

18. From the group consisting of buffers, salts, enhancers, chelating agents, detergents, blocking agents, and combinations thereof.

18. Any of claims 1 to 17, further comprising at least one additional component selected from the group consisting of 2. A hybridization buffer according to claim 1.

19. Further containing NaCl and 3-(N-morpholino)propanesulfonic acid (MOPS) A hybridization buffer according to any one of claims 1 to 18.

20. an enhancer at a concentration of about 20% (w / v) or less; Salt, and Buffer Further comprising: the sulfone solvent is sulfolane at a concentration of about 10% (v / v); The sulfonic acid polymer or salt thereof is at a concentration of about 30% (w / v); 20. A hybridization buffer according to any one of claims 1 to 19.

21. A hybridization buffer according to any one of claims 1 to 20, and at least A hybridization composition comprising at least one nucleic acid probe.

22. 1. A method for hybridizing nucleic acid sequences comprising: At least sufficient to hybridize at least one nucleic acid probe to a target nucleic acid sequence. The at least one nucleic acid probe and the hybridization buffer are then subjected to the hybridization for a period of time. applying to a target nucleic acid sequence, said hybridization buffer comprising from about 5 to about 100 μl of A sulfone solvent at a concentration of about 30% (v / v) and a compound of formula (I), (Ia), (II), or (IIa ) or (III): 【Chemistry 2】 [In the formula, R １ is hydrogen or C １～６ aliphatic, and L, if present, is a covalent bond or C １～６ is aliphatic, R ２ is a covalent bond, if present, C １～６ Aliphatic, Phenyl, or a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms; R ２ is one or two SO ３ H group or its salt, with the proviso that R ２ If is a covalent bond, one Only SO ３ H group or its salt is present, R ２ is -R ３ , halogen, -CN , -NO ２ , -OR ３ , -N(R ３ ) ２ , and -SR ３ 1 to 2 independently selected from R ３ are independently hydrogen or C １～６ Alki and n is an integer greater than 10. or a sulfonic acid polymer of formula (I), (Ia), (II), (IIa) or (II I), wherein the sulfonic acid polymer or salt thereof has a molecular weight of about 5 is present at a concentration of about 50% (w / v).

23. 23. The method of claim 22, wherein the target nucleic acid sequence is present in an in situ biological sample.

24. 2. The method of claim 1 , further comprising the step of denaturing the target nucleic acid sequence at a temperature of about 65° C. or higher.

24. The method according to claim 22 or 23.

25. A nucleic acid probe comprising a nucleic acid sequence sufficient to hybridize to said target nucleic acid sequence.

25. The method of any one of claims 22 to 24, wherein the period of time is 4 hours or less.

26. Hybridization of the at least one nucleic acid probe to the target nucleic acid is 50 26. The method of any one of claims 22 to 25, carried out at a temperature of not more than °C.

27. Obtaining said hybridization buffer from storage at a temperature below 8° C.; The hybridization buffer is then added to the probe prior to combining the probe with the hybridization buffer. The hybridization buffer is added to the at least one Combining with a species nucleic acid probe 27. The method of any one of claims 22 to 26, further comprising:

28. The sulfone solvent is dimethyl sulfone, diphenyl sulfone, methylphenyl sulfone, tetramethylene sulfone (sulfolane), tetramethylene sulfone (sulfolane), and mixtures thereof 28. The method of any one of claims 22 to 27, wherein

29. 29. The method of any one of claims 22 to 28, wherein the sulfone solvent is sulfolane. Law.

30. 1. A method for preparing a hybridization composition comprising: The hybridization buffer is incubated at a certain temperature for at least 2 months, or storing for 4, 6, 8, 12, 16, 18 or 24 months; The hybridization buffer is then combined with at least one nucleic acid probe. forming a hybridization composition; A method comprising:

31. 31. The method of claim 30, wherein the hybridization buffer is stored at a temperature above 0°C. Method of posting.

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