MXPA98003063A - Methods for preparing solid supports for hybridization and reducing non-specified fund - Google Patents

Abstract

The present invention relates to a method related to solid supports for hybridization. The present invention provides methods for preparing solid supports and their use in hybridization analysis, so that the non-specific background on solid supports is reduced. The non-specific background reduction allows the detection of low levels of specific binding that could normally be masked by the non-specific binding. The methods can be applied to a variety of target ligands and probes, including nucleic acids such as oligonucleotide

Description

METHOD FOR PREPARING SOLID SUBSTITUTES FOR HIDING AND REDUCING BACKGROUND NON-PECIFY Technical Field The present invention is generally directed to methods for preparing solid supports for use in hybridization reactions. This invention relates more particularly to processes for preparing solid hybridization supports such that the non-specific background is reduced by chemical treatment of the supports. BACKGROUND OF THE INVENTION Hybridization technology is a powerful tool for identifying pairs of molecules with a complementary relationship to each other. This technology has been applied to a variety of types of molecules, including n-ucleic acids and proteins. For example, nucleic acid hybridization is a well-known and documentary method for identifying specific nucleic acid sequences. Hybridization of n-nucleic acids is based on the evaluation of bases of complementary nucleic acid strands. When single-stranded nucleic acids are incubated in appropriate buffer solutions, the complementary base sequence forms stable double-stranded molecules. The presence or absence of said evaluation can be detected by a number of different methods known in the art. Most of the previously described hybridization assays involve multiple steps such as the hybridization techniques described by Dunn and Hassel in Cell 12:23 (1977). The normal hybridization analysis protocol for detecting a target nucleic acid in a complex population of nucleic acids can generally be described as follows. The target nucleic acids are separated by size in a gel matrix (electrophoresis), or cloned and isolated, or subdivided into pools or used in a complex population. The target nucleic acids are then transferred, or located or in some way immobilized on a solid support, such as a nylon membrane or a nitrocellulose membrane. (This "immobilization" can also be referred to as "disposition"). The immobilized nucleic acids are then subjected to a heating step by UV radiation which irresistibly immobilizes the nucleic acid. The membranes are then immersed in one or more of the "blocking agents" including Denhardt's reagent (Denhardt, Biochem. Biophys, Res. Comm. 23: 641 (1996)), heparin (Singh and Jones, Nucleic Acids Res. 12: 5627 (1984)), and fat-free dry milk (Jones et al., Gene Anal. Tech. 1: 3 (1984)). Frequently, these reagents are used in combinations with single-stranded DNA and detergents such as sodium dodecyl sulfate (SDS). In Northern blot analysis of non-abundant sequences or hybridizations using RNA probes, or single copy Southern hybridizations, Denhardt's reagent is generally used with 0.5% SDS and 100 micrograms / ml of denatured fragmented DNA (50x of Denhardt's reagent consists of 1% w / v of Ficoll (type 400, Pharmacia), 1% w / v of polyvinylpyrrolidone, 1% w / v of bovine serum albumin (fraction 5). Blocking agents are generally included In the prehybridization step and the hybridization steps when nitrocellulose is used, however, when nucleic acid is immobilized on nylon membranes, the blocking agents are generally omitted from the hybridization solution given to the high concentrations of protein with strengthening. The probe with its white nucleus The last problem is particularly noticeable when using short probes, such as oligonucleotides. then they are usually tested with labeled "signal" nucleic acid under stringent hybridization conditions. The signal nucleic acids are then frequently detected using a conjugated enzyme in which the conjugated enzyme has a member of a pair of ligands. The signal nucleic acid contains the other member of the pair of ligands. The unbound enzyme is washed and the membrane is immersed in a solution of substrates. The signal is then detected by colorimetric means, by fluorescence or chemiluminescence, depending on the type of substrate. In short, the hybridization analysis protocol can be summarized as follows: the target nucleic acid is immobilized in the following manner on a solid support. The solid support is treated with blocking agents to prevent parasitic binding (non-specific binding); also known as "background") of probes. The solid support is then probed and the signal is detected by a variety of means. The use of a blocking agent to reduce non-specific binding is essential for a number of reasons, including being able to detect low levels of specific binding that could be masked by non-specific binding if the latter is blocked. Unfortunately, the use of traditional blocking agents such as those described above has never been a reproducible method. Although volumes have been written on the subject, it is virtually impossible to "block" uniformly, for example a 20.32 cm or 30.48 cm piece of nitrocellulose or nylon membrane using a combination or cocktail of blocking reagents or Ficoll compounds (type 400, Pharmacia), polyvinyl pyrrolidone, bovine serum albumin (fraction 5, Sigma), fragmented single nucleic acid, dairy products, etc. The methods of the present invention as described herein overcome this limitation of previous methods. Due to the problems associated with current approaches for reducing non-specific backgrounds in hybridization reactions, there is a need in the art for new methods. The present invention meets this need and also provides other related advantages. SUMMARY OF THE INVENTION In summary, the present invention provides methods for preparing solid hybridization supports and their use in hybridization reactions. In one aspect, the present invention provides a method for preparing solid hybridization supports that reduce non-specific backgrounds, comprising the steps of: (a) contacting a solid support for hybridization with a ligand under conditions sufficient to immobilize the target ligand to the solid support; and (b) reacting the solid support containing the immobilized white ligand with a compound of conditions sufficient to block non-specific sites, thereby producing a blocked solid support containing immobilized white ligand, the compound having the formula: (I) (II) (VII) (VIII) where R-. - R? 7 are independently selected from H, OH; CHs, CH2-CH3, CH = CH-CH3, X, CH2X, CHX2, CH2-CH2X, CH2-CHX2, CX3, CX2-CX3, CX2-CX2-CX3 and C (= O) CH3 and R1 and R3 or R2 and Rs or Rs and R6 can be taken together as = CH2, and Rs and Re can be taken together as = O, and wherein each X is independently selected from halogen. In a preferred embodiment, the method further includes, after step (b), a step comprising substantially removing all said compound that has not reacted with the blocked solid support containing immobilized white ligand. Also within the present invention there is provided equipment that provides a solid support, containing immobilized white ligand, prepared with any of the above methods. In another aspect, the present invention provides a method for reducing a non-specific background in hybridization reactions, comprising the steps of: (a) contacting a solid support for hybridization with a ligand under conditions sufficient to immobilize the target ligand to solid support; and (b) reacting the solid support containing the immobilized target ligand with a compound under conditions sufficient to block non-specific sites, thereby producing a blocked solid support containing immobilized white ligand, the compound having the formula: (i) (II) ( DI) (IV) (VII) (VIII) wherein R *, - R1 7 are independently selected from H, OH, CH3, CH2-CH3, CH = CH-CH3, X, CH2X, CHX2, CH2-CH2X, CH2-CHX2, CX3, CX2-CX3, CX2-CX2-CX3 and C (= O) CH3 and R1 and R3 or R2 and R5 or Rs and Rep are taken together as = CH2, and Rs and Rβ can be taken together as = O, and where each X it is independently selected from halogen; (c) contacting the blocked solid support containing immobilized white ligand with a probe under conditions sufficient for specific binding to the immobilized target ligand; and (d) detecting the presence of the probe on solid support, thus determining the hybridization between the target ligand and the probe. In a preferred embodiment, the method further includes, between steps (b) and (c), a step comprising substantially removing any compound that has not been reacted with the blocked solid support by keeping the target ligand immobilized. In another preferred embodiment, the method further includes, between steps (c) and (d), a step comprising substantially removing all of the probe that has not been bound to said immobilized target ligand. In another preferred embodiment, the method additionally includes between steps (b) and (c), a step comprising substantially removing all of the compound that has not been reacted with the blocked solid support containing the immobilized white ligand and additionally included among Steps (c) and (d), a step comprising substantially eliminating the entire probe that has not bound to the immobilized target ligand.

In preferred embodiments, the compound is according to Formula I wherein R-, and R2 are CH = CH-CH3, X, CH2X, CHX2, CH2-CH2X, CH2-CHX2, CX3, with each X being independently selected from halogen In other preferred embodiments, the compound is according to formula II wherein R- > and R4 are H; or T is CH3 and R2-R are H; or R < Is CH3 and R2-R are H, or R2 and R are H and R, and R3 were taken as = CH2, or R *, and R2 are H and R3 and R are C (= O) CH3. In other preferred embodiments, the compound is according to formula III wherein R? -R ,, are H; or R-. is CH3 and R2 is H; or R, and R2 are CH3; or R, is X and R2 is H; or R1 and R2 are X; with each X it is independently selected from halogen. In other preferred embodiments of compounds, according to formula IV wherein R? -R6 are H; or R1 and R5 are H and R6 is CH3; or Rt and R3 are CH3 and R2, R, Rs and Re are H; or RI-R4 are H and R5 and Re are CH3; or R1-R4 are H and Rs is CH3 and Re is CH CH3; or Rn-Re are X; or R, and R6 so H and R3 and R are C (= O) CH3 and Rs and Re are taken together as = O. In other preferred embodiments, the compound is according to formula VI wherein R7-R9 are H. In other preferred embodiments, the compound is according to formula VII wherein R? 0-? 3 are H. In other embodiments preferred compounds is according to formula VIII wherein R14 and R17 are X and R15 and R1S are H; or R14-R 7 are X; or R14 is OH and R15 and R17 are H. These and other aspects of the present invention will be apparent by reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION As noted above, the use of traditional blocking agents has never been a reproducible method to uniformly block specific binding to solid hybridization supports. The methods of the present invention overcome this limitation of the methods of the prior art by achieving a uniform level of blocking of non-specific sites on solid hybridization supports such as nitrocellulose or nylon membranes. The present invention represents a significant improvement in the field of hybridization in that the non-specific background reduction substantially increases the signal-to-noise ratio. This allows, for example, the replacement of radiolabeled probes with chemically labeled probes that are compatible with fluorescence or chemiluminescence signal systems. In addition, by eliminating the need for traditional blocking agents, the present invention has the additional advantages of decreasing the risk of nuclease or protease contamination and decreasing the cost of hybridizations that are carried out on a large scale. Hybridization assays are useful for identifying molecules that are capable of binding to a selected molecule. As used herein, the term "hybridization" refers to the specific binding between any two molecules including, for example, the union between two nucleic acid molecules. Such hybridization analyzes usually involve a series of steps that can be described generally as before. In short, a molecule, ie a white ligand, is immobilized on a solid support. The solid support is treated with a blocking agent to decrease the specific binding of a probe molecule. The solid support containing the immobilized target molecule is probed with a candidate binding partner (ie a probe molecule). The presence or absence of the probe molecule bound to the solid support is detected, thus determining whether hybridization has occurred between the target ligand and the probe. As noted above, before the probing step, a solid support is prepared so that a white molecule is immobilized thereto and the specific binding sites are blocked. A variety of solid supports can be used as the hybridization matrix within the present invention and are well known in the art. Any solid support able to immobilize a white ligand without significantly damaging its ability to bind to a binding partner is adequate. Solid supports having preactivated surfaces to immobilize a white ligand are well known to those in the art and are commercially available. Solid supports include both porous and non-porous solid supports. Solid non-porous supports include porous membranes. Examples of porous membranes include nitrocellulose membranes (e.g., Schleicher and Schuell, Keene, N H) and nylon membranes (eg, Amersham, Arlington Heights, I L, or Schleicher and Schuelle, Keene, N H). Solid non-porous supports include microbeads, glass surfaces and fused silica. Examples of non-porous microbeads include magnetic beads, polystyrene, Teflon®, nylon, silica and latex. Magnetic beads can be obtained from PerSeptive Diagnostics (Cambridge, MA) or Dynal (Oslo, Norway). Latex, silica and other types of beads can be obtained from Polisciences (Warrington, PA). Where the white ligand is a nucleic acid, the particularly preferred solid supports are a nitrocellulose membrane, a nylon membrane or a glass surface. Where the white ligand is a protein, the particularly preferred solid supports are a nitrocellulose membrane or a nylon membrane. A white ligand was immobilized on a solid support such as those described above. A variety of molecules can be used as a white ligand within the present invention. Any molecule capable of immobilization on a solid support is suitable. White ligands include nucleic acids, proteins and small organic or bio-organic molecules (e.g., natural products). As used herein, the term "nucleic acid" includes deoxyribonucleic acid (AD N that includes genomic DNA and cDNA), ribonucleic acid (including RNA, mRNA, rRNA and tRNA), oligonucleotides and nucleic acid analogs. As used herein, the term "protein" includes proteins (such as enzymes and antibodies), polypeptides, peptides (ie, more than two amino acids) complexes of proteins and amino acid analogues. "Proteins" that are negatively charged are white ligands of preferred proteins. White ligands can be obtained in a variety of ways including commercial sources, purification of biological sources, recombinant production and synthetic chemical preparation. By selecting a white ligand such as those described above, it is immobilized on a solid support. A variety of immobilization procedures can be used within the present invention and are well known to those skilled in the art. Any procedure and mobilization that deposits or binds a white ligand without significantly damaging its ability to join an appropriate binding partner. Traditional means for immobilization on a solid support include heat or UV radiation. For immobilization by heating (e.g., baking under vacuum), temperatures typically vary from about 50 ° to 100 ° C for a period of about 1 minute to 60 minutes. For immobilization by irradiation, the UV power typically varies from about 1,000-1,000,000 microjoules / cm 2 for a period of about 10 seconds to 5 minutes. By subjecting a solid support in the presence of a white ligand to one of a variety of said reaction conditions, the white ligand is immobilized irreversibly on solid support. As noted above, the present invention provides a different method for blocking non-specific binding sites on solid supports prepared for use in hybridization reactions. Non-specific binding sites allow the binding of a candidate binding partner (probe) or a solid support in a manner that is independent of the white ligand immobilized on solid support. The nonspecific binding of a probe or a solid support having immobilized white ligand gives the appearance that the probe is a binding partner of the target ligand. Within the present invention, after immobilization of a white ligand on a solid support, the solid support is reacted with one or more compounds such as those set forth below to block nonspecific binding sites on the solid support. Compounds that can be used in the present invention include the anhydrides described by the following formulas I-VI I I: (I) (II) n) (IV) (VII) (VIII) The compounds represented by the formulas I-VI I I have the substituents R-i to R17 as noted above. Each Ri to R17 is independently selected (i.e., the selection of a substituent can be made without considering the selection of any other substituents) from the substituents which include the following: H, OH, CH3, CH2-CH3, CH = CH -CH3, X, CH2X, CHX2 > CH2-CH2X, CH2-CHX2, CX3, CX2-CX3, CX2-CX2-CX3 and C (= O) C H3. Each X is independently selected from halogen, ie F, Cl, Br and I. In addition, substituents attached to the same carbon in the ring can be taken together as = CH or = O. For example, any or all of R < ? and R3, R2 and R or R5 and R6 can be taken together as = CH2. Similarly, for example, Rs and RT can be taken together as = 0. A variety of anhydrides are commercially available (e.g., Aldrich, Milwaukee, Wl) or can be synthesized using methods known in the art (see, procedures described in March and references cited therein. Advanced Organic Chemistry, 2nd edition, McGraw-Hill, New York, N.Y. (1977)). It will be apparent that those in the art when they have the present disclosure, that variations on the above compounds are contemplated by, and can be used in, the practice of the present invention and are within the spirit and scope of the invention. A suitable compound is reacted with a solid support containing immobilized white ligand under conditions to block non-specific sites. In summary, a compound is generally mixed with a solvent, such as polar solvent. The solution is added to the solid support for a time ranging from about several minutes to several hours at a temperature generally ranging from about room temperature to below the boiling point of the solvent. For example, succinic anhydride is mixed to produce a fine concentration of about 0.01 mg to 10 mg per ml. For a solvent such as m-pyrrole, acetonitrile, or other polar solvents, containing from about 0.01 to 0.5 M sodium borate at a pH of about 7 to 9. The reaction with the solid support is allowed to proceed for about 2 to 60. minutes at a temperature of about 20 ° to 37 ° C. Where a particular white ligand can react with the compound and such a reaction could significantly affect the ability of the target ligand to bind to a probe, it may be desirable to protect it against said reaction between the target ligand and the compound. This can be achieved in a variety of ways. ways including protecting the target ligand or adjusting the conditions under which the compound is reacted with a solid support containing immobilized white ligand. For example, the pH can be adjusted to a pH that allows substantial reaction of the solid support with the compound, but allows the substantial reaction of the white ligand with the compound. Alternatively, for example, functional groups on the white ligand that can react with the compound can be reversibly protected. The white ligand is reacted with one or more protective chemicals that prevent reaction with the compound and the protecting groups are removed from the white ligand, after the compound has been reacted with the solid support containing immobilized white ligand. Techniques for the reversible protection of functional groups on molecules are known to those skilled in the art (e.g., methods described in Greene and references cited therein: TW Greene, Protective Groups in Organic Synthesis, John Wiley and Sons, New York, N.Y. (1981)). As shown above, both non-aromatic and aromatic compounds can be used within the present invention. Examples of compounds include: proprionic anhydride, butyric anhydride, isobutyric anhydride, valeric anhydride, anhydride (s) - (+) - methylbutyric, trimethylacetic anhydride, hexanoic anhydride, heptanoic anhydride, decanoic anhydride, lauric anhydride, palmitic anhydride, stearic anhydride, docosanoic anhydride, crotonic anhydride, anhydride methacrylic, oleic anhydride, linoleic anhydride, chloroacetic anhydride, iodoacetic anhydride, dichloroacetic anhydride, trifluoroacetic anhydride, chlorodifluoroacetic anh ídrido, trichloroacetic anhydride, succinic anhydride, pentafluoroporpionico anhydride, heptafluorobutyric anhydride, methylsuccinic anhydride, 2,2-dimethylsuccinic anhydride cis-1 , 2-cyclohexanedicarboxylic acid, trans-1,1-cyclohexanedicarboxylic anhydride, italic anhydrides, itaconic anhydride, 2-duodecen-1-ylsuccinic anhydride, dicarboxylic anhydrides, cis-aconic anhydrides, s-acetyl mercaptosuccinic anhydride, (+) - diacetyl-L anhydride - tartaric, malic anhydrides, citraconic anhydride, 2,3-dimethylmaleic anhydride, malic anhydrides, glutaric anhydrides, benzoic, 2,3-diphenylmaleic, 2-phenylgutaric, homofalic, isatoic, N-methylisatoic, 5-chloroisatoic, italic, cyclic acid 2 -sulfobenzoic, 4-methylphthalic, 3,6-difluorophthalic, 3,6-dicylophthalic, 4,5-dichlorophthalic, tetrafluorophthalic anhydrides, tetrabromophthalic, 3-hydroxyphthalic, carboxylic anhydrides, 3-nitroftalic, 4-nitroftalic, diphenic, and naphthalic anhydrides . Preferred compounds include: crotonic anhydride, chloroacetic anhydride, dichloroacetic anhydride, trifluoroacetic anhydride, chlorodifluoroacetic anhydride, trichloroacetic anhydride, pentafluoropropionic anhydride, heptafluorobutyric anhydride, succinic anhydride, methylisuccinic anhydride, 2,2-dimethylsuccinic anhydride, itaconic anhydride, maleic anhydride, citraconic anhydride , 2,3-dimethylmaleic anhydride, 1-cyclopentene-1,1-dicarboxylic anhydride, 3,4,5,6-tetrahydroftalic anhydride, bromomaleic anhydride, dichloromaleic anhydride, glutaric anhydride, 3-methylglutaric anhydride, 2,2-dimethylgutaric anhydride , 3,3-dimethylglutaric anhydride, 3-ethyl-3-methylglutaric anhydride, hexafluuroplutaric anhydride, 3-5-diacetyltetrahydropyran-2,4,6-trione, diglycolic anhydride, 3,6-difluorophthalic anhydride, 3,6-dichlorophthalic anhydride , tetrafluroftalic anhydride, tetrachlorophthalic anhydride, tetrabromophthalic anhydride, anhydride or 3-hydroxyphthalic and 2,3-dibromomaleic anhydride. Before using a "blocked" solid support (containing a white ligand) in a hybridization reaction, it may be desirable to remove substantially all of the unreacted blocking compound with the solid support. A variety of forms can be used to perform this removal within the context of the present invention. For example, the unreacted compound can be washed, such as by rinsing the solid support with a solution that does not contain the compound. Similarly, the unreacted compound can be removed by physical removal of the solid support from the reaction solution. Alternatively, the unreacted compound can be removed by a chemical cooling reaction. For example, the unreacted anhydride can be converted to a non-reactive form of hydrolysis or reaction with an amine which is added to the reaction solution. Any of these techniques can be used with another or in combination. The removal of unreacted compound can be performed immediately after immobilization or after a time of intervention. If the initial time interval is sufficiently long, no action may be required as to whether the anhydride may have been hydrolyzed to a non-reactive form. In addition, there may be many situations where the presence of the unreacted compound does not interfere with the particular use of a solid support containing immobilized white ligand.

Following the preparation of a solid support containing immobilized target ligand and blocked non-specific sites, the solid support is contacted with a probe (candidate binding partner) to determine whether the probe can hybridize to the target ligand. Any of the molecules described above for use as a target ligand can be used as a probe. Consequently, the probes include nucleic acids, proteins and small organic or bio-organic molecules (e.g., natural products). For example, where the probes are nucleic acids, the nucleic acids can be obtained from the entire sequence and portions thereof of an organism genome, messenger RNA (mRNA) or cDNA. Once the appropriate sequences are determined, the DNA probes are preferably chemically synthesized using commercially available methods and equipment (e.g., Applied BioSystems, Foster City, CA). For example, the solid phase method can be used to produce short probes between 15 and 150 bases (Caruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:41 1 (1982)). The hybridization medium will generally contain normal buffer solutions and detergents. A pH buffer solution such as sodium citrate, Tris HCl, PI PES H EPES can be used at a concentration of about 0.01 M to 0.2 M. The hybridization medium will usually also contain from about 0.01% to 0.5% of an ionic detergent. or non-ionic such as sodium dodecyl sulfate (SDS) or Sarkosyl (Sigma St. Louis, Missouri), EDTA of between about 1 to 10 mm and NaCl of about 0.1 to 1 M. Other additives, such as preservatives, can be included. volume exclusion which includes a variety of polar water soluble agents such as anionic polyacrylate or polymethacrylate and charged saccharide polymers such as dextran sulfate and the like. The hybridization assays of the present invention can be carried out by any method known to those skilled in the art or analogous to the immunoassay methodology given the guidelines presented herein. Hybridization assays are typically carried out at temperatures ranging from about 4 ° C to 70 ° C for periods that normally range from about 1 hour to 72 hours. Hybridization temperatures are known to depend on the concentrations of salts, chaotropes and the like present in solutions that support hybridizations. The preferred methods of analysis are sandwich analysis and variations thereof and the analysis of competition or displacement. Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach, Hames and Higgins (eds.) I RL Press (1985); Gall and Pardue, Proc. Nati Acad. Sci. USA. 63: 378 (1969) and John et al., Nature 223: 583 (1969). The specificity (strength) of hybridization (ie, probe binding of white ligands) can be controlled by a number of different manners known in the art (e.g., Molecular Cloning: A Laboratoy Manual, Sambrook and others, (eds), Cold Spring Harbor Press (1989).) For example, specificity can be controlled by varying the salt concentration, incubation time and / or incubation temperature.The specificity control can be exercised at the point of binding reaction either in a washing step or in both For example, the conditions in the incubation of a probe with a white ligand immobilized on a solid support can be such that only strong binding is allowed. binding reaction may be less strong, but a wash (after strong binding) may be under strong conditions to control the specificity of any probe that remains bound to the solid support via the white ligand or immobilized Within the context of the present invention, the specificity of the binding of a probe to the immobilized target ligand can be controlled in any of a variety of ways, including those described above. For example, after hybridization at an appropriate temperature and time for the particular hybridization solution used, a solid support to which the nucleic acid probe-nucleic acid complex binds, is introduced into a solution that normally contains similar reagents (e.g., NaCl, pH regulating solutions, organic solvents and detergents) as provided in the hybridization solutions. These reagents may be in similar concentrations as in the hybridization solutions, but often at a lower concentration when high strength is required in the hybridization. The washing time can vary from approximately several minutes to several hours. The hybridization or the washing medium can be strong. After appropriate strong washing, the correct hybridization complex can now be detected according to the nature of the label. The determination of the degree of hybridization can be carried out by any of the methods well known in the art. If there is no detectable hybridization, then the degree of hybridization is zero. Hybridization can be detected "directly" (ie, where the probe contains a reporter group) or "indirectly" (ie, where the reporter group is on a molecule used to detect the presence of a probe). Various labels (signals) are suitable for use within the present invention. The labels act as reporter groups to detect double formation between a white sequence and its complementary signal sequence. A reporter group as used herein is a group which has a physical or chemical characteristic that can be observed, measured or detected. Detectability can be provided by such characteristics as color change, luminescence, fluorescence or radioactivity; or it can be provided by the ability of the reporter group to serve as a ligand recognition site. Typically, for example, labeled signal nucleic acid probes are used to detect nucleic acid hybridization. In addition, nucleic acids (signal probes) can be labeled by one of several methods normally used to detect the presence of hybridized polynucleotides. For example, labeled nucleic acid probes include double-stranded DNA labeled by critical point translation, single-stranded DNA prepared by primer extension of an oligonucleotide fortified with a recombinant M13 bacteriophage, radiolabeled oligonucleotide probes or synthetic oligonucleotide probes. labeled with biotin or digoxin, or synthesized RNA in vitro with RNA polymerases that depend on prokaryotic DNA (e.g., bacteriophage ART polymerases SP6, T7, or T3). Methods for synthesizing and using these probes are described, for example in Molecular Cloning, A Laboratoy Manual, Sambrook et al. (Eds.), Cold Spring Harbor Press (1989). The most common method of detection is the use of autoradiography with probes labeled with 3 H, 125 I, 35 S, 14 C, 32 P and the like. Other brands include ligands that bind to labeled antibodies, fluorophores, chemiluminescent enzymes, and serve as members of specific binding pairs for a labeled ligand. It will be evident to the experts that the choice of label depends on the requirements of sensitivity, ease of conjugation with the probe, stability requirements and availability requirements. Non-isotopic probes can be directly labeled with signal (such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates) or indirectly labeled by conjugation with a ligand capable of binding to a portion having a detectable signal attached thereto. For example, biotin bound covalently to a probe can bind to streptavidin which covalently binds to a detectable signal, such as an enzyme or photoreactive compound. The combination ligand and receptor can vary widely. Where a ligand has a natural "receptor" (i.e., ligands such as biotin, thyroxine and cortisol), it can be used together with its labeled receptor present in nature. Alternatively, a hapten or antigen may be used in combination with a suitably labeled antibody. Non-radioactive probes are often marked by indirect means. Generally a molecule of ligands is covalently bound to the probe. The ligand then binds to a molecule of antiligands which is inherently detectable or is covalently bound to a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Ligands and antiligands can vary widely. Where a ligand has a natural antiligand, for example, biStin, thyroxine and cortisol, it can be used together with antiligands present in nature. Alternatively, any haptenic or antigenic compound can be used together with an antibody. Enzymes suitable for use as signals include hydrolases (particularly phosphatases), esterases, ureases, glycosidases, oxidoreductases (particularly perioxidases). Suitable fluorescent signals include fluorescein and its derivatives, rhodamine and its derivatives, dansyl umbelliferone and the like. Useful chemiluminescers within the claimed invention include luciferin, luminol and 1,2-dioxetanes. The amount of labeled probe that is present in the hybridization solution can vary widely. In general, substantial molar excesses of probe on the amount of white ligand will be employed in order to increase the rate of binding of the probe to the target ligand. The means for detecting the signal are determined by the selected signal. For example, where the label is a radioisotope, the support surface containing captured and labeled probe complexes - by attaching blank can be exposed to an X-ray film analyzed in a scintillation or gamma counter. Where the label is fluorescent, the complexes are irradiated with light of a particular wavelength and are absorbed by the labeled complex, resulting in the emission of light of a lower wavelength which is detected. Where the label is an enzyme, the complexes are incubated with an appropriate substrate for the enzyme and the signal generated, for example, it can be a color precipitate, a compound or soluble colored or fluorescent material, or photons generated by bioluminescence or chemiluminescence. . The probe can be conjugated directly to the mark. For example, where the probe is radioactive, the probe in association with its complement is exposed to X-ray film. Where the label is fluorescent, the sample is detected by irradiating first with light of a particular wavelength and the emission is detected. collected by a detector (Freifelder, Physical Biochemistry, W. H. Freeman &; Con. (1982), p. 537). When the label is an enzyme, the sample is detected by incubation on an appropriate substrate for the enzyme. The signal generated can be a color precipitate, a soluble colored or fluorescent material or photons generated by bioluminescence or chemiluminescence. For example, the alkaline phosphatase will dephosphorylate the indoxyl phosphate which will be precipitated in a reduction reaction to convert the tetrazolium salts to a highly colored and insoluble formazan. The label may also allow indirect detection of the hybridization complex. For example, when the label is a hapten or antigen, the sample can be detected using antibodies. In these systems, a signal is generated by linking the fluorescent or enzyme molecules to an antibody and in some cases, by binding to a radioactive compound (Tijssen, "Practice and Theory of Enzyme Immunoassays", Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg (eds.), Elsevier (1985), p.9). Prior to detection of the probe, it may be convenient to remove substantially all of the probe that has not bound to the immobilized target ligand. A variety of ways can be used to perform this removal within the context of the present invention. For example, the unattached probe can be washed, such as by wiping the solid support. Similarly, the unbound probe can be removed by physical removal of the solid support from the hydration reaction solution. In turn, the unbound probe can be removed by a cooling reaction (ie, the unbound probe is converted to a shape that is no longer detectable). Any of these techniques can be used with another or all of them in combination. The following examples are offered by way of illustration and not limitation. EXAMPLES EXAMPLE 1 Demonstration of Fund Reduction with Nitrocellulose Blocked Succinic Anhydride Compared to Denhardt Blocked Niyrocellulose Using a Colorimetric Reporter In this example, the DNA is plotted on a nitrocellulose filter, immobilized, probed with a complementary biotinylated oligonucleotide. The hybridized signal oligonucleotide is then detected with streptavidin / alkaline phosphatase using a colorimetric substrate. Chemical means to block nitrocellulose are compared with traditional blocking agents. The nitrocellulose with plastic bottom (Schleicher and Schuell, Part # 76489, Keene, N H) was cut into 1 x 3 cm pieces. The nitrocellulose was briefly soaked in 50% ethanol by immersing one end and removing it through the ethanol solution. The sheets were washed three times rapidly in water and then soaked with shaking in 2 x SSC (20 X SSC is 175.3 g of NaCl and 88.2 g of dissolved sodium citrate in a final volume of 1 liter of water at pH 7.0) during 5 minutes. 100 ng of double-stranded DNA lamba (dsDNA) was plotted at intervals of 300 microns. The DNA was immobilized irreversibly by UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). To denature the AD Ndh, the slides were wetted with 50% EtOH, then washed 2x with water. The sheets were then soaked in denaturing solution (0.1 N NaOH, 0.05 M EDTA) at room temperature for 1 minute, rinsed quickly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 mM EDTA ), washed once in water and dried the sheets with paper towels. The DNA was again treated with UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). The slides were rehydrated with 50% ethanol for 5 minutes and washed twice with water. The sheets were slid in 2 containers for separate "lock" procedures. A container contained sheets of nitrocellulose that were blocked with the chemical agent succinic anhydride. To block the sheets with succinic anhydride, 2.5 grams of succinic anhydride was dissolved in 25 ml of m-pyrrole and 125 ml of 0.1 M Na-Borate pH 8.5 was added. The solution was mixed well and then added to the sheets. The incubation with gentle mixing was 10 minutes. The sheets were then washed 5 times with 0.01 M Tris and 0.005 DTA. The sheets in the second container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400, Pharmacia), 5 grams of poiivinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water at a volume total of 500 ml) in 90 ml of water and adding herring sperm DNA from a single strand fragmented to a final concentration of 100 micrograms / μl. The solution was mixed well and then added to the sheets. Incubation with gentle mixing for 30 minutes. The sheets were then washed 5 times with 0.01 M Tris, pH, and 0.005 EDTA. The biotinylated oligonucleotide (BMD 469 (GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arlington Heights, IL) The hybridizations were heated at 42 ° C for 60 minutes The slides were then rinsed four times with 1 x SSC / 0.1% SDS for 1 minute per wash The slides were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Sarkosyl) The streptavidin / alkaline phosphatase conjugate (Vector, Burlingame, CA) was diluted (1: 10,000) in wash solution.The solution was then applied to the sheets for 1 hour at room temperature with shaking. then rinsed four times with wash solution, rinsed once with detection buffer (0.1 M NaCl, 0.01 M Tris pH 8.5, 0.05 M MgCl 2) for 5 minutes.The alkaline phosphatase substrate was prepared by dissolving one tablet of BCI P / N BT (Sch leicher and Schuell, part # 78349, Keene, N H) in 30ml of dH2O. The reaction was carried out for 4 hours at room temperature. The sheets were then rinsed with water and dried. Table 1 Blocking of Anhydride Blockade of Denhardt Succínico Background color in a 1 7 scale of 1 -10 The results (Table 1) indicated a significant lower level of "background" color on the sheets treated with succídic anhydride, interspersed with the sheets blocked with Denhardt's reagent. EXAMPLE 2 Demonstration of Background Reduction with Nitrocellulose Blocked with Succinic Anhydride compared to Nitrocellulose Blocked with Sol. By Denhardt using a Fluorescent Reporter In this example, the DNA is plotted on a nitrocellulose filter, immobilized, probed with a complementary biotinylated oligonucleotide. The hybridized signal oligonucleotide is detected with estrepatavidin / alkaline phosphatase using 4-methyl-umbelliferyl phosphate (4-hydroxy-methyl coumarin). Chemical means to block nitrocellulose are compared with traditional blocking agents. The nitrocellulose with plastic bottom (Schleicher and Schuell, Part # 76489, Keene, N H) was cut into 1 x 3 cm pieces. The nitrocellulose was briefly soaked in 50% ethanol by immersing one end and removing it through the ethanol solution. The sheets were washed three times rapidly in water and then soaked with shaking in 2 x SSC for 5 minutes. 100 ng of double-stranded DNA lamba (dsDNA) was plotted at intervals of 300 microns. The DNA was immobilized irreversibly by UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). To denature the dsDNA, the films were wetted with 50% EtOH, then washed 2x with water. The sheets were then soaked in denaturing solution (0.1 N NaOH, 0.05 mM EDTA) at room temperature for 1 minute, rinsed rapidly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 M EDTA ), washed once in water and dried the sheets with paper towels. The DNA was again treated with UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). The slides were rehydrated with 50% ethanol for 5 minutes and washed twice with water. The sheets containing DNA were labeled differently than the control sheets that did not contain DNA. The sheets were slid in 2 containers for separate "lock" procedures. A container contained sheets of nitrocellulose that were blocked with the chemical agent succinic anhydride. To block the sheets with succinic anhydride, 2.5 grams of succinic anhydride were dissolved in 25 ml of m-pyrrole and 125 ml of 0.1 M Na-Borate pH 8.5 were added. The solution was mixed well and then added to the sheets. The incubation with gentle mixing was 10 minutes. The sheets were then washed 5 times with 0.01 M Tris and 0.005 EDTA. The sheets in the second container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400, Pharmacia), 5 grams of polyvinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water at a volume total of 500 ml) in 90 ml of water and by adding herring sperm DNA from a single fragmented thread to a final concentration of 100 micrograms / ml. The solution was mixed well and then added to the sheets. Incubation with gentle mixing for 30 minutes. The sheets were then washed 5 times with 0.01 M Tris, pH, and E DTA 0.005. The biotinylated oligonucleotide (BMD 469 (GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arl ington Heights, I L). Hybridizations were heated at 42 ° C for 60 minutes. The sheets were then rinsed four times with 1 x SSC / 0.1% SDS for 1 minute per wash. The sheets were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Sarkosyl). The streptavidin / alkaline phosphatase conjugate (Vector, Burlingame, CA) was diluted (1: 10,000) in wash solution. The solution was then applied to the sheets for 1 hour at room temperature with stirring. The sheets were then rinsed four times with wash solution, rinsed once with detection buffer (0.1 M NaCl, 0.01 M Tris pH 8.5, 0.05 M MgCl 2) for 5 minutes. The sheets were then incubated individually with 1 ml of 0.05 mM 4-methyl-umbelliferyl phosphate (4-hydroxy-methyl coumarin). The reaction was carried out for 4 hours at room temperature. Part of the solution was removed (150 microliters) and placed in a black microtiter plate / Dynatek Laboratories, Chantilly, VA.). The plates were read directly using a Fluoroskan II fluorometer (Flow Laboratoties, McLean, VA.) Using an excitation wavelength of 360 nm and monitoring the aa emission at 456 nm. Table 2 Sheet # Blockade of Anhydride Blocking Denhart Succinic + DNA 425 rfu 619 rfu -ADN 20 rfu 395 rfu The results (Table 2) indicated a significant lower level of "background" color on the sheets treated with succinic anhydride compared to the sheets blocked with Denhardt's reagent. EXAMPLE 3 Demonstration of Background Reduction with Nitroceluose Blocked with Succinic Anhydride Compared to Denhardt Sun Blocked Nitrocellulose Using a Chemiluminescent Reporter In this example, the DNA is plotted on a nitrocellulose filter, immobilized, probed with a biotinylated oligonucleotide complementary. The hybridized signal oligonucleotide is detected with estrepatavidin / alkaline phosphatase and using the chemoluminescent substrate Lumingen. Chemical means to block nitrocellulose are compared with traditional blocking agents. The nitrocellulose with plastic bottom (Schleicher and Schuell, Part # 76489, Keene, N H) was cut into 1 x 3 cm pieces. The nitrocellulose was briefly soaked in 50% ethanol by immersing one end and removing it through the ethanol solution. The sheets were washed three times rapidly in water and then soaked with shaking in 2 x SSC for 5 minutes. 100 ng of double-stranded DNA lamba (dsDNA) was plotted at intervals of 300 microns. The DNA was immobilized irreversibly by UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). To denature the dsDNA, the slides were wetted with 50% EtOH, then washed 2x with water. The sheets were then soaked in denaturing solution (0.1 N NaOH, 0.05 mM EDTA) at room temperature for 1 minute, rinsed rapidly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 mM EDTA) , they were washed once in water and the sheets were dried with paper towels. The DNA was again treated with UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). The slides were rehydrated with 50% ethanol for 5 minutes and washed twice with water. The sheets containing DNA were labeled differently than the control sheets that did not contain DNA. The sheets were slid in 2 containers for separate "lock" procedures. A container contained sheets of nitrocellulose that were blocked with the chemical agent succinic anhydride. To block the plates with succinic anhydride, 2.5 grams of succinic anhydride were dissolved in 25 ml of m-pyrrole and 125 ml of 0.1 M Na-Borate pH 8.5 were added. The solution was mixed well and then added to the sheets. The incubation with gentle mixing was 10 minutes. The sheets were then washed 5 times with 0.01 M Tris and 0.005 EDTA. The sheets in the second container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400, Pharmacia), 5 grams of polyvinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water at a volume total of 500 ml) in 90 ml of water and by adding herring sperm DNA from a single fragmented thread to a final concentration of 100 micrograms / ml. The solution was mixed well and then added to the sheets. Incubation with gentle mixing for 30 minutes. The sheets were then washed 5 times with 0.01 M Tris, pH, and 0.005 EDTA. The biotinylated oligonucleotide (BMD 469(GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arlington Heights, IL). Hybridizations were heated at 42 ° C for 60 minutes. The sheets were then rinsed four times with 1xSSC / 0.1% SDS for 1 minute per wash. The sheets were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Sarkosyl). The streptavidin / alkaline phosphatase conjugate (Vector, Burlingame, CA) was diluted (1: 10,000) in wash solution. The solution was then applied to the sheets for 1 hour at room temperature with stirring. The sheets were then rinsed four times with wash solution, rinsed once with detection buffer (0.1 M NaCl, 0.01 M Tris pH 8.5, 0.05 M MgCl 2) for 5 minutes. The sheets were then incubated individually with 1 ml of Lumingen (lumingen Inc., Detroit, Mi). The reaction was carried out for 4 hours at room temperature. Part of the solution was removed (200 microiitres) and placed in a 4 mm x 40 mm polypropylene tube. The signal was measured using a Turner TD 20e luminometer (Turner Designs, Sunnyvale, CA) with an integration time of one minute. Table 3 Sheet # Blockade of Anhydride Blocking Denhart Succinic + DNA 680 rfu 725 rfu -ADN 200 rfu 580 rfu The results (Table 3) indicated a significant lower level of "background" color on the sheets treated with succinic anhydride compared to the sheets blocked with reagent from Denhardt. EXAMPLE 4 Demonstration of Background Reduction with Nylon and Nitrocellulose Membranes Using Succinic Anhydride as the Agent of Blocking (the presence of a hybridized probe was detected using a colorimetric reporter) In this example, the DNA was immobilized on different types of filters, probed with a complementary biotinylated oligonucleotide. The hybridized signal oligonucleotide is detected with estrepatavidin / alkaline phosphatase using a colorimetric substrate.

The chemical means of blocking nitrocellulose are compared with traditional blocking agents. Membrane filters used in this example include Protran Nitrocellulose, nitrocellulose with plastic bottom (part # 76489), Optitran ™ nitrocellulose, Nytran® nylon membrane (Schleicher and Schuell, Deene, N H) and nylon membranes Hybond ™ -N and Hybond ™ -N + (Amersham, Arlington Heights, I L, 60005).

Nitrocellulose and nylon membrane filters were cut into 1 x 1 cm pieces. The nitrocellulose was briefly soaked in 50% ethanol by immersing one end and removing it through the t-solution of ethanol. The sheets were washed three times rapidly in water and then soaked with shaking in 2 x SSC for 5 minutes. 100 ng of double-stranded DNA lamba (dsDNA) was plotted at intervals of 300 microns. The DNA was immobilized irreversibly by UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). To denature the dsDNA, the slides were wetted with 50% EtOH, then washed 2x with water. The sheets were then soaked in denaturing solution (0.1 N NaOH, 0.05 mM EDTA) at room temperature for 1 minute, rinsed quickly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 mM EDTA) , they were washed once in water and the sheets were dried with paper towels. The DNA was again treated with UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). The slides were rehydrated with 50% ethanol for 5 minutes and washed twice with water. The sheets were slid in 2 containers for separate "lock" procedures. A container contained sheets of nitrocellulose that were blocked with the chemical agent succinic anhydride. To block the plates with succinic anhydride, 2.5 grams of succinic anhydride was dissolved in 25 ml of m-pyrrole and 125 ml of 0.1 M Na-Borate pH 8.5 was added. The solution was mixed well and then added to the sheets. The incubation with gentle mixing was 10 minutes. The sheets were then washed 5 times with 0.01 M Tris and 0.005 EDTA. The sheets in the second container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400), Pharmacia), 5 grams of polyvinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water to a total volume of 500 ml) in 90 ml of water and adding herring sperm DNA from a single strand fragmented at a final concentration of 100 micrograms / ml. The solution was mixed well and then added to the sheets. Incubation with gentle mixing for 30 minutes. The sheets were then washed 5 times with 0.01M Tris, pH, and 0.005 EDTA. The biotinylated oligonucleotide (BMD 469 (GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arlington Heights, IL). Hybridizations were heated at 42 ° C for 60 minutes. The sheets were then rinsed four times with 1xSSC / 0.1% SDS for 1 minute per wash. The sheets were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Tween 20). The streptavidin / alkaline phosphatase conjugate (Vector, Burlingame, CA) was diluted (1: 10,000) in wash solution. The solution was then applied to the sheets for 1 hour at room temperature with stirring. The sheets were then rinsed four times with wash solution, rinsed once with detection buffer (0.1 M NaCl, 0.01 M Tris pH 8.5, 0.05 M MgCl 2) for 5 minutes. The alkaline phosphatase substrate was prepared by dissolving a BCIP / NBT tablet (Schleicher and Schuell, part # 78349, Keene, NH) in 30 ml of dH2O. The reaction was carried out for 0.5 to 4 hours at room temperature. The sheets were then rinsed with water and dried. Table 4 Denhardt Succinic Block Anhydride Block Membrane type Potran Nitrocellulose 3 Plastic Nitrocellulose 6 Optitran ™ nitrocellulose 3 Nytran® Hybond ™ Niylon membrane Hybond ™ Niyl membrane -N + Niylon membrane Where the depth of the background graduated on a scale of 1 to 10, 1 representing a surface color without change (white) and 10 representing a surface intensely colored (dark brown). EXAMPLE 5 Demonstration of Background Reduction with Nitrocellulose Blocked with Propionic Anhydride, Butyric Anhydride, Difluorophthalic Anhydride, Compared with Denhardt Sun Blocked Nitrocellulose Using a Colorimetric Reporter In this example, three types of anhydrides were compared. To compare the type of anhydrides, DNA was plotted on a cellulose filter, immobilized and probed with a complementary biotinylated oligonucleotide. The hybridized signal oligonucleotide was then detected with streptavidin / horseradish peroxidase (SA / PR) using a colorimetric substrate (4-methoxy-naphthol, 4MN). Chemical means to block nitrocellulose are compared with traditional blocking agents. Nitrocellulose with plastic bottom (Schleicher and Schuell, Part # 76489, Keene, NH) was cut into 1 x 3 cm pieces. The nitrocellulose was briefly soaked in 50% ethanol by immersing one end and removing it through the ethanol solution. The sheets were washed three times rapidly in water and then soaked with shaking in 2 x SSC for 5 minutes. 100 ng of double-stranded DNA lamba (dsDNA) was plotted at intervals of 300 microns. The DNA was irreversibly immobilized by UV irradiation (120,000 microjoules / cm 2 for a period of 30 seconds). To denature the dsDNA, the slides were wetted with 50% EtOH, then washed 2x with water. The sheets were then soaked in denaturing solution (0.1 N NaOH, 0.05 mM EDTA) at room temperature for 1 minute, rinsed quickly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 mM EDTA) , they were washed once in water and the sheets were dried with paper towels. The DNA was again treated with UV irradiation (120,000 microjoules / cm2 for a period of 30 seconds). The slides were rehydrated with 50% ethanol for 5 minutes and washed twice with water. The sheets were slid in 5 containers for separate "lock" procedures. Each container confined the nitrocellulose sheets that were blocked with the chemical agents propionic anhydride, butyric anhydride, difluorophthalic anhydride or succinic anhydride. To block the sheets with propionic anhydride, butyric anhydride, difluorophthalic anhydride or succinic anhydride the respective compound was dissolved in 25 ml of m-pyrrole at a concentration of 1 molar. Then an equal volume of 0.1 M Na Borate was added, pH 8.5. The solution was mixed well and then added to the sheets. The incubation with gentle mixing was 10 minutes. The sheets were then washed 5 times with 0.01 M Tris and 0.005 EDTA. The sheets in the fifth container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400, Pharmacia), 5 grams of polyvinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water at a volume total of 500 ml) in 90 ml of water and by adding herring sperm DNA from a single fragmented thread to a final concentration of 100 micrograms / ml. The solution was mixed well and then added to the sheets. Incubation was with mixing for 30 minutes. The sheets were then washed 5 times with 0.01 M Tris, pH, and 0.005 EDTA. The biotinylated oligonucleotide (BMD 469 (GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arlington Heights, I L). Hybridizations were heated at 42 ° C for 60 minutes. The sheets were then rinsed four times with 1 x SSC / 0.1% SDS for 1 minute per wash. The sheets were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Sarkosyl). The streptavidin / PR conjugate (Vector, Burlingame, CA) was diluted (1: 2,000) in wash solution. The solution was then applied to the sheets for 1 hour at room temperature with stirring. The sheets were then rinsed four times with wash solution, rinsed once with phosphate pH buffer (PBS) for 5 minutes. The PR substrate was prepared by placing 25 mg of 4-methoxy-naphthol in 7.5 ml of MeOH which in turn was added to 42.5 ml of PBS containing 33 μl of 30% hydrogen peroxide. The reaction was carried out for 15 minutes at room temperature. The sheets were then rinsed with water 5 times and dried. The results indicated a significant lower level of "background" color in the sheets treated with one of the anhydrides compared to the sheets blocked with Denhart's reagent. Type of Blocking Step Relative Color of Background (Scale 1 10) Propionic Anhydride 2 Butyric Anhydride 2 Difluorphthalic Anhydride 3 Anhydride Succinic 1 Denhardt 7 The scale of the background is on a scale of 1 to 10, 1 with the intensity of color being lighter and 10 being the background more obscure or intense. The results indicate that the background is substantially reduced when the nitrocellulose sheets are chemically blocked with any of the 4 types of anhydrides compared to Denhardt's block. Although proprionic anhydride, butyric anhydride and difluorophthalic anhydride are not as effective as succinic anhydride, they are an improvement over Denhardt's solution. EXAMPLE 6 Demonstration of the Reduction of Background with Slides of Glass Blocked with Propionic Anhydride, Butyric Anhydride, Difluorophthalic Anhydride, Compared to Denhardt's Sun-Blocked Nitrocellulose Using a Colorimetric Reporter In this example, three types of anhydrides were compared using a solid, non-porous support (glass slides). To compare the type of anhydrides, the oligonucleotides were covalently immobilized on the slides using an intcing reagent and probed with a complementary biotinylated oligonucleotide. The hybridized signal oligonucleotide was then detected with streptavidin / horseradish peroxidase (SA / PR) using a colorimetric substrate (4-methoxy-naphthol, 4M N). Chemical means to block nitrocellulose are compared with traditional blocking agents (Denhardt's solution). The slides (Sectionlock ™, purchased from Polysciences, Warrington, PA) were washed three times in water and then incubated for 4 hours in 2 x SSC. 100 ng of double-stranded DNA lamba was plotted on the slide using a micropipette. The DNA was immobilized irreversibly by UV irradiation (120,000 microjoules / cm 2 for a period of 30 seconds). To denature the dsDNA, the slides were wetted with 50% EtOH, then washed 2x with water. The slides were then soaked in denaturing solution (0.1 N NaOH, 0.05 mM EDTA) at room temperature for 1 minute, rinsed quickly with water, washed three times in neutralization solution (0.1 M Tris pH 7.2, 0.05 mM EDTA) . The slides were slid in 5 containers for separate "blocking" procedures. Each container contained a slide that was blocked with one of the following chemical agents: propionic anhydride, butyric anhydride, difluorophthalic anhydride or succinic anhydride. To block the sheets with propionic anhydride, butyric anhydride, difluorophthalic anhydride or succinic anhydride the respective compound was dissolved in 25 ml of m-pyrrole at a concentration of 1 molar. Then an equal volume of 0.1 M Na Borate was added, pH 8.5. The solution was mixed well and then added to the slides. The incubation with gentle mixing was 10 minutes. The slides were then washed 5 times with 0.01 M Tris and 0.005 EDTA. The slides in the fifth container were blocked 5x with Denhardt's solution. This was done by placing 10 ml of 50x stock solution of Denhardt's solution (5 grams of Ficoll (type 400, Pharmacia), 5 grams of polyvinylpyrrolidone, 5 grams of bovine serum albumin (Fraction 5, Sigma) and water at a volume total of 500 ml) in 90 ml of water and by adding herring sperm DNA from a single fragmented thread to a final concentration of 100 micrograms / ml. The solution was mixed well and then added to the slides. Incubation was with mixing for 30 minutes. The slides were then washed 5 times with 0.01 M Tris, pH, and 0.005 EDTA. The biotinylated oligonucleotide (BMD 469 (GTTTAACATACTTTCATTT) was added to a final concentration of 10 ng / ml in 1000 μl of rapid hybridization solution (Rapid-Hyb, Amersham, Arlington Heights, I L). Hybridizations were heated at 42 ° C for 60 minutes. The slides were then rinsed four times with 1 x SSC / 0.1% SDS for 1 minute per wash. The sheets were then washed 2x with Wash Solution (0.01 M Tris pH 7.2, 0.1 M NaCl, 0.005 M EDTA, 0.1% Sarkosyl). The streptavidin / PR conjugate (Vector, Burlingame, CA) was diluted (1: 2,000) in wash solution. The solution was then applied to each group of slides for 1 hour at room temperature with shaking. The slides were then rinsed four times with wash solution, rinsed once with phosphate pH buffer (PBS) for 5 minutes. The PR trate was prepared by placing 25 mg of 4-methoxy-naphthol in 7.5 ml of MeOH which in turn was added to 42.5 ml of PBS containing 33 μl of 30% hydrogen peroxide. The reaction was carried out for 15 minutes at room temperature. The slides were then rinsed with water 5 times and dried. The results indicated a significant lower level of "background" color on the slides treated with one of the anhydrides compared to the slides blocked with Denhart's reagent.

Type of Blocking Step Relative Color of Background (Scale 1 10) Propionic anhydride 1 Butyric anhydride 2 Difluorphthalic anhydride 2 Anhydride Succinic 1 Denhardt 4 The scale of the background is on a scale of 1 to 10, 1 with the intensity of color being lighter and 10 being the background more obscure or intense. The results indicate that the background is tantially reduced when the nitrocellulose sheets are chemically blocked with any of the 4 types of anhydrides compared to Denhardt's block. Although propionic anhydride, butyric anhydride and difluorophthalic anhydride are not as effective as propionic anhydride or succinic anhydride, they are an improvement over the Denhardt solution. All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was incorporated specifically and individually by reference. From the foregoing, it will be apparent that, although the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention.

Claims (20)

1. REVIVAL D ICATIONS 1. A method for preparing solid hybridization supports that reduces non-specific background, comprising the steps of: (a) contacting a solid support for hybridization with a white ligand under conditions sufficient to immobilize said target ligand to said solid support; and (b) reacting a solid support containing immobilized white ligand with a compound under conditions sufficient to block non-specific sites on said solid support, thereby producing a blocked solid support containing immobilized white ligand, said compound having the formula: (I) (ID (VII) where Ri-R1 7 are independently selected from H, OH, CH3, CH2-CH3, CH = CH-CH3, X, CH2X, CHX2, CH2-CH2X, CH2-CHX2, CX3, CX2- CX3, CX2-CX2-CX3 and C (= 0) CH3 and R, and R3 or R2 and R5 or Rs and e can be taken together as = CH2, and Rs and e can be taken together as = O, and where each X is selected independently of halogen.
2. 2. A method for reducing non-specific background in hybridization reactions, comprising steps (a) and (b) according to claim 1 and further including the steps of: (c) contacting said blocked solid support containing white ligand immobilized with a probe under conditions sufficient for specific binding to said immobilized target ligand; and (d) detecting the presence of said probe on said solid support, thus determining the hybridization between said target ligand and said probe.
3. The method of claim 1 or 2, further including, after step (b), a step comprising substantially removing all of the compound that did not react with said blocked solid support containing immobilized white ligand.
4. The method of claim 2, further including, between steps (c) and (d), a step comprising substantially eliminating the entire probe that did not bind to said immobilized target ligand.
5. 5. The method of claim 2, further comprising, between steps (b) and (c), a step comprising substantially removing all of the compound that was not reacted with the blocked solid support containing immobilized white ligand and additionally including, among the Steps (c) and (d), a step comprising substantially removing all of the probe that did not bind to said immobilized target ligand.
6. The method of any of claims 1, 2, 3, 4 or 5, wherein said white ligand is a nucleic acid.
7. The method of claim 6, wherein said solid support is a nitrocellulose membrane, a nylon membrane or a glass surface.
8. The method of any of claims 1, 2, 3, 4, or 5, wherein said white ligand is a protein.
9. The method of claim 8, wherein said solid support is a nitrocellulose membrane or a nylon membrane.
10. 10. The method of any of claims 2, 3, 4 or 5, wherein said white ligand is a nucleic acid and said probe is a nucleic acid or a protein.
11. The method of claim 10, wherein said solid support is a nitrocellulose membrane, a nylon membrane or a glass surface.
12. 12. The method of any of claims 2, 3, 4 or 5, wherein said white ligand is a protein and said probe is a protein or a nucleic acid.
13. 13. The method of claim 12, wherein said solid support is a nitrocleulose membrane or a nylon 14 membrane.
14. The method of any of claims 1-13, wherein the compound is according to formula I wherein Ri and R are both CH = CH-CH3 or CH2X or CX3 or CX2-CX3 or CX2-CX-CX3, with each X independently selected from halogen.
15. 15. The method of any of claims 1-13, wherein the compound is according to formula II wherein R? -R4 are H; or R-- is CH3 and R2-R4 are H; or R2 and R are H and Ri and R3 are taken together as = CH2; or Ri and R2 are H and R3 and R4 are C (= O) CH3.
16. 16. The method of any of claims 1-13, wherein the compound is according to formula III wherein R? -R are H; or R! is CH3 and R2 is H; or R *, and R2 are CH3; or Ri is X and R2 is H; or Ri and R2 are X; with each X being independently selected from halogen.
17. The method of any of claims 1-13 wherein the compound is according to formula IV wherein R? -R6 are H, or R1-R5 are H and R6 is CH3; or R, and R3 are CH3 and R2, R4 > Rs and Re are H; or R? -R are H and R5 and R6 are CH3; or R1-R4 are H and R5 and is CH3 and R6 is CH2CH3; or R-i-Re are X; or R1 and R2 are H and R3 and R4 are C (= O) CH3 and Rs and Re are taken together as = 0.
18. 18. The method of any of claims 1-13, wherein the compound is according to formula VI wherein R7-R9 are H or the compound is according to formula VII wherein R10-R13 are H.
19. 19. The method of any of claims 1-13, wherein the compound is according to formula VIII wherein R14 and R17 are X and R15 and Ri6 are H; or R14-Ri7 are X; or R14 is OH and R15-R17 are H.
20. 20. A kit comprising a solid support prepared according to a method of claim 1 or 3. SUMMARY A method related to solid supports for hybridization is described. The present invention provides methods for preparing solid supports and their use in hybridization analysis, so that the non-specific background on solid supports is reduced. The non-specific background reduction allows the detection of low levels of specific binding that could normally be masked by the non-specific binding. The methods can be applied to a variety of target ligands and probes, including nucleic acids such as oligonucleotides.