JPH10510994A - Method for immobilizing oligonucleotide on solid support material and method using oligonucleotide bound to support - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for immobilizing an oligonucleotide directly on a support. The method comprises the steps of contacting the oligonucleotide solution with a solid support material and drying the oligonucleotide solution on the support material. The oligonucleotide and the solid support to which it is immobilized can be used as a capture reagent to immobilize a nucleic acid sequence that is complementary to the immobilized oligonucleotide. In this way, the hybridization ability of the directly immobilized oligonucleotide is maintained. Also provided is a method for measuring the presence or amount of a nucleic acid sequence in a test sample.

Description

DETAILED DESCRIPTION OF THE INVENTION          Method for immobilizing oligonucleotide on solid support material and          Method using a support-bound oligonucleotide   This is a co-pending U.S. Patent Application No. 0, filed September 22, 1994. It is a continuation-in-part application of 8 / 311,462.Field of the invention   The present invention relates to oligonucleotides. In particular, the present invention relates to short oligonucleotides. It relates to the fixation of otide to a support material.Background of the Invention   Ligase linkage as described in European patent application EP-A-320-308 Gap rigger described in Reaction Method (LCR), EP-A-439-182 Zelen Chain Reaction (GLCR) and US Pat. Nos. 4,683,202 and 4 Such as the polymerase chain reaction (PCR) described in US Pat. Amplification reactions are well known in the art. Such a nucleic acid amplification method, for example, Has become a useful clinical diagnostic tool to construct analytical methods to detect infected organisms in samples. ing. Amplification is also useful in research and development and forensic fields I found sex.   Nucleic acid amplification techniques typically generate a copy of the target nucleic acid sequence and The presence or amount of the copy can be detected using immunological analysis techniques. An example For example, a copy of the target sequence can, for example, specifically hybridize to a copy of the target sequence. Coated oligonucleotide (variably referred to as capture oligonucleotide) Contact with a {capture reagent} consisting of a substantially solid support material, such as a suspension of dispersed microparticles Can be done. In this way, the product of the target or amplification reaction is Hybridization of copy and capture oligonucleotide to capture reagent Can be fixed. Once the target sequence is immobilized on the capture reagent, these Separates the solid support from the reaction mixture, such as by washing or filtration This allows easy separation from excess reactants. Fixed to capture reagent The presence or amount of amplified sequence that would have been determined depends on the captured target sequence and {complex}. The contact can be detected. The complex is fixed to the capture reagent Binds to a specific binding pair member that also binds specifically to the amplified target sequence It can consist of a detectable part. Detecting the presence of a detectable part Thus, the presence or amount of the target sequence can be measured.   One known method of immobilizing oligonucleotides on a support material is a chemical cross-linking agent. Is used. Typically, the crosslinker covalently links the oligonucleotide to the support material. To form a bond that binds the oligonucleotide to the support material. For example, rice No. 4,948,882 discloses covalently linking an oligonucleotide to a solid support. Disclosed are compounds that can be used to However, Oligonu Chemically cross-linking a nucleotide to a support material typically involves linking the oligonucleotide This is a time-consuming method that requires modification of constituent base pairs.   Saiki, R. K. etc,Proc . Natl. Acad. Sci. USA86: 6230-6234 (1989). Another method of immobilizing the oligonucleotides on the support material is to use including. The tail is typically an oligonucleotide of about 15 base pairs or more in length. It is an extension. The oligonucleotide tail binds preferentially to the solid support. As with the crosslinker, the oligonucleotide is free from the support material and hybridized. Make it available for zation. Unfortunately, the tail itself is a nucleic acid Sometimes interfere with the ability of oligonucleotides to specifically hybridize to nucleic acid sequences. Harm.   As evident by the above method of immobilizing oligonucleotides on a support material, Relatively short oligonucleotides, having from about 5 to about 50 base pairs, are oligonucleotide Directly binds to solid support without compromising otide hybridization capacity It has been acknowledged that it cannot be done. Therefore, oligonucleotides are supported. Known methods of binding to support materials include the indirect binding of an oligonucleotide to a support material. Combine. The oligonucleotide itself binds to the support material by indirect binding. And, in theory, hybridizes freely to another nucleic acid sequence.Summary of the Invention   The present application relates to a method for immobilizing oligonucleotides directly and non-covalently on a support material. Has been stated. According to this method, the oligonucleotide is immobilized on the support material quickly. Achieved quickly and without chemical modification to the bases that make up the oligonucleotide Is done. Importantly, directly immobilized oligonucleotides The ability of the application has not been impaired. Oligonucleotides and their immobilized The support material is a nucleic acid sequence that is complementary to the oligonucleotide bound to the support material. Can be used as a capture reagent.   This method comprises contacting a solution of the oligonucleotide with a solid support material, Drying the oligonucleotide solution on the substrate. Oligos in solution The nucleotides range in length from about 5 nucleotides to about 30 nucleotides. Good. In addition, the affinity of the oligonucleotide for the support It has been found that it can be enhanced by modifying the tide. This method is And may further include a firing step and / or a coating step.   The complex is contacted with the solid support material and the oligonucleotide immobilized thereon. A measurable sequence as an indicator of the presence or amount of immobilized oligonucleotide. The presence of the oligonucleotide bound to the support by detecting The amount can be detected.   According to another aspect, a solid support material and an oligonucleotide immobilized thereon Can be brought into contact with the complex and provide a measurable signal Can be detected as an indicator of quantity.   According to yet another aspect, the support-bound oligonucleotide is an immobilized oligonucleotide. Contains nucleic acid sequences that are complementary to nucleotides Form a hybridization complex upon contact with a suspected test sample be able to. The hybridization complex is then contacted with the complex The measurable signal can be determined by the presence of a complementary nucleic acid sequence in the test sample or It can be detected as an indicator of quantity.BRIEF DESCRIPTION OF THE FIGURES   Fig. 1 illustrates the principle of total internal reflectance (TIR). Show.   2A, 2B and 2C are perspective views of a waveguide device, respectively. It is a side view and a sectional view. FIG. 2C is an enlarged cross-section taken along line CC of FIG. 2B only. That's a big deal.   3, 4A-4D, 5A-5B, 6A-6C, and 7A-7B are described herein. Using a capture reagent comprising an oligonucleotide immobilized on a support material as taught in 4 is a photograph showing a result obtained by an analysis. Details of the data for these photos Examples 1 to 4 are shown.Detailed description of the invention   Despite previous teachings, surprisingly, from about 5 nucleotides to about 50 nucleotides. Oligonucleotides comprising nucleotides Impairs the hybridization capacity of the immobilized oligonucleotide Can be directly and non-covalently bonded to a solid support material without the need. Oh The mechanism by which lignonucleotides attach directly to solid surfaces is not fully understood. However, direct binding of oligonucleotides to the surface of the solid It means that the otide is fixed to the support material in a manner similar to adsorption. In addition, Direct binding of gonucleotides to solid surfaces affects crosslinkers, tails or immobilization No additional nucleic acid sequence is required. Importantly, as taught herein. If the oligonucleotide binds directly to the surface of the solid, Specifically hybridizing or hybridizing to a complementary nucleic acid sequence.   Oligonucleotides immobilized as indicated herein may be, for example, 2 ' -Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA- Complementary nucleic acid sequences (such as described in WO 93/25706) and the like. To capture or, rather, to immobilize nucleotides on a fixed support Can be used. Once the complementary sequence is If bridging, its existence Can be detected using methodology well known in the art.   For example, including a solid support having oligonucleotides directly immobilized thereon. A capture reagent comprising a test sample can be contacted with a test sample. The test sample is , A nucleus capable of specifically hybridizing with an immobilized oligonucleotide Any liquid believed to contain an acid sequence. Capture reagent and test sample The sample, if present in the test sample, is a hybrid of nucleic acid and oligonucleotide. Soybeans, thereby enabling the formation of hybridization complexes Under suitable time and conditions. Hybridization The complex, if any, specifically binds to the hybridization complex. Contact with the complex for a time and under conditions sufficient to allow it can. The presence of the nucleic acid sequence that may have been present in the test sample or A signal can be detected as an indicator of quantity.   Immobilized oligonucleotides as described herein can be used in a {one-step} analytical format Can also be used. According to such a format, the immobilized oligonucleotide A test sample suspected of containing nucleic acids that are complementary to the pull Binds to nucleic acid sequences that may be present in it to form a complex / nucleic acid complex The composite can be contacted for a time and under conditions suitable to allow it. Ah Alternatively, the nucleic acid that may be present in the test sample contains a detectable moiety. You may go out. The nucleic acid sequence may be, for example, where labeled nucleotides are incorporated into the target sequence. Labeling or detection with a detectable moiety A composite can be formed with the ejectable portion. Compound containing detectable moiety / The nucleic acid complex or nucleic acid is then contacted with a support-bound oligonucleotide. Complex / nucleic acid / oligonucleotide complex or nucleic acid / oligonucleotide complex The body can be formed. Next, the presence of the nucleic acid sequence present in the test sample is determined. The signal can be detected as an indicator of presence or amount.   In a preferred embodiment, the presence of the oligonucleotide in the test sample is rapidly determined. Provide a way to detect. According to this aspect, the oligonucleotide contains Probable sample can be contacted with support material and present in test sample Oligonucleotides, which may be immobilized on a support material as indicated herein Can be Next, the oligonucleotide to which the complex was immobilized The conjugate was immobilized under time and under conditions that allowed it to bind to the nucleotide. It can be contacted with an oligonucleotide. Then present in the test sample Signal as an indicator of the presence or amount of oligonucleotides that may have Can be detected.   Oligonucleotides immobilized as indicated herein, for example, The time of contact with the sample, complex / nucleic acid complex or complex is not critical. However, such contact times should be kept to a minimum, for example, less than 30 minutes. Preferably, more preferably less than 15 minutes, most preferably less than 10 minutes.   That the complex will contain a detectable moiety bound to the specific binding pair member Will be understood by those skilled in the art. The detectable part is variably called a signal. Any compound with detectable and measurable physical or chemical properties Or conventional detectable chemical groups. Such detectable Groups are enzymatically active such as enzymes and enzyme substrates, prosthetic families or coenzymes Fluorescent molecules such as fluorophores and fluorophores; chromophores and Light-emitting molecules such as luminescent, chemiluminescent and bioluminescent materials; phosphorescence Body molecules; specifically bindable ligands such as biotin and avidin; Radioactive isotopes; Toxins; Drugs; Haptens; Polysaccharides Class; polypeptides; liposomes; colored or fluorescent particles; colored or fluorescent microparticles Particles: colloidal particles such as selenium colloid or gold colloid , But is not limited to these. Further, the detectable portion may be, for example, Assigned to the same person as filed on July 13, 1993, which is hereby incorporated by reference. No. 6,078,095, which is incorporated by reference in its entirety, and which are described in co-pending U.S. patent application Ser. No. 091,149. A plurality of fluorophores fixed to such a polymer can be included. Detectable part The detectable physical or chemical properties associated with the More detectable. Specific binding member is a well-known term and is usually Pairing, i.e., one of the molecules is specific to the other molecule via chemical or physical means Means members of two different molecules that bind to For specific binding pairs of antigen and antibody In addition, other specific binding pairs include avidin and biotin, antibodies and hapten, complementary Nucleotide sequence or complementary nucleic acid sequence such as DNA, RNA or PNA Sequence, enzyme cofactor or substrate and enzyme, peptide sequence and this sequence Or antibodies specific to all proteins, dyes and protein binders, peptides and specific proteins Binding agents (eg, ribonuclease, S-peptide and ribonuclease S- Protein) and the like, but are not limited thereto. In addition, Thus, an analog of the original binding member, for example, a member or analyte that is an analyte-analog Can include binding members made by recombinant techniques or molecular engineering. You. Thus, PNA is a specific binding member of DNA or RNA. Coupling structure If the member is an immunoreactant, this may be, for example, monoclonal or polyclonal. Ronal antibodies, recombinant proteins or antibodies, chimeric antibodies, mixtures thereof Or a fragment. The detectable moiety is the specific binding characteristic of the specific binding member. Any chemical means or method that does not destroy the detectable properties of the detectable moiety And / or can bind to a specific binding pair member via physical means.   Preferably, oligonucleotides are applied to the surface of the glass using the methods provided herein. Immobilized, which is then incorporated herein by reference.  Scattering Optical Waveguide Method for Detecting Specific Binding Even US Patent Application Ser. Used for waveguide formats as taught in application Ser. No. 08 / 311,462. Waveguide device for immunoassay or hybridization type format The capabilities of the device are known in the art and include total internal reflection (TIR) as described with respect to FIG. It is based on a phenomenon called. A denser medium 12 (ie, a higher refractive index N₁To ), The denser and less sparse media 16 (ie, lower refractive index). N_TwoThe light 10 impinging on the interface 14 between the two) has a critical angle equal to:         θ_C= Arc sine (N_Two/ N₁The critical angle θ defined by_CBigger Angle θ_RIf it hits the boundary at TIR operates by the principle of shooting. Under these conditions, A known electromagnetic waveform results. As shown in FIG. 1, the light associated with denser media The electric field forms a sinusoid 18 that stands perpendicular to the interface. A damped wave is a more sparse medium Penetrates the body 16 but its energy E is the distance from the interface as shown at 20 Dissipates exponentially as a function of Z. {Depth of transmission} (d_p-Shown at 22 in FIG. The parameter known as the Defined as the distance from the interface at the interface to 0.368 times the energy value You. [E = (e^-1) ・ E₀D as the depth of_pSutherland et al. To defineJ . Immunol . Meth. , 74: 253-265 (1984)reference]. The penetration depth is calculated as follows:   Factors that tend to increase the depth of transmission are: angle of incidence θ_RIncrease; of two media A matching index of refraction (ie, N_Two/ N₁-> 1); and increase in wavelength λ You. For example, a quartz TIR element (N₁= 1.46) in aqueous medium (N_Two= 1.34) Medium The critical angle θ_CIs 66 ° (= arc sine 0.9178). 50 0 nm light is θ_R= 70 ° (ie greater than the critical angle) If d_pIs about 270 nm.   In addition, TIR uses light scattering detection by a technique called scatter total internal reflection ({STIR}). It has been used in connection. See, for example, U.S. Pat. No. 4,979,821 to Schutt et al. And 5,017,009 and WO 94/00763 (Akzo N.V.). According to this technique, a beam of light travels across the surface of the TIR element at an appropriate angle. And the light energy is completely reflected except for the damping wave It is. Red blood cells, colloidal gold or latex specifically bound within the depth of penetration Particles scatter light, and the scattered light is detected by a light detection device.   FIGS. 2A-2C illustrate a waveguide including a planar waveguide element 32 and a parallel planar plate 34. The wave tube device 30 will be specifically described. Therefore, the waveguide elements are parallel surfaces 36 and And a terminal 40 for receiving light. Similarly, plates 34 are parallel Surfaces 42 and 44. The waveguide element 32 and the plate 34 Surface 38 and plate surface 42 delimit a narrow groove 46. They are kept together in a spaced parallel fashion. This element and plate are An adhesive component 48 consisting of a double adhesive tape disposed along the edge of the plate. Can be held together by various components. Groove 46 is preferably through it Relatively small to allow capillary movement of the fluid sample. For example, the height is about 1m m, preferably less than about 0.1 mm.   Element 32 is a plastic, such as polycarbonate, acrylic or polystyrene. It should be made of an optically transparent material such as plastic, glass, quartz. all To achieve internal reflection, As is known in the art, the refractive index of the waveguide is greater than the refractive index of the sample fluid. Need to be larger. In the aqueous solution sample, the refractive index n is about 1.33, The waveguide typically has an index of refraction greater than 1.35, typically about 1.5 or more. . The waveguide may be a single piece of plastic or glass, for example, a standard Russ microscope slides or coverslips can be used.   Plate 34 can be made of a similar material. From FIGS. 2A and 2B As can be seen, the light receiving end 40 of the waveguide element 32 is stray from the light source 54. It is located in the narrow slit 50 of the mask 52 to minimize the effect of light. Stray Light minimization is also improved by the use of light absorbing materials.   The light source 54 that produces the projection light is located in the visible, ultraviolet, and near infrared spectra. It may be an electromagnetic energy source containing energy. Therefore, kana is Is more widely interpreted and is not limited to the visible range unless detection is made visible . Invisible wavelengths are detected for specific wavelengths, as is well known in the art. Detected by the detector. Light can be monochromatic, polychromatic, or collimated Polarized light, even if not collimated It need not be polarized light. Preferred light sources include lasers, light emitting diodes, and flashes. Light bulbs, arc lamps, incandescent lamps and fluorescent discharge lamps are included. Illuminates waveguide element The light source used for this may be a low wattage helium-neon laser. For portable disposables such as those described in Example 1 below, the light source is a pocket Small incandescent light bulbs powered by batteries, such as those used in flashlights There may be. Preferably, the light source has a potential for changing the intensity of the light source. Including meter parts. Alternatively, use a filter and / or lens to Can be adjusted to an appropriate level.   Measuring light scattering caused by light scattering labels (LSL) using a detection device Can be. As best seen in FIG. 2A, the LSL may be, for example, a fixed orientation. Between specifically binding members, such as those between gonucleotides and homologous DNA sequences To the surface 38 of the waveguide element 32 through the interaction of LSL Scatters incident light elastically, i.e. without substantially absorbing light energy Is a molecule or substance, often a particle. A typical LSL is Colloy Colloidal metal and non-metal labels such as gold or selenium; red blood cells; Tex, polystyrene, polymethyl acrylate, polycarbonate or the same Colored plastic particles made of the same materials. Such specific markers Knowledge size is 10 nm to 10 μm, typically 50 to 500 nm, and is preferred. In the range from 70 to 200 nm. The larger the particles, the greater the light scattering effect, , Since this applies to both bound and bulk liquid particles, Ground also increases with particle size. Suitable particles LSL are available from Bangs Laborato ries, Inc., Carmel, IN, USA.   Measuring the degree of light scattering caused by LSL using instruments and visual detection means be able to. Light scattering events traversing all waveguides are also caused by the eyes and brain of the observer. Are CCD cameras that form images that are digitized and processed using a computer. The measurement can be performed substantially simultaneously by the photodetector including the camera.   As mentioned above, according to the present invention, the immobilization of the oligonucleotide on the support material comprises: Contact the support material with the oligonucleotide solution and dry the solution on the support material Consisting of   The support or solid support to which the oligonucleotide can be immobilized is Well known in the industry, Materials that are soluble are included. The porous material can be a solid support, and For example, paper; nylon; and cellulose and nitrocellulose. Such cellulose derivatives can be mentioned. Smooth polymerized and non-polymerized materials Are also suitable support materials, such as, for example, polycarbonate, polystyrene. Plastics and derived plastics such as ren and polypropylene; magnetic Or non-magnetic metals; including but not limited to quartz and glass There is no. Preferably, quartz, glass or nitrocellulose is used as support material I have. Solid supports include, but are not limited to, test tubes, micro Interwell, sheet, film, strip, bead, microparticle, chip, slurry It can be used in many shapes known to those skilled in the art, including ids, coverslips, etc. You.   An oligonucleotide according to the invention refers to a DNA, RNA or PNA sequence. Taste is a matter of course. Oligonucleotide chains immobilized on a support material Length is primarily a matter of choice for one of ordinary skill in the art, and is typically DNA, RNA or the complementary sequence of PNA. Orient that is fixed The length of a gonucleotide is typically from about 5 to It is about 50 bases, but preferably, the length of the oligonucleotide to be immobilized is about 5 to about 30 bases, more typically about 10 to about 25 bases.   Oligonucleotide synthesis is fairly routine using automated synthesizers. You. If desired, an automated synthesizer may be used to modify terminal amines or other groups. Oligonucleotides can be produced. Useful overview of coupling chemistry The theory is Goodchild,Bioconjugate Chemistry, 1 (3): 165-187 (1990) It is.   Modified oligonucleotides are more solid than unmodified oligonucleotides. High affinity for the support. Methodologies for modifying oligonucleotides are well known And chemical groups such as amines or fluoresce for oligonucleotides. Addition of haptens such as cein may be mentioned. Such modifications may involve support material It does not provide a covalent bond between the oligonucleotides, but still binds to the support. Have been found to increase the affinity of certain oligonucleotides. Modification is It is particularly effective if it is made at the 3 'or 5' end of a gonucleotide.   The oligonucleotide solution in contact with the solid support is It can comprise a lignonucleotide. Oligonucleotide concentration in solution The degree is primarily a matter of choice for those skilled in the art, and is typically Based on how leotide is used. Usually, the oligonucleotide solution is about 1 μM to about 1 mM, preferably about 20 μM to about 250 μM oligonucleotide Has a tide concentration. However, as mentioned above, the modified oligonucleotides Has a higher affinity for solid supports than unmodified oligonucleotides I know that. Thus, the modified oligonucleotide is It can be used in a lower concentration range than nucleotides.   The pH of the oligonucleotide solution is from about 6.5 to about 8.0, preferably about 7. It may be from 0 to about 7.5. Furthermore, the oligonucleotide solution is preferably Salt water, the sodium chloride concentration of such a solution can vary greatly But typically between about 75 mM and 2M, preferably between about 100 mM and about 1M, up to Also preferably from about 120 mM to about 500 mM.   Optionally, a buffer system can be included in the oligonucleotide solution. Buffer Stems are well known and usually contain compounds that resist changes in the hydrogen ion concentration of the solution. Comprising an aqueous solution. Buffer Examples of systems include, for example, acetic acid, boric acid, phosphoric acid, phthalic acid, citric acid, carbonic acid. And the like, but not limited thereto. Not only. Preferably moderate Sodium or sodium phosphate, more preferably about 10 Is sodium phosphate, most preferably from about 10 mM to about 175 m Comprising.   The amount of oligonucleotide solution to be applied or spotted on a solid support is For example, capture enough complementary sequences to allow detection of the captured sequence or conjugate. They need to be large enough to capture. This is due, in part, to the capture oligonucleotide. It depends on the density of the support material that fixes the metal. For example, as small as 150 μm in diameter Area can be used. Many sites on the support material are oligonucleotide solutions In the case of spotting at such a small area, such a small area is preferable. Practical under size The limit is about 1 μm in diameter. Visual detection is sufficient to detect without magnification Larger area, for example, at least about 1 to about 50 mm^Two; 1cm^TwoUp to or larger. Manufacturing costs and users There is no upper limit on the size except as required for convenience.   Evaporation is preferred once the oligonucleotide solution is in contact with the solid support This is a drying method and can be performed at room temperature (about 25 ° C.). Complementary, if desired Significantly inhibits the ability of oligonucleotides to specifically hybridize to Unless otherwise, evaporation can be performed at elevated temperatures.   The method for immobilizing oriconucleotides on a solid support further comprises the steps of: "Baking" the above oligonucleotide solution. The oligonucleotide solution residue is removed from about 60 ° C. to about 95 ° C., preferably from about 70 ° C. Subjecting to a temperature of 80 ° C. Firing time is not important and is preferred Or about 15 minutes to about 90 minutes. For example, nitrocellulose Calcination is particularly preferred when using a porous support material.   If a porous support material is used, the coating step is optional for the methods provided herein. Can be used to form a smooth polymeric or non-polymeric support. When used for coating, The process is particularly preferred. Overcoats are typically present in support materials and fluid samples. Treat the support material to prevent possible non-specific interactions between complementary sequences Consisting of Cover or prevent the oligonucleotide solution before it dries on the support It is preferred to provide quality. Suitable inhibitors are casein, zein, bovine serum Albumin (BSA), 0.5% sodium dodecyl sulfate (SDS), and 1X To 5X solution of Denhardt (1X Denhardt's solution is 0.02% Ficoll, 0.02% % Polyvinylpyrrolidone and 0.2 mg / ml BSA). other Inhibitors can include detergents and long-chain water-soluble polymers.   Casein has been found to be a preferred inhibitor, and Sigma Chemical, Available from St Louis, MO. Casein is a “meta-soluble” protein (eg, No. 5,120,643 to Ching et al., Which is incorporated herein by reference. Belongs to a family of proteins known as You. Such treatments include acid or alkali treatments, resulting in complete protein cleavage And / or partial hydrolysis. Casein is about 23,60 0 (bovine β-casei Milk protein having a molecular weight of (casein) as used herein. Alkali-treated casein can be obtained from both US Patent 5,120,643. The partially hydrolyzed mixture by alkali treatment as described in Example 1 Point. An electrophoresis gel of casein thus treated (20% polyacrylamide) TBE) is primarily associated with the diffuse band below this marker. A mixture of fragments having a molecular weight of less than 15,000. ExampleEmbodiment 1 FIG. DNA hybridization analysis A. DNA waveguide creation   DNA waveguides for the detection of human genetic mutations causing cystic fibrosis The substrate was made from a 1 cm square. Oligonucleotides can be replicated on reactive surfaces. Fixed on glass to provide a number of capture sites. In particular, CAT01 to CAT09 ( Nine different oligonucleotides, designated SEQ ID NOs: 1-9), were CAT # is occupied by the same number on a standard push-button telephone for use on the phone surface A 3 × 3 array pattern was formed so as to correspond to the position. DNA spots are directly About 2mm in diameter Approximately 2 mm apart. Sequences of CAT01 to CAT09 (SEQ ID NOS: 1-9) The mutation sites are shown in Table 1.1.   Human genetic mutations are indicated by standard notation. For example, $ F508 is a sac 508 position of the cystic fibrosis transmembrane conductance regulatory polypeptide Refers to the deletion of three base pairs at the position (J. Zielenski et al.Genomics 10: 214-228, 199 1). {WT} indicates the wild type or normal sequence at this position. The DNA solution is S prepared by ynthecell (Columbia, MD) and 1:20 in PBS (phosphate buffered). Saline, pH 7.4) Dilute in buffer and round drill bit about 1 mm in diameter It was provided on the glass surface of the waveguide using the cutting edge. Transfer DNA to a clean glass surface Or Was fixed on the surface of glass coated with 0.05% casein for hybridization. The results of the session were indistinguishable. Of the DNA provided on the glass surface of the waveguide Final concentrations range from high values of 14 μM for CAT02 to low values of 0.9 μM for CAT08. And measured by comparison with the starting material concentration received from Synthecell. Was. After application, the DNA solution is conditioned at room temperature or at a relative humidity of about 35% to 80%. On an airy day, incubate at 50-70 ° C until dry (about 10 minutes). Dried on the chips in a mortar. According to this procedure, 9 pixels are added to the above 3 × 3 array. Pot II or hybridization capture sites were formed. Hippo in different glass The slip created a groove to hold the composite solution. Two cover slips , Stacked on offset, about 16mm wide and about 75μm thick (double-sided tape Double-sided tape (Arcare 7710B, Adhesives Research)  inc., Glen Rock, Penn). The groove has a volume of about 25 μl Had. B. Hybridization   Nine types with a biotin label at the 3 'end to evaluate the performance of DNA waveguides Additional oligonucleotide CAT21B CAT29B (SEQ ID NOS: 10-18) was synthesized by Synthecell. For testing The sequence of the DNA oligonucleotide is shown in Table 1.2.   Oligonucleotides are CAT21, which is complementary to CAT01 (SEQ ID NO: 1) B (SEQ ID NO: 10), CAT22B (complementary to CAT02 (SEQ ID NO: 2) SEQ ID NO: 11), others and CAT complementary to CAT09 (SEQ ID NO: 9) 29 (SEQ ID NO: 18). The concentration was CAT25B (distribution 473 μM as high as column number 14) to 15 as CAT27B (SEQ ID NO: 16). It changed to as low as 1 μM. 1 μl of each of the 9 DNA samples was mixed with 1 ml of the hybrid Dization In buffer (1% casein, 10 mM Tris pH 7.4, 15 mM NaCl) To 9 different DNA waveguides at room temperature (about 23 ° C.) For about 5 minutes. Clean the surface of the DNA waveguide with PBS using a washing bottle. Washed and then stored under PBS until detection of hybridization. C. Hybridization detection   The waveguide is illuminated with a light source consisting of a 150 watt incandescent bulb with a slit gap of about 2 mm. Irradiated from Light is guided so that the light enters the end of the waveguide that receives light with a thickness of 2 mm. The tube was inserted into the light source (see FIG. 2A). Slide the waveguide at about 45 ° to the mask. Inserted into the unit.   Hybridization of nine different biotin-labeled DNAs was performed -Detected in the waveguide by light scattered from the biotin complex. 546 nm absorption maximum U.S. Pat. No. 4,954,45 to Yost et al., Having a 32 OD concentration at large wavelengths. A composite was prepared using colloidal selenium particles as described in paragraph 2. 2. 5 μl of anti-biotin antibody (polyclonal rabbit anti-biotin antibody, PBS 1.13mg / ml in pH 7.4-U.S. Ser. No. 08 / 196,8, which is incorporated herein by reference. Selenium of Mushahwar et al. Corresponding to EP 0 160 900 B1) Addition to 1 ml colloid followed by 30 μl bovine serum albumin (20% w / v A selenium composite is prepared by adding powdered BSA dissolved in water to form a solution. Was. Apply 50 μl of the composite solution to the surface of the DNA waveguide and direct light toward the waveguide. Selenium binding to various DNA capture sites was observed. Positive hybridization Was observed at many sites within one minute. Wash the DNA waveguide with PBS Remove excess selenium conjugate and illuminate to achieve waveguide scattering And imaged. This visible signal is transferred to a standard 8-bit CCD (charge-coupled device). ) Recording using a camera (Cohu model 4815 Cohu, Inc., San Diego, CA). Picture Digital representation of the image is displayed on a Compac DeskPro 386 / 20e (Compaq Computer Corporation , Houston, TX) frame grabber (Imaging Technology Incorporated, PC VISI ON plus Frame Grabber Board; Woburn, Mass). Digitized image Convert the image data file and print the image on a 300 dpi resolution printer. Publishers PaintBrush software (ZSoft Corp., Atlanta, Georgia). The printed image is shown in FIG.   Single image measurement of all patterns of DNA hybridization using waveguide And the DNA sequence of the oligo provided to the waveguide was determined. CAT21 B (SEQ ID NO: 10) and CAT22B (SEQ ID NO: 11) (the first two CAT01 (SEQ ID NO: 1) and CAT02 (sequence The DNA capture sites other than No. 2) and these oligonucleotides can be ignored. Due to the sequence homology, the hybridization pattern is relatively simple. Was. However, in the case of CAT23B-CAT29B (SEQ ID NOS: 12-18) In case, significant sequence homology is attributable to more complex binding patterns.Embodiment 2. FIG. Detection of oligonucleotides immobilized directly on nitrocellulose A. Immobilization of oligonucleotides on nitrocellulose   The synthetic oligonucleotide was haptenized using conventional methodology and the resulting The sequences are shown in Table 2.1 below. These oligonucleotides are then individually 20X SSC buffer (3M sodium chloride, 342 mM sodium citrate) , pH 7.0) and diluted (1: 1) to 150 μM of each of the three oligonucleotides. A solution was obtained.   About 0.3 μl of each oligonucleotide solution was added to Schleicher &Schuell; Keene , 0.45 μm and 0.5 μm nitrocellulose available from . Spotted approximately in the middle of a 4 cm x 5 cm piece. Nitto oligonucleotide After being subjected to rocellulose, the nitrocellulose pieces were placed in an oven at 80 ° C. for 20 minutes. Heated. B. Detection of immobilized oligonucleotide   Rabbit anti-biotin selenium colloidal complex Diluted 1: 1 in distilled water before addition to the antibody (OD at 546 nm maximum wavelength). 15 was obtained, except that (15 was obtained). With the result Complex 1: 3 in 3% casein dissolved in mM NaCl, pH 7.8). One end of each piece of nitrocellulose on which the oligonucleotide was immobilized had 3 0 μl of the diluted conjugate was provided. Before observing when the faint red dot appears, The composite was moved along the length of the nitrocellulose (about 2 minutes). After about 5 minutes, Red dot on all nitrocellulose pieces in the area where the oligonucleotide is fixed Was observed.Embodiment 3 FIG. Orientation using oligonucleotides directly immobilized on nitrocellulose Gonucleotide capture A. Oligonucleotide immobilization   In this example, the oligonucleotide was immobilized on nitrocellulose and the complementary Single-stranded or double-stranded DNA sequences (Genosys, Woodlands, TX; and Sy nthecell, Columbia, MD). Double-stranded DNA One strand consists of a sequence complementary to the immobilized oligonucleotide. Nitrocell The oligonucleotides immobilized on the loin pieces can be found in Table 3.1.   A 150 μM solution of the oligonucleotide was added to 5 μm nitrocellulose (Schleicher & Schuell) by spotting on an approximately 0.4 cm x 5 cm piece Otide was fixed. The immobilization procedure consists of adding the oligonucleotide solution to a nitrocellulose And then heated as described in Example 2 except that these pieces were heated at 75 ° C. The order is the same. B. Hybridization of immobilized oligonucleotides with their homologs N   First, a double-stranded configuration consisting of a homologous oligonucleotide and a complementary oligonucleotide Rows (both of which are hereafter referred to as running oligonucleotides) are 100 μl The concentration was from M to 500 μM. 10 mM running oligonucleotide Diluted in 1% casein in Tris, 15 mM NaCl, pH 7.4. Was. The sequences of the running oligonucleotides are shown in Table 3.2. Can be seen.   Add 30 μl of running oligonucleotide to one end of the nitrocellulose piece This oligonucleotide was passed through the region containing the immobilized oligonucleotide. Hybridization was achieved by moving over (about 5 minutes) . Hybridization of running oligonucleotide with immobilized oligonucleotide Detection of the is achieved in one of two ways. One method is to make the piece purple Place it below the outside line and observe the position of the immobilized oligonucleotide by the fluorescent signal. It included. The other method uses a selection oligonucleotide mixed with a running oligonucleotide. Contact the colloidal complex with the end of the nitrocellulose strip to fix the oligonucleotide. The complex is moved past the position of the leotide and the immobilized oligonucleotide Involving observing the position by visual signal. Immobilized oligonucleotide Area The signals of Thus, the presence of the immobilized oligonucleotide in its original position was indicated. Selenkoroy Complex was bound to selenium colloid and running oligonucleotide Consists of an antibody specific for the label. The conjugate is treated with an anti-fluorescein antibody and an anti-bio It was prepared as in Example 2 above using an otin antibody.   FIG. 4 shows that SEQ ID NO: 23 has moved past the fixed SEQ ID NO: 1 position. It is a photograph under an ultraviolet lamp of a nitrocellulose piece. FIG. 4A shows 100n When M concentration SEQ ID NO: 23 was used as a running oligonucleotide, 5 specifically illustrates the fluorescent signal generated at the position of the oligonucleotide. FIG. 4 (b ) Used a 1 μM concentration of SEQ ID NO: 23 as a running oligonucleotide. In case, specifically indicate the fluorescent signal generated at the position of the immobilized oligonucleotide . FIG. 4 (c) shows that a 1.6 μM concentration of SEQ ID NO. When used as a probe, the fluorescent signal generated at the position of the immobilized oligonucleotide Is specifically shown. FIG. 4 (d) shows that SEQ ID NO. When used as a oligonucleotide, it occurs at the position of the immobilized oligonucleotide. Fluorescent This is a specific example of the signal. More specific by the signals observed in FIGS. 4 (a)-(d) As shown in the figure, the running oligonucleotide was immobilized on the immobilized oligonucleotide. Hybridized in the region originally provided with the peptide.   FIG. 5 shows that SEQ ID NO: 22 was immobilized on nitrocellulose and SEQ ID NO: 25 (double-stranded DNA) at a concentration of 24 (single-stranded DNA) and 20 μM Running buffer (10 mM Tris, 15 Another one-step format used at a 1:30 dilution in mM NaCl, pH 7.4). The results obtained from the test are shown below. Selenium colloid anti-fluorescein composite (actual Hybridization was visually detected using (as prepared in Example 2) . FIG. 5 (a) shows that SEQ ID NO: 24 and the selenium colloid conjugate have SEQ ID NO: 22. Shows the result after moving past a fixed position. Similarly, FIG. Shows that SEQ ID NO: 25 and the selenium colloid complex are fixed, while SEQ ID NO: 22 is fixed. 3 shows the result after moving past a certain position. According to FIGS. 5 (a) and 5 (b) As specifically shown, hybridization of single-stranded DNA (SEQ ID NO: 22) Was detected, but double-stranded DNA (SEQ ID NO: 25) No hybridization was detected.   FIG. 6 shows that SEQ ID NO: 1 was immobilized on three pieces of nitrocellulose, No. 26 was used as the running oligonucleotide. Show physically. FIG. 6 (a) shows that the running oligonucleotide is nitrocellulose. The results obtained when 100 nM concentration is applied to one end are specifically shown. FIG. ) Indicates that the running oligonucleotide was added to the end of the nitrocellulose piece at a concentration of 1 nM. The results obtained in the case where the sample was provided are specifically shown. FIG. 6 (c) shows the running oligonucleotide. Obtained when nucleotide was applied to the end of a nitrocellulose piece at a concentration of 0.1 nM. The results are specifically shown. Running oligonucleotides and immobilized oligonucleotides Hybridization with tides was performed using selenium colloid anti-biotin as described above. The conjugate (as prepared in Example 2) was used to detect visually. Fig. 6 (a)- As shown by 6 (c), observed in the region where SEQ ID NO: 1 was initially fixed Hybridization occurred in all three pieces as indicated by the signal .Embodiment 4. FIG. Oligonucleotides using DNA & PNA directly fixed on glass Capture A. Oligonucleotides and PNAs fixed directly to glass   Oligonucleotides were 22 mm x 22 m obtained from Corning m on a glass coverslip to capture homologous oligonucleotides Using. SEQ ID NO: 27 is a DNA oligonucleotide fixed to a glass cover slip. SEQ ID NO: 28 is PN immobilized on a glass coverslip. A oligonucleotide. SEQ ID NO: 27 was synthesized using conventional automation techniques. And listed in Table 4.1 below. SEQ ID NO: 28 from Millipore (Bedford, MA) Purchased and listed in Table 4.2 below.   Two dilutions of the oligonucleotide sequence were prepared. PNA with phosphate buffered saline A solution containing PNA at a concentration of 44 μM and 11 μM diluted in water (PBS). A liquid was obtained. The DNA was diluted with PBS to a concentration of 37 μM and 14 μM oligo. A solution containing nucleotides was obtained. 1 μl of each dilution is then added to a glass cap. Dispensed on burslip. Before heating the coverslip at 60 ° C. for 20 minutes The NA and DNA solutions were dried on coverslips at room temperature. Cover slip Is heated, then cooled to room temperature, and alkali-treated casein dissolved in HPLC water. Was coated with a 0.05% solution. Continue coating for 1 minute and remove excess casein by HPLC Rinse off coverslip with water. Forcibly remove the remaining liquid on the cover slip. Dried in the wind. B. Manufacture of optical waveguides   Using double-sided tape, cover slip with oligonucleotide sequence spotted A second 22 mm x 22 mm glass coverslip was secured (slightly centered). Away from). The groove formed between the two coverslips is about 0.75μ m and held approximately 25 μl of liquid reagent. In this way two waveguides Manufactured. C. Hybridization and detection   DNA complementary to SEQ ID NO: 27 (DNA) and SEQ ID NO: 28 (PNA) The sequence (Genosys) was used as the sample oligonucleotide. This sample oligonucleotide The otide was named SEQ ID NO: 29 and is listed in Table 4.3 below.   Separate dilutions of SEQ ID NO: 29 were prepared using buffers containing different concentrations of sodium phosphate. Prepared in solution. SEQ ID NO: 29 was prepared using 1.5 mM phosphate buffer and 150 mM phosphoric acid. Diluted to 170 nM in acid buffer. Enough to fill the waveguide groove The amount of each dilution was then dispensed into the two waveguides created above. The waveguide, It was then incubated for 5 minutes at room temperature in a humid room with 100% humidity. ink After incubation, the liquid in the waveguide was treated with anti-biotin prepared as described above in Example 1. Replace with a selenium colloid composite, and treat the composite with a 10% alkaline treatment in distilled water. Diluted 1: 1 with casein. Immediately after the replacement, the waveguide is set to 150 as in Example 1. Watts inserted into the light source.   FIG. 7 specifically shows the result obtained when the waveguide is inserted into the light source. FIG. ) Is the spot pattern used to fix various concentrations of SEQ ID NO: 27 and SEQ ID NO: 28. It is a legend showing a turn. FIG. 7 (b) shows the results obtained when the sample was diluted in 1.5 mM phosphate buffer. 29 shows the waveguide result of SEQ ID NO: 29 in this case. As shown by FIG. Null is strongest in the section spotted with 44 μM PNA. FIG. The result of the waveguide of SEQ ID NO: 29 when diluted with 0 mM phosphate buffer is shown. FIG. 7 ( As shown by c), the signal was the spot spotted with 44 μM PNA. Again, but the binding affinity of the DNA spot is greater than the 1.5 mM dilution. No.   Although the above examples illustrate some specific aspects of the present invention, the present invention However, the present invention is not limited to these specific embodiments. Rather, books to be protected The invention is set forth in the appended claims.

Claims (1)

[Claims] 1. A method of non-covalently immobilizing an oligonucleotide on a support material, comprising: (a) contacting the oligonucleotide solution with a solid support material, wherein the oligonucleotide comprises 5 to 30 nucleotides; and b) drying the solution on the solid support material. 2. 2. The method of claim 1, wherein said solution has an oligonucleotide concentration of 1 [mu] M to about 1 mM. 3. 2. The method of claim 1, wherein the solution further comprises a pH of 6.5 to 8.0, 75 mM to 2M sodium chloride and a buffer system. 4. The method of claim 3, comprising sodium citrate or sodium phosphate. 5. 2. The method of claim 1, wherein, after said drying, said method further comprises heating said solid support material at a temperature of 60C to 95C. 6. The method of claim 1, wherein the oligonucleotide further comprises an amine group at the 5 'or 3' end. 7. 2. The method of claim 1, wherein said oligonucleotide is a peptide nucleic acid. 8. After step (b), the method further comprises: (i) contacting the solid support with the complex; and (ii) detecting a measurable signal as an indicator of the presence or amount of the oligonucleotide. The method of claim 1 comprising: 9. After step (b), the method further comprises: (i) contacting the solid support with the complex; and (ii) detecting a measurable signal as an indicator of the presence or amount of the complex. The method of claim 1 comprising: 10. After step (b), the method further comprises: (i) contacting the solid support with a test sample suspected of containing a nucleic acid sequence complementary to the oligonucleotide to form a hybridization complex (Ii) contacting the complex with the complex; and (iii) detecting a measurable signal as an indicator of the presence or amount of the nucleic acid sequence.