WO1996019587A2 - Procedes d'immobilisation d'oligonucleotides sur des materiaux de support et procedes d'utilisation des oligonucleotides fixes auxdits supports - Google Patents

Procedes d'immobilisation d'oligonucleotides sur des materiaux de support et procedes d'utilisation des oligonucleotides fixes auxdits supports Download PDF

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WO1996019587A2
WO1996019587A2 PCT/US1995/016627 US9516627W WO9619587A2 WO 1996019587 A2 WO1996019587 A2 WO 1996019587A2 US 9516627 W US9516627 W US 9516627W WO 9619587 A2 WO9619587 A2 WO 9619587A2
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Prior art keywords
oligonucleotides
seq
oligonucleotide
immobilized
support material
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PCT/US1995/016627
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English (en)
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WO1996019587A3 (fr
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Tsung-Hui K. Jou
Joanell V. Hoijer
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Abbott Laboratories
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Priority to EP95943480A priority Critical patent/EP0795032A2/fr
Publication of WO1996019587A2 publication Critical patent/WO1996019587A2/fr
Publication of WO1996019587A3 publication Critical patent/WO1996019587A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates to oligonucleotides.
  • the invention relates to the immobilization of short oligonucleotides to support materials.
  • Amplification reactions such as the ligase chain reaction (LCR) which is described in European Patent Applications EP-A-320-308, the gap ligase chain reaction (GLCR) which is described in EP-A-439-182, and the polymerase chain reaction (PCR) which is described in U.S. Patents Numbered 4,683,202 and 4,683,195 are well known in the art.
  • LCR ligase chain reaction
  • GLCR gap ligase chain reaction
  • PCR polymerase chain reaction
  • Amplification reactions have also found utility in research and development fields as well as forensic fields.
  • Nucleic acid amplification techniques typically generate copies of a target nucleic acid sequence and the presence or amount of the target sequence copies can be detected using immunological assay techniques.
  • target sequence copies can be contacted with a "capture reagent" which comprises a substantially solid support material such as, for example, a suspension of microparticles coated with an oligonucleotide (variably referred to as a capture oligonucleotide) which specifically hybridizes with the target sequence copies.
  • a capture reagent which comprises a substantially solid support material such as, for example, a suspension of microparticles coated with an oligonucleotide (variably referred to as a capture oligonucleotide) which specifically hybridizes with the target sequence copies.
  • a capture reagent which comprises a substantially solid support material such as, for example, a suspension of microparticles coated with an oligonucleotide (variably referred to as a capture oligonucleotide) which
  • the target sequences are immobilized to the capture reagent, they can easily be separated from, for example, extraneous reactants, by separating the solid support from the reaction mixture such as by washing or filtration.
  • the presence or amount of the amplified sequences which may be immobilized to the capture reagent can be detected by contacting the captured target sequence with a "conjugate".
  • a conjugate can comprise a detectable moiety which is attached to specific binding pair member that also specifically binds the amplified target sequences which are immobilized to the capture reagent By detecting the presence of the detectable moiety, the presence or amount of the target sequences can be determined.
  • One known method of immobilizing an oligonucleotide to a support material uses chemical crosslinking agents.
  • crosslinking agents covalently bind a support material and an oligonucleotide to form a linking arm which attaches the oligonucleotide to the support material.
  • U.S. Patent No. 4,948,882 discloses compounds which can be employed to covalently link an oligonucleotide to a solid support material.
  • chemically crosslinking an oligonucleotide to a support material generally is a time consuming process which requires modifications to the base pairs comprising the oligonucleotide.
  • Another method of immobilizing an oligonucleotide to a support material which is described in Saiki, R.K., et al., Proc. Natl. Acad. Sci.
  • Tails are extensions of oligonucleotides that are typically around fifteen base pairs or more in length.
  • An oligonucleotide's tail preferentially binds solid support material and, similarly to a crosslinking agent, leaves the oligonucleotide free of the support material and available for hybridization.
  • tails which are themselves nucleic acids, sometimes interfere with the oligonucleotide's ability to specifically hybridize to a nucleic acid sequence.
  • the present application describes a method for directly and non- covalently immobilizing an oligonucleotide to a support material.
  • immobilization of an oligonucleotide to a support material is effected quickly and without chemical modifications to the bases comprising the oligonucleotide.
  • the hybridization capacity of a directly immobilized oligonucleotide is not impaired.
  • Oligonucleotides and support materials to which they are immobilized can be employed as capture reagents for immobilizing nucleic acid sequences which are complementary to the oligonucleotides bound to the support material.
  • the method comprises the steps of contacting a solution of oligonucleotides with a solid support material and drying the oligonucleotide solution upon the support material.
  • the oligonucleotides in solution can be in the range of between about 5 nucleotides and about 30 nucleotides in length. Additionally, it has been discovered that the affinity of an oligonucleotide for a support material can be enhanced by modifying the oligonucleotides.
  • the method may further comprise a baking step and/or an overcoating step.
  • the presence or amount of the support bound oligonucleotides can be detected by contacting the solid support material, and the oligonucleotides immobilized thereon, with a conjugate and detecting a measureable signal as an indication of the presence or amount of the immobilized oligonucleotides.
  • the solid support material, and the immobilized oligonucleotides thereon can be contacted with a conjugate and a measurable signal can be detected as an indication of the presence or amount of the conjugate.
  • support bound oligonucleotides can be contacted with a test sample suspected of containing nucleic acid sequences which are complementary to the immobilized oligonucleotides to form hybridization complexes.
  • the hybridization complexes can then be contacted with a conjugate and a measureable signal can be detected as an indication of the presence or amount of any complementary nucleic acid sequences in the test sample.
  • FIG 1 illustrates the principles of total internal reflectance (ILK).
  • Figure 2A, 2B and 2C are, respectively, perspective, side and cross section views of a waveguide device.
  • Figure 2C is an enlarged cross section taken alone line C-C of Figure 2B.
  • Figures 3, 4A-4D, 5A-5B, 6A-6C, and 7A-7B are printed representations of results obtained for assays using a capture reagent comprising oligonucleotides which were immobilized to a support material as taught herein. Details of these printed data are found in Example 1 through Example 4.
  • an oligonucleotide comprising between about 5 nucleotides and about 50 nucleotides can be directly and non-covalently attached to a solid support material without impairing the hybridization capacity of the immobilized oligonucleotide. While the mechanism by which oligonucleotides direcdy adhere to a solid surface is not completely understood, directly attaching an oligonucleotide to a solid surface means that an oligonucleotide becomes immobilized to a support material in a manner similar to adsorption.
  • oligonucleotides can specifically pair or hybridize with a complementary nucleic acid sequence.
  • Oligonucleotides which are immobilized as taught herein can be employed to capture or otherwise immobilize complementary nucleic acid sequences such as, for example, 2'-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA - as described WO 93/25706) and the like to support materials to which the oligonucleotides are immobilized.
  • complementary nucleic acid sequences such as, for example, 2'-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA - as described WO 93/25706) and the like to support materials to which the oligonucleotides are immobilized.
  • complementary sequences hybridize to the immobilized oligonucleotides, their presence can be detected using methodologies well known in the art.
  • a capture reagent comprising a solid support having oligonucleotides directly immobilized thereon can be contacted with a test sample.
  • the test sample can be any liquid suspected of containing a nucleic acid sequence which can specifically hybridize with the immobilized oligonucleotides.
  • the capture reagent and test sample can be contacted for a time and under conditions suitable for allowing nucleic acids in the test sample, if any, and the oligoncucleotides to hybridize and thereby form hybridization complexes.
  • the hybridization complexes if any, can be contacted with a conjugate for a time and under conditions sufficient to enable the conjugate to specifically bind any hybridization complexes.
  • a signal can then be detected as an indication of the presence or amount of any nucleic acid sequences which may have been present in the test sample.
  • Immobilized oligonucleotides as taught herein can also be employed in a "one-step" assay configuration. According to such a configuration, a test sample suspected of containing nucleic acids which are complementary to the immobilized oligonucleotides can be contacted with a conjugate for a time and under conditions suitable for allowing the conjugate to bind any nucleic acid sequences which may be present in the test sample to form conjugate/nucleic acid complexes.
  • the nucleic acids which may be present in a test sample may comprise a detectable moiety.
  • Nucleic acid sequences can be labeled or conjugated with a detectable moiety through, for example, nick translation whereby labeled nucleotides are incorporated into a target sequence.
  • Conjugate/nucleic acid complexes or nucleic acids which comprise a detectable moiety can then be contacted with the support bound oligonucleotides to form conjugate/nucleic acid/oligonucleotide complexes or nucleic acid/oligonucleotide complexes.
  • a signal can then be detected as an indication of the presence or amount of any nucleic acid sequences present in the test sample.
  • a method for quickly detecting the presence of an oligonucleotide in a test sample is provided.
  • a sample which is suspected of containing oligonucleotides can be contacted with a support material and the oligonucleotides which may be present in the test sample can be immobilized to the support material as taught herein.
  • a conjugate can then be contacted with the immobilized oligonucleotides for a time and under conditions for allowing the conjugate to bind the immobilized oligonucleotides.
  • a signal can then be detected as an indication of the presence or amount of any oligonucleotides which may have been present in the test sample.
  • oligonucleotides which are immobilized as taught herein are contacted with, for example, a test sample, conjugate/nucleic acid complexes, or a conjugate is not important. However, it is preferred that such a contact period be kept to a minimum for example less than 30 minutes, more preferably less than 15 minutes and most preferably less than 10 minutes.
  • a conjugate may comprise a detectable moiety attached to specific binding pair member.
  • Detectable moieties may include any compound or conventional detectable chemical group having a detectable and measurable physical or chemical property variably referred to as a signal.
  • detectable groups can be, but are not intended to be limited to, enzymatically active groups such as enzymes and enzyme substrates, prosthetic groups or coenzymes; spin labels; fluorescent molecules such as fluorescers and fluorogens; chromophores and chromogens; luminescent molecules such as luminescers, chemiluminescers and bioluminescers; phosphorescent molecules; specifically bindable ligands such as biotin and avidin; electroactive species; radioisotopes; toxins; drugs; haptens; polysaccharides; polypeptides; liposomes; colored or fluorescent particles; colored or fluorescent microparticles; colloidal particles such as selenium colloid or gold colloid; and the like.
  • a detectable moiety can comprise, for example, a plurality of fluorophores immobilized to a polymer such as that described in co-owned and co-pending U.S. Patent Application Serial No. 091,149 filed on July 13, 1993, which is herein incorporated by reference.
  • the detectable physical or chemical property associated with a detectable moiety can be detected visually or by an external means.
  • Specific binding member is a well known term and generally means a member of a binding pair, i.e., two different molecules where one of the molecules through chemical or physical means specifically binds to the other molecule.
  • binding pairs include, but are not intended to be limited to, avidin and biotin, antibody and hapten, complementary nucleotide sequences or complementary nucleic acid sequences such as DNA, RNA or PNA, an enzyme cofactor or substrate and an enzyme, a peptide sequence and an antibody specific for the sequence or an entire protein, dyes and protein binders, peptides and specific protein binders (e. g., ribonuclease, S-peptide and ribonuclease S- protein), and the like.
  • avidin and biotin antibody and hapten
  • complementary nucleotide sequences or complementary nucleic acid sequences such as DNA, RNA or PNA
  • an enzyme cofactor or substrate and an enzyme an enzyme cofactor or substrate and an enzyme
  • a peptide sequence and an antibody specific for the sequence or an entire protein dyes and protein binders, peptides and specific protein binders (e. g., ribonuclease, S-peptide and
  • binding pairs can include members that are analogs of the original binding member, for example, an analyte-analog or a binding member made by recombinant techniques or molecular engineering.
  • PNAs are specific binding members for DNA or RNA.
  • the binding member is an immunoreactant it can be, for example, a monoclonal or polyclonal antibody, a recombinant protein or recombinant antibody, a chimeric antibody, a mixture(s) or fragment(s) of the foregoing.
  • Detectable moieties can be attached to specific binding pair members through any chemical means and/or physical means that do not destroy the specific binding properties of the specific binding member or the detectable properties of the detectable moiety.
  • the method herein provided is employed to immobilize oligonucleotides to a glass surface which is then employed in a waveguide configuration such as that taught in co-owned and co-pending U.S. Patent Application Serial No.08/311,462 filed on September 22, 1994 and entitled "Light Scattering Optical Waveguide Method for Detecting Specific Binding Events” which is herein incorporated by reference.
  • a waveguide device's ability to be employed in an immunoassay or hybridization type format is based upon a phenomenon called total internal reflection (TIR) which is known in the art and is described with reference to Figure 1.
  • TIR total internal reflection
  • TER operates upon the principle that light 10 traveling in a denser medium 12 (i.e.
  • Factors that tend to increase the penetration depth are: increasing angle of incidence, ⁇ R; closely matching indices of refraction of the two media (i.e. N2/N 1 -> 1); and increasing wavelength, ⁇ .
  • ⁇ R angle of incidence
  • N2/N 1 -> 1 closely matching indices of refraction of the two media
  • ⁇ increasing wavelength
  • TTR has also been used in conjunction with light scattering detection in a technique referred to as Scattered Total Internal Reflectance ("STIR").
  • Scattered Total Internal Reflectance See, e.g., U.S. Patents 4,979,821 and 5,017,009 to Schutt, et al and WO 94/00763 (Akzo N. V.).
  • a beam of light is scanned across the surface of a TIR element at a suitable angle and the light energy is totally reflected except for the evanescent wave.
  • Particles such as red blood cells, colloidal gold or latex specifically bound within the penetration depth will scatter the light and the scattered light is detected by a photodetection means.
  • Figures 2A-2C illustrate a waveguide device 30 comprising a planar waveguide element 32 and a parallel planar plate 34.
  • the waveguide element thus has parallel surfaces 36 and 38 as well as a light-receiving edge 40.
  • the plate 34 has parallel surfaces 42 and 44.
  • the waveguide element 32 and the plate 34 are held together in spaced parallel fashion, such that the element surfaces 38 and the plate surface 42 define a narrow channel 46.
  • the element and plate may be held together by any convenient means, including adhesive means 48 consisting of double stick tape disposed along the edges of the element and plate.
  • the channel 46 is preferably rather small so as to enable capillary transfer of a fluid sample therethrough.
  • the height should be less than about 1mm, preferably less than about 0.1mm.
  • the element 32 should be made of an optically transparent material such as glass, quartz, plastics such as polycarbonate, acrylic, or polystyrene.
  • the refractive index of the waveguide must be greater than the refractive index of the sample fluid, as is known in the art for effecting total internal reflectance.
  • the refractive index, n is about 1.33, so the waveguide typically has a refractive index of greater than 1.35, usually about 1.5 or more.
  • the waveguide may be a piece of plastic or glass, for example, a standard glass microscope slide or cover slip may be used.
  • the plate 34 may be constructed of similar materials. As seen in Figures
  • the light receiving end 40 of the waveguide element 32 is disposed in a narrow slit 50 of a mask 52 in order to minimize the effects of stray light originating from the light source 54. Minimization of stray light is also improved by the use of light absorbing materials.
  • Light source 54 for generating the incident light beam may be a source of electromagnetic energy, including energy in the visible, ultra-violet, and near-IR spectra. The term "light” is thus construed quite broadly and is not confined to the visible range, except in cases where detection is made visually.
  • Non-visible wavelengths are detected by detectors optimized for the particular wavelength as is well known in the art
  • the light may be monochromatic or polychromatic, collimated or uncollimated, polarized or unpolarized.
  • Preferred light sources include lasers, light emitting diodes, flash lamps, arc lamps, incandescent lamps and fluorescent discharge lamps.
  • the light source used to iUuminate the waveguide element can be a low wattage helium-neon laser.
  • the light source can be a small incandescent light bulb powered by a battery, such as is used in pocket flashlight
  • the light source includes potentiometer means for varying the intensity of the light source.
  • filters and/or lenses may be employed to adjust the intensity to a suitable level.
  • Detection means may be employed to determine light scattering produced by a light scattering label (LSL).
  • LSL light scattering label
  • a LSL may be immobilized to surface 38 of waveguide element 32 via interactions between specific binding members such as, for example, that between an immobilized oligonucleotide and a cognate DNA sequence.
  • a LSL is a molecule or a material, often a particle, which causes incident light to be scattered elastically, i.e. substantially without absorbing the light energy.
  • Exemplary LSLs include colloidal metal and non-metal labels such as colloidal gold or selenium; red blood cells; and dyed plastic particles made of latex, polystyrene, polymethylacrylate, polycarbonate or similar materials.
  • the size of such particulate labels ranges from 10 nm to 10 ⁇ m, typically from 50 to 500 nm, and preferably from 70 to 200 nm.
  • Suitable particle LSLs are available from Bangs Laboratories, Inc., Carmel, IN, USA.
  • Instrumentation and visual detection means may be employed to determine the degree of light scattering produced by a LSL.
  • Light scattering events across the entire waveguide can be monitored essentially simultaneously, whether by the eye and brain of an observer or by photodetection devices including CCD cameras which form images that are digitized and processed using computers.
  • immobilizing oligonucleotides to support materials comprises contacting a support material with an oligonucleotide solution and drying the solution upon the support material.
  • Support materials or solid supports to which oligonucleotides can be immobilized are well known in the art and include materials that are substantially insoluble. Porous materials can serve as solid supports and may include, for example, paper, nylon; and cellulose as well as its derivatives such as nitrocellulose. Smooth polymeric and nonpolymeric materials are also suitable support materials and include, but are not intended to be limited to, plastics and derivatized plastics such as, for example, polycarbonate, polystyrene, and polypropylene; magnetic or non-magnetic metal; quartz and glass. Preferably, quartz, glass or nitrocellulose is employed as a support material.
  • Solid supports can be used in many configurations well known to those skilled in the art including, but not limited to, test tubes, microtiter wells, sheets, films, strips, beads, microparticles, chips, slides, cover slips, and the like.
  • Oligonucleotides according to the invention will be understood to mean a sequence of DNA, RNA or PNA.
  • the length of an oligonucleotide which is immobilized to a support material is largely a matter of choice for one skilled in the art and is typically based upon the length of a complementary sequence of, for example, DNA, RNA, or PNA which will be captured.
  • an immobilized oligonucleotide is typically between about 5 and about 50 bases, preferably, the length of an immobilized oligonucleotide is between about 5 and about 30 bases, more typically between about 10 and about 25 bases.
  • Synthesis of oligonucleotides is fairly routine using automated synthesizers. If desired, automated synthesizers can produce oligonucleotides which are modified with terminal amines or other groups. A useful review of coupling chemistries is found in Goodchild, Bioconjugate Chemistry. 1(3): 165- 187 (1990).
  • Modified oligonucleotides may have a greater affinity for solid supports than unmodified oligonucleotides.
  • Methodologies for modifying an oligonucleotide are well known and may include the addition of chemical groups such as amines, or haptens such as fluorescein to an oligonucleotide. Such modifications do not provide for covalent linkages between a support material and an oligonucleotide, but nevertheless have been found to increase the affinity of oligonucleotides for support materials. Modifications are particularly effective when made to the 3' or 5' ends of an oligonucleotide.
  • the oligonucleotide solution that is contacted with a solid support may comprise oligonucleotides which are in solution.
  • concentration of the oligonucleotides in the solution is largely a matter of choice for one skilled in the art and is typically based upon how the immobilized oligonucleotides will be employed.
  • the oligonucleotide solution will have an oligonucleotide concentration of between about 1 ⁇ M and about 1 mM, preferably between about 20 ⁇ M and about 250 ⁇ M.
  • modified oligonucleotides have been found to have a greater affinity for support materials than unmodified oligonucleotides.
  • modified oligonucleotides can be employed in lower concentration ranges than unmodified oligonucleotides.
  • the pH of the oligonucleotide solution may be between about 6.5 and about 8.0, preferably between about 7.0 and about 7.5.
  • oligonucleotide solutions are preferably saline and the sodium chloride concentration of such a solutions can vary greatly but is typically between about 75 mM and about 2 M, preferably between about 100 mM and about 1 M, and most preferably between about 120 mM and about 500 mM.
  • Buffering systems may optionally be included in the oligonucleotide solution.
  • Buffering systems are well known and generally comprise aqueous solutions of compounds which resist changes in a solution's hydrogen ion concentration.
  • Examples of buffering systems include, but are not intended to be limited to, solutions of a weak acid or base and salts thereof such as, for example, acetates, borates, phosphates, phthalates, citrates, carbonates and the like.
  • the buffering system comprises between about 5 mM and about 250 mM tris®, sodium citrate, or sodium phosphate, more preferably between about 10 mM and about 200 mM tris®, sodium citrate, or sodium phosphate and most preferably between about 10 mM and about 175 mM tris®, sodium citrate, or sodium phosphate.
  • the amount of oligonucleotide solution which is applied or "spotted" upon a solid support need be large enough only to capture sufficient complementary sequences to enable detection of, for example, a captured sequence or conjugate. This is dependent in part on the density of support material to which the capture oligonucleotide is immobilized. For example, areas of as little as 150 ⁇ m in diameter may be employed. Such small areas are preferred when many sites on a support material are spotted with oligonucleotide solution(s).
  • the practical lower limit of size is about l ⁇ in diameter. For visual detection, areas large enough to be detected without magnification are desired; for example at least about 1 to about 50 mm 2 ; up to as large as 1 cm 2 or even larger. There is no upper size limit except as dictated by manufacturing costs and user convenience.
  • evaporation is the preferred drying method and may be performed at room temperature (about 25°C). When desired, the evaporation may be performed at an elevated temperature, so long as the temperature does not significantly inhibit the ability of the oligonucleotides to specifically hybridize with complementary sequences.
  • the process of immobilizing oligonucleotides to a solid support may further comprise "baking" the support material and the oligonucleotide solution thereon. Baking may include subjecting the solid phase and oligonucleotide solution residue, to temperatures between about 60°C and about 95°C, preferably between about 70°C and about 80°C.
  • the baking time is not critical and preferably lasts for between about 15 minutes and about 90 minutes. Baking is particularly preferred when porous support materials such as, for example, nitrocellulose are employed.
  • Overcoating typically comprises treating the support material so as to block non-specific interactions between the support material and complementary sequences which may be in a fluid sample. It is preferred that the overcoating or blocking material is applied before the oligonucleotide solution has been dried upon the support material.
  • Suitable blocking materials are casein, zein, bovine serum albumin (BSA), 0.5% sodiumdodecyl sulfate (SDS), and IX to 5X Denhardt's solution (IX Denhardt's is 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.2 mg/ml BSA).
  • Other blockers can include detergents and long-chain water soluble polymers.
  • Casein has been found to be a preferred blocking material and is available from Sigma Chemical, St Louis, MO. Casein belongs to a class of proteins known as "meta-soluble" proteins (see, e.g., U.S. Patent 5,120,643 to Ching, et al, incorporated herein by reference) which are preferably treated to render them more soluble. Such treatments include acid or alkaline treatment and are believed to perform cleavage and/or partial hydrolysis of the intact protein.
  • Casein is a milk protein having a molecular weight of about 23,600 (bovine beta- casein), but as used herein, "casein” or “alkaline treated” casein both refer to a partially hydrolyzed mixture that results from alkaline treatment as described in example 1 of US Patent 5,120,643.
  • An electrophoresis gel (20% polyacrylamide TBE) of the so-treated casein shows a mixture of fragments predominantly having molecular weight less than 15,000, as shown by a diffused band below this marker.
  • DNA waveguides for the detection of human genetic mutations that cause cystic fibrosis were constructed from glass substrates 1 cm square.
  • Oligonucleotides were immobilized to the glass to provide multiple capture sites in the reactive surface.
  • CATOl through CAT09 (SEQ ID Nos. 1 - 9) were applied to the glass surface of the waveguide to form a 3 x 3 array pattern such that the CAT# corresponded to the position occupied by the same number on a standard touch-tone telephone.
  • DNA spots were about 2 mm in diameter and about 2 mm apart.
  • the sequence and mutation site of CATOl through CAT09 (SEQ ID Nos. 1 - 9) are shown in
  • the DNA solutions were allowed to dry on the chip at room temperature or, on humid days between about 35% and 80% relative humidity, in an incubator set at 50-70 °C until dry (about 10 minutes). This procedure formed nine "spots" or hybridization capture sites in the 3 x3 array described above.
  • Another glass cover slip created a channel to hold the conjugate solution.
  • the two cover slips were offset and held together by double-sided tape (Arcare 7710B, Adhesives Research Inc., Glen Rock, Penn) so as to form a channel approximately 16 mm wide and approximately 75 ⁇ m thick (the thickness of the double sided tape). The channel held approximately 25 ⁇ l in volume.
  • CAT21B (SEQ ID No. 10) is complementary to CATOl (SEQ ID No. 1)
  • CAT22B (SEQ ID No. 11) is complementary to CAT02 (SEQ ID No. 2)
  • et cetera to CAT29 (SEQ ID No. 18) which is complementary to CAT09 (SEQ ID No. 9).
  • concentrations varied from a high of 473 ⁇ M for CAT25B (SEQ ID No. 14) to a low of 151 ⁇ M for CAT27B (SEQ ID No. 16).
  • Each of the nine DNA samples were diluted 1 ⁇ l into 1 ml of hybridization buffer (1% casein, 10 mM Tris pH 7.4, 15 mM NaCl), and a different one was applied to each of the nine different DNA waveguides and incubated at room temperature (approximately 23 °C ) for 5 minutes.
  • the surface of the DNA waveguides were washed with PBS using a wash bottle and then stored under PBS until detection of hybridization.
  • the waveguide was illuminated with a light source comprising a 150 watt incandescent bulb with a ca. 2 mm slit aperture.
  • the waveguide was inserted into the light source slit so that light was shone into the 2 mm thick light receiving edge of the waveguide (see figure 2A).
  • the waveguide was inserted into the slit at approximately 45° relative to the mask.
  • Hybridization of the nine different biotin labeled DNA's was detected in the waveguide by light that was scattered from a selenium anti-biotin conjugate.
  • Colloidal selenium particle as described in US Patent No. 4,954,452 to Yost, et al. having a 32 O.D. concentration, at the absorption maximum wavelength of 546 nm, was used to manufacture the conjugate.
  • the selenium conjugate was prepared by addition of 2.5 ⁇ l of anti-biotin antibody (polyclonal rabbit anti- biotin antibody, 1.13 mg/ml in PBS, pH 7.4- see EP 0 160 900 B I to Mushahwar, et al., corresponding to US Serial No.
  • This visual signal was recorded using a standard 8 bit CCD (charged coupled device) camera (Cohu model 4815 Cohu, Inc., San Diego, CA).
  • a digital representation of the image was created using a frame grabber (Imaging Technology Incorporated, PC VISION plus Frame Grabber Board; Woburn, Mass) in a Compac DeskPro 386/20e (Compaq Computer Corporation, Houston, TX).
  • the digitized image data file was converted and imported into Publishers PaintBrush software (ZSoft Corp., Atlanta, Georgia) from which the image was printed on a 300 dpi resolution printer.
  • the printed image is shown as Figure 3.
  • the entire pattem of DNA hybridization was detected using the waveguide in a single image measurement and allowed determination of the DNA sequence of the oligo applied to the waveguide.
  • Synthetic oligonucleotides were haptenated using conventional methodologies and the resulting sequences are shown below in Table 2.1. The oligonucleotides were then individually diluted (1:1) in 20X SSC buffer (3 M sodium chloride, 342 mM sodium citrate, pH 7.0) to yield three 150 ⁇ M solutions of each oligonucleotide. TABLE 2.1
  • oligonucleotide solution Approximately 0.3 ⁇ l aliquots of each oligonucleotide solution were dotted to the approximate middle of individual 0.4 cm x 5 cm strips of 0.45 ⁇ m and 0.5 ⁇ m nitrocellulose available from Schleicher & Schuell; Keene, NH. After the oligonucleotides were applied to the nitrocellulose, the nitrocellulose strips were baked in an oven at 80°C for 20 minutes.
  • a rabbit anti-biotin selenium colloid conjugate was prepared as in Example 1 except the colloid was diluted 1:1 in distilled water (to yield an OD of 15 at a maximum wavelength of 546 nm) before it was added to the polyclonal antibody.
  • the resulting conjugate was diluted 1:3 in 3% casein dissolved in tris® buffered saline (TBS - 100 mM tris, 150 mM NaCl, pH 7.8). 30 ⁇ l of the diluted conjugate was applied to one end of each of the nitrocellulose strips which had the oligonucleotides immobilized thereon.
  • the conjugate was allowed to migrate along the length of the nitrocellulose strips (approximately 2 minutes) before an observation was made at which point a faint red dot was developing. After approximately 5 minutes, a red dot was observed on all of the nitrocellulose strips in the area where the oligonucleotides had been immobilized.
  • oligonucleotides were immobilized to nitrocellulose and employed to capture complementary single stranded DNA sequences or double stranded DNA sequences (obtained from Genosys, Woodlands, TX; and Synthecell, Columbia, MD).
  • One strand of the double stranded DNA comprised a sequence complementary to the immobilized oligonucleotides.
  • the oligonucleotides which were immobilized to the nitrocellulose strips can be found in Table 3.1. TABLE 3.1
  • the oligonucleotides were immobilized to nitrocellulose by dotting 150 ⁇ M solutions of the oligonucleotides onto approximately 0.4 cm x 5 cm strips of 5 ⁇ m nitrocellulose (Schleicher & Schuell).
  • the immobilization procedure was the same as the procedure set forth above in Example 2 except that after the oligonucleotide solutions were applied to the nitrocellulose strips, the strips were baked at 75°C.
  • the cognate oligonucleotides and the double stranded sequences comprising a complementary oligonucleotide were at a concentration of between 100 ⁇ M and 500 ⁇ M.
  • the running oligonucleotides were diluted in 1% casein dissolved in 10 mM tris, 15 mM NaCl, pH 7.4.
  • the sequences of the running oligonucleotides can be found in table 3.2.
  • Hybridization was achieved by applying 30 ⁇ l of the running oligonucleotides to one end of the nitrocellulose strips and allowing the oligonucleotides to migrate past the region containing the immobilized oligonucleotides (approximately 5 minutes). Detecting hybridization of the running oligonucleotides to the immobilized oligonucleotides was accomplished in one of two ways. One method included placing the strips under UN. light and observing the situs of the immobilized oligonucleotides for a fluorescent signal.
  • the other method included contacting the ends of the nitrocellulose strips with a selenium colloid conjugate mixed with the running oligonucleotides, allowing the conjugate to migrate past the situs of the immobilized oligonucleotides, and observing the situs of the immobilized oligonucleotides for a visible signal.
  • a signal in the region of the immobilized oligonucleotides indicated the hybridization of the running oligonucleotides and therefore the presence of the immobilized oligonucleotides in their original position.
  • the selenium colloid conjugates comprised selenium colloid and an antibody specific for the label attached to the running oligonucleotide.
  • FIG. 4 is a photograph of nitrocellulose strips under a UN. lamp after SEQ ID No. 23 migrated past the situs of immobilized SEQ ID No. 1.
  • Figure 4(a) illustrates the fluorescent signal generated at the situs of the immobilized oligonucleotides when a 100 nM concentration of SEQ ID No. 23 was employed as the running oligonucleotide.
  • Figure 4(b) illustrates the fluorescent signal generated at the situs of the immobilized oligonucleotides when a 1 ⁇ M concentration of SEQ ID No. 23 was employed as the running oligonucleotide.
  • Figure 4(c) illustrates the fluorescent signal generated at the situs of the immobilized oligonucleotides when a 1.6 ⁇ M concentration of SEQ ID No. 23 was employed as the running oligonucleotide.
  • Figure 4(d) illustrates the fluorescent signal generated at the situs of the immobilized oligonucleotides when a 3.3 ⁇ M concentration of SEQ ID No. 23 was employed as the running oligonucleotide.
  • the running oligonucleotides hybridized to the immobilized oligonucleotides in the region where they were originally applied.
  • Figure 5 illustrates the results obtained from another one step format where SEQ ID No. 22 was immobilized to nitrocellulose and SEQ ID No. 24 (single stranded DNA), at a 20 ⁇ M concentration, and SEQ ID No. 25 (double stranded DNA), at a 20 ⁇ M concentration, were used as the running oligonucleotides at a 1 :30 dilution in the running buffer (1 % casein dissolved in 10 mM tris, 15 mM NaCl, pH 7.4).
  • a selenium colloid anti-fluorescein conjugate (as prepared in Example 2) was employed to visually detect hybridization.
  • Figure 5(a) shows the results after SEQ ID No.
  • FIG. 5(b) shows the results after SEQ ID No. 25 and the selenium colloid conjugate had migrated past the situs where SEQ ID No. 22 had been immobilized.
  • Figures 5(a) and 5(b) hybridization was detected for the single stranded DNA (SEQ ID No. 22) but no hybridization was detected for the double stranded DNA (SEQ ID No. 25).
  • Figure 6 illustrates the results obtained when SEQ ID No. 1 was immobilized to three nitrocellulose strips and various concentrations of SEQ ID No. 26 were employed as a running oligonucleotide.
  • Figure 6(a) illustrates the results obtained when the running oligonucleotide was applied to the end of a nitrocellulose strip at a 100 nM concentration.
  • Figure 6(b) illustrates the results obtained when the running oligonucleotide was applied to the end of a nitrocellulose strip at a 1 nM concentration.
  • Figure 6(c) illustrates the results obtained when the running oligonucleotide was applied to the end of a nitrocellulose strip at a 0.1 nM concentration.
  • Hybridization of the running oligonucleotide to the immobilized oligonucleotide was detected visually, as above, using a selenium colloid anti-biotin conjugate (as prepared in Example 2). As shown by Figures 6(a)-6(c), hybridization occurred on all three strips as indicated by the signals observed in the regions where SEQ ID No. 1 was initially immobilized.
  • Oligonucleotides were immobilized to 22 mm x 22 mm glass cover slips from Corning and employed to capture a cognate oligonucleotide. SEQ ID No.
  • SEQ ID No. 28 was the PNA oligonucleotide immobilized to the glass cover slip.
  • SEQ ID No. 27 was synthesized using convenitonal automated techniques and is listed below in Table 4.1.
  • SEQ ID No. 28 was purchased from Millipore
  • the PNA was diluted in phosphate buffered saline (PBS) to yield solutions containing PNA concentrations of 44 ⁇ M and 11 ⁇ M.
  • the DNA was diluted in PBS to yield solutions containing oligonucleotide concentrations of 37 ⁇ M and 14 ⁇ M. 1 ⁇ l aliquots of each dilution were then dispensed onto glass cover slips.
  • the PNA and DNA solutions were allowed to dry onto the cover slip at room temperature before the cover slip was baked at 60°C for 20 minutes. After the cover slip was baked, it was cooled to room temperature and overcoated with a 0.05% solution of alkaline treated casein dissolved in HPLC water. The overcoating lasted for one minute and excess casein was rinsed from the cover slip with HPLC water. Residual liquid on the cover slip was dried with forced air.
  • a second 22 mm x 22 mm glass cover slip was secured (slightly off center) to the cover slips which had been spotted with the oligonucleotide sequences.
  • the channel formed between the two cover slips was approximately 0.75 ⁇ m deep and held approximately 25 ⁇ l of a liquid reagent. Two waveguides were produced in this manner.
  • sample oligonucleotide 27 (DNA) and SEQ ID No. 28 (PNA) was employed as a sample oligonucleotide.
  • the sample oligonucleotide is designated SEQ ID No. 29 and is listed below in Table 4.3.
  • SEQ ID No. 29 biotin-TCACTCACCAACCTC Separate dilutions of SEQ ID No. 29 were made in buffers containing different concentrations of sodium phosphate. SEQ ID No. 29 was diluted to 170 nM in 1.5 mM phosphate buffer and 150 mM phosphate buffer. An aliquot, large enough to fill the waveguide's channel, of each dilution was then dispensed into the two waveguides produced above. The waveguides were then incubated at room temperature for 5 minutes in a humidity chamber at 100% humidity.
  • the liquid in the waveguide was displaced with an anti-biotin selenium colloid conjugate which was prepared as above in Example 1 then the conjugate was diluted 1:1 with 10% alkaline treated casein in distilled water. Immediately upon displacement, the waveguide was inserted into a 150 watt light source as in Example 1.
  • Figure 7 illustrates the results obtained when the waveguide was inserted into the light source.
  • Figure 7(a) is a legend showing the dotting pattem used for the immobilization of the various concentrations of SEQ ID No. 27 and SEQ ID No. 28.
  • Figure 7(b) shows the waveguide results for SEQ ID No. 29 when diluted in 1.5 mM phosphate buffer. As shown by Figure 7(b), the signal was the greatest in the area where 44 ⁇ M PNA was dotted.
  • Figure 7(c) shows the waveguide results for SEQ ID No. 29 when diluted in 150 mM phosphate buffer. As shown by Figure 7(c), the signal was again the greatest in the area dotted with the 44 ⁇ M PNA but the binding affinity for the DNA spots was greater than in the 1.5 mM dilutions.
  • MOLECULE TYPE synthetic DNA
  • FEATURE FEATURE
  • MOLECULE TYPE synthetic DNA
  • FEATURE (A) NAME/KEY: 3' amine

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Abstract

Procédé d'immobilisation directe d'un oligonucléotide sur un matériau support consistant à mettre en contact une solution d'oligonucléotides avec un matériau support solide puis à dessécher ladite solution sur le support. Les oligonucléotides et leur support d'immobilisation peuvent servir de réactifs de capture pour l'immobilisation des séquences nucléotidiques complémentaires des oligonucléotides immobilisés. On maintient de cette façon la capacité d'hybridation des oligonucléotides directement immobilisés. Sont également présentés des procédés de détermination de la présence ou du volume de séquences nucléotidiques dans un échantillon d'essai.
PCT/US1995/016627 1994-12-22 1995-12-20 Procedes d'immobilisation d'oligonucleotides sur des materiaux de support et procedes d'utilisation des oligonucleotides fixes auxdits supports WO1996019587A2 (fr)

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EP1133574A1 (fr) * 1998-11-25 2001-09-19 The Perkin-Elmer Corporation Procedes et appareil pour hybridation en continu
KR100459865B1 (ko) * 2001-11-26 2004-12-03 아람 바이오시스템 주식회사 유전적 염기서열 고정 방법
WO2005094370A2 (fr) * 2004-03-29 2005-10-13 The General Hospital Corporation Compositions de complexe d'oligonucleotides et procedes d'utilisation de celles-ci comme outils de modification de genes
US7232688B2 (en) 1997-01-23 2007-06-19 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device
US9669376B2 (en) 2000-10-30 2017-06-06 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate

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US6133436A (en) 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7232688B2 (en) 1997-01-23 2007-06-19 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
EP1133574A1 (fr) * 1998-11-25 2001-09-19 The Perkin-Elmer Corporation Procedes et appareil pour hybridation en continu
EP1133574A4 (fr) * 1998-11-25 2005-11-30 Applera Corp Procedes et appareil pour hybridation en continu
US9669376B2 (en) 2000-10-30 2017-06-06 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
KR100459865B1 (ko) * 2001-11-26 2004-12-03 아람 바이오시스템 주식회사 유전적 염기서열 고정 방법
WO2005094370A2 (fr) * 2004-03-29 2005-10-13 The General Hospital Corporation Compositions de complexe d'oligonucleotides et procedes d'utilisation de celles-ci comme outils de modification de genes
WO2005094370A3 (fr) * 2004-03-29 2005-12-29 Gen Hospital Corp Compositions de complexe d'oligonucleotides et procedes d'utilisation de celles-ci comme outils de modification de genes
US8314226B2 (en) 2004-03-29 2012-11-20 The General Hospital Corporation Oligonucleotide complex compositions and methods of use as gene alteration tools
US9617535B2 (en) 2004-03-29 2017-04-11 The General Hospital Corporation Oligonucleotide complex compositions and methods of use as gene alteration tools
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device

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