WO1994000600A1 - Supports solides pour dosages d'hybridation de l'acide nucleique - Google Patents

Supports solides pour dosages d'hybridation de l'acide nucleique Download PDF

Info

Publication number
WO1994000600A1
WO1994000600A1 PCT/US1993/006044 US9306044W WO9400600A1 WO 1994000600 A1 WO1994000600 A1 WO 1994000600A1 US 9306044 W US9306044 W US 9306044W WO 9400600 A1 WO9400600 A1 WO 9400600A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
polymer
amine
nylon
poly
Prior art date
Application number
PCT/US1993/006044
Other languages
English (en)
Inventor
Jeffrey Van Ness
Charles R. Petrie
John C. Tabone
Nicolaas M. J. Vermeulen
Michael W. Reed
Original Assignee
Microprobe Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Microprobe Corporation filed Critical Microprobe Corporation
Priority to AU45441/93A priority Critical patent/AU4544193A/en
Publication of WO1994000600A1 publication Critical patent/WO1994000600A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates generally to the field of nucleic acid hybridization assays for detecting specific polynuc * eotide sequence-? and, more par 4 - larly, o compos ions and methods for covalent.
  • Nucleic acid hybridization is a nowr ,od fc identifying specific sequences of nucleic acid, jbrid- ⁇ :.n is based upon base pairing between complements- - lcleic a strands.
  • jbrid- ⁇ :.n is based upon base pairing between complements- - lcleic a strands.
  • comp l ementary base sequence air to form double stranded stable molecules. The presence o absence of such pairing may be detected by several different methods described in. the art.
  • hybridization assays involve multiple steps, for example the hybridization technique described by Dunn, et al., Cell 12:23-36 (1977) (incorporated herein by reference) , wherein a sandwich-type assay consists of a first hybridization between a "target" nucleic acid and a "capture” nucleic acid probe that has been immobilized on a solid support and a second hybridization between a "signal" nucleic acid probe, typically labeled with a radioactive isotope, and a different region of the immobilized target nucleic acid. The hybridization of the signal probe may then be detected by, for example, autoradiography.
  • European Patent Application 86112899.9 (both incorporated herein by reference) have described treatment of a nylon support with an alkylating agent to introduce amidine groups onto the surface of the nylon.
  • the derived nylon surface possesses the capacity to noncovalently bind single stranded nucleic acids.
  • the noncovalently bound nucleic acids are then used as probes to detect specific target nucleic acids in solution.
  • compositions comprising an activated oligonucleotide, which can be covalently attached to a polymer-coated support such as a bead are described.
  • a polymer-coated support such as a bead
  • cyanuric chloride a homotrifunctional reagent, activates the oligonucleotide, or derivatives of cyanuric chloride.
  • the covalently immobilized oligonucleotides on polymer-coated beads or similar structures can serve as nucleic acid probes, and hybridization assays can be conducted wherein specific target nucleic acids are detected in complex biological samples.
  • the beads can be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.
  • immobilized oligonucleotides on a polymer-coated bead can be of the same or different nucleic acid sequences.
  • the bead solid supports of the present invention possess the following advantages over known membrane or bead supports, in which capture nucleic acid sequences are noncovalently attached.
  • the capture rate of target nucleic acid sequences is improved 5 to 25-fold, and 100% of the capture nucleic acid sequence is available for hybridization with a complementary sequence; ⁇ ⁇ -ond, the quantity of immobilized capture nucleic acid c be increaf . approximately 20-fold on an apparent surface ⁇ _ ⁇ a basis; third, greater ease of manufacturing exists; fourth, the bead possesses covalently immobilized, capture nucleic acid sequences (oligonucleotides) and can withstand denaturation temperatures in excess of 90°C for 10 or more minutes; and, finally, a multisite dipstick can be constructed, leading to miniaturization of a detection device. All of these advantages contribute to greater sensitivities when, for example, a sandwich assay format is used.
  • Dipsticks are also included in the present invention. These dipsticks comprise a nonporous solid support and a means for attaching the beads or similar structures discussed above. Additionally, multiple beads possessing oligonucleotides with different sequences or specificities can be closely aligned on a multisite dipstick, giving rise to an indicator card that can detect a multiplicity of pathogens in a single biological example, for example, to identify bacterial and viral agents.
  • beads or similar structures in the dipstick format achieves a significant decrease in nonspecific background levels of signal systems because of the simple pressure fit by which the bead or similar structure is placed in the dipstick, as compared with membrane supports and the like, which necessarily must be sandwiched between two supports or glued or attached in place.
  • Fig. 1 is a configuration for a dipstick of the present invention
  • Fig. 2 is a multisite indicator card that can be used for periodontitis diagnosis
  • Fig. 3 are high performance liquid chromatography (HPLC) profiles showing that cyanuric chloride reacts selectively with the 5'-tethered amine of an oligonucleotide, and not with sugars or bases thereof.
  • compositions useful in nucleic acid hybridization assays comprise a dichlorotriazine oligonucleotide and other activated oligonucleotides covalently attached to a polymer-coated bead or similar structure. Additionally, processes for covalently immobilizing an oligonucleotide on a solid support, preferably a bead, are included.
  • these processes comprise the steps of treating a solid support with an alkylating agent; reacting the treated solid support with an amine-containing polymer, whereby the polymer covalently coats the solid support; activating an oligonucleotide with a monofunctional or multifunctional reagent, preferably the homotrifunctional reagent, cyanuric chloride (i.e., 2,4,6-trichlorotriazine) ; conjugating the activated oligonucleotide and the polymer-coated solid support.
  • the unreacted amines are then blocked by acylatation to impart the proper surface charge to the solid support surface.
  • solid support refers to any surface that is transferable from solution to solution or forms a structure for conducting oligonucleotide-based assays, and includes beads, membranes, microtiter wells, strings, plastic strips, or any surface onto which nucleic acid probes may be immobilized.
  • beads encompasses any type of solid or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material onto which a nucleic acid can be covalently immobilized. As such, the term also includes string or strings.
  • a bead that is spherical in shape is employed in the present compositions, and a preferred diameter range for such beads is from about 0.01 inch to about 0.5 inch, more preferably from about 0.06 inch to about 0.09 inch (corresponding to commercially available 3/32 inch nylon beads), and most preferably about 0.09 inch (corresponding to commercially available 3/32 inch nylon beads) . Additionally, it is preferred that the beads are unpolished or, if polished, roughened before treating with an alkylating agent.
  • the solid support will typically be comprised of a polymeric material containing derivatizable functional groups [e.g., poly(p-aminostyrene) ] or polymeric soild supports that can be activated (e.g., nylon beads, cyanuric chloride activated cellulose commercially available) .
  • a polymeric material containing derivatizable functional groups e.g., poly(p-aminostyrene)
  • polymeric soild supports that can be activated (e.g., nylon beads, cyanuric chloride activated cellulose commercially available) .
  • preferred solid support compositions include nylon, polystyrene, glass, polypropylenes, polystyrene/glycidyl ethacrylate latex beads, latex beads containing amino, carboxyl, sulfonic and/or hydroxyl groups, polystyrene coated magnetic beads containing amino and/or carboxylate groups, glass, teflon, plastic and the like.
  • Procedures for preparing the different solid phases will be different depending on the functional groups available on the solid support and the polymer coat.
  • the chemistries to covalently couple compounds with the same or different functional grouops are well known in the art. See, e.g., Ji, Method. Enzymol. 91:580-609 (1983), which is incorporated herein by reference.
  • a nylon bead or beads, or any composition or structure of nylon is first derived (prepared) by treating the bead with an alkylating agent. Alkylating agents used in this manner react with amides present in the nylon to form a reactive imidate ester. Preferred alkylating agents are described below.
  • the activated nylon surface is reacted with a polymer containing an amine group, and the coated bead is then reacted with cyanuric chloride or derivatives as appropriate.
  • the appropriate chemistries well known to the skilled artisan, are selected to coat the bead.
  • To immobilize the aminohexyl tailed oligonucleotides to the polymer coated solid support requires the appropriate chemistries, depending on the functional groups.
  • Most solid support surfaces described above can be modified to introduce polar groups on the surface by a process referred to as the Coronal Discharge Process. Using this process, a support, e.g., plastic, is subjected to sparks of electricity in the presence of ozone to introduce carboxyl groups.
  • Preferred alkylating agents include, but are not limited to, dialkyl sulfates, alkyl triflates, alkyldiphenyl sulfonium salts, alkyl perchlorates, and, preferably, trialkyloxoniu salts.
  • the latter includes the lower alkyl salts, trimethyloxonium and triethyloxoniu salts.
  • the salt counterion can be selected from the group consisting of hexachloroantimonate, hexafluorophosphate, and tetrafluoroborate, with the last named counterion being preferred; however, other salt counterions can also be used and will be apparent to one skilled in the relevant art.
  • a solvent for the alkylating agent is important for the present invention.
  • a solvent should be employed that does not dissolve or render tacky nylon during the alkylation.
  • Non-nucleophilic organic solvents such as dichloromethane, dimethylsulfoxide, tetrahydofuran, N-methyl - pyrrolidone, and others are appropriate solvents. In particular, N-methyl-pyrrolidone is preferred.
  • the resulting imidate esters on the bead surface are then reacted under suitable conditions with an amine-containing polymer, whereby amidine residues are formed. Any primary or secondary amine-containing polymer can be employed to form amidine residues, thus covalently immobilizing the polymer onto the surface of the bead.
  • PEI Poly(ethyleneimine)
  • polyallylamine polyallylamine
  • polyvinylamine are preferred examples.
  • PEI is available in a variety of molecular weights, ranging from about 600 to about 100,000 Daltons, although preferably a polymer of about 30,000 MW is used to coat a solid phase.
  • a PEI polymer of 10,000 MW contains about 58 primary, 116 secondary and 58 tertiary amine groups, and is a bush-like structure.
  • polymers which introduce multiple functional groups which increases the number of oligonucleotides that can be immobilized on a polymer coated surface include cellulose propionate, poly(aerylamide-acrylie acid 10% carboxyl) , poly(acryhydrazide) , poly(p-aminostyrene) , poly(ethylene-methacrylic acid), poly-L-lysine (MW 40-60,000), poly(vinylsulfonic acid) and the like. Standard chemistries are used to immobilize polymers (amine, carboxyl, sulfonyl and hydroxyl containing) to amine, carboxyl, sulfonyl or hydroxyl containing solid supports.
  • a preferred solvent used to dissolve a polymer during the conjugation of the polymer to an' activated nylon bead is N-methyl-pyrrolidone.
  • Nylon can also be partially hydrolyzed to yield reactive amine or carboxyl groups that can be reactive with amine- or carboxyl-containing polymers.
  • any carboxyl moieties coating the surface of a solid support can be coated with amine-containing polymers using similar chemistries described above.
  • any other polymer capable of being derived with any primary or secondary aliphatic or aromatic amine is suitable for the present invention.
  • the units of such a polymer are joined together by direct polymerization process bonding or by coupling agent linking.
  • Direct polymerization produces interbonding of available chemical groups or backbone moieties in adjacent units.
  • oxidative enzymes can be used to polymerize monomer units by oxidative cross-linking.
  • a coupling agent derived from a bifunctional or multifunctional organic cross-linking agent, can bond with the appropriate chemical group or backbone moiety of the units.
  • the term “coupling agent” denotes the linkage group after bonding and the term “cross-linking reagent” denotes the linkage compound before bonding.
  • the cross-linking reagent has generic formula:
  • reactive groups A, B, and E are independently selected from the group consisting of hydrogen, a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an iminoester, a primary amine, an aldehyde group, an ⁇ -halo methylcarbonyl group, a hydrazine group, an acyl hydrazide group, an azide group, and an N-maleimide group, wherein at least two of A, B, and E are other than hydrogen.
  • R 1 is an aliphatic group of at least two carbons or an aromatic or heterocycle group of at least six carbons.
  • Multifunctional cross-linking reagents with more than three reactive groups that are similar to A, B, and E are also within the scope of the present invention. These additional reactive groups will be independently selected from the foregoing definitions of A, B, and E.
  • Choice of the reactive groups will depend upon the selection of the chemical groups or backbone moieties of the polymer units (monomeric units) that are to be linked. Each type of chemical group or backbone moiety will react with the appropriate reactive group or groups.
  • an amine group will react with a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an acyl imidazolide, an N-(carbonyloxy) imide group, an iminoester, or an aldehyde group.
  • An oxidized 1,2-diol group (a dialdehyde) will react with a primary amine, a hydrazine group, an azide, or an acyl hydrazide group.
  • a carbonyl group will react with a primary amine, a hydrazine group, or an acyl hydrazide group.
  • a mercaptan group will react with a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an acyl imidazolide, or an N-(carbonyloxy) imide.
  • a carbon-hydrogen bond will react with an azide (nitrene) .
  • the solid supports are thus coated with the selected polymer, including multifunctional polymers, that contains a large number of activatable primary and secondary amines.
  • polymer-coated solid supports are then conjugated with activated oligonucleotides or other substances of interest using similar or identical chemistries to those described above.
  • an amine-containing polymer can be covalently linked through the amine to a protein by activating the protein with a homofunctional or heterofunctional reagent and conjugating the activated protein to the polymer.
  • the protein may be a receptor, ligand, enzyme, hormone, interleukin, antibody, etc.
  • the polymer is coupled to a solid support, such as a bead, membrane, microwell, centrifuge tube, or string.
  • oligonucleotides refer to short nucleic acid sequences that are approximately 12 to 100 bases in length. Such oligonucleotides can be used as capture probes in hybridization assays and are preferably chemically synthesized using commercially available methods and equipment. For example, the solid phase phosphoramidite method can be used to produce short probes of between 15 and 50 bases having a molecular weight of less than 16,000 daltons. For the synthesis of oligonucleotides, see Caruthers, et al., Cold
  • nucleotide sequence When synthesizing an oligonucleotide probe for a specific target nucleic acid, the choice of nucleotide sequence will determine the specificity of the test. For example, by comparing DNA sequences from several bacterial isolates, one can select a sequence for bacterial detection that is either type-specific or genus-specific. Comparisons of DNA regions and sequences can be achieved using commercially available computer programs.
  • the preferred capture oligonucleotides for use in the present invention are synthetic oligonucleotides from about 16 to about 100 bases in length.
  • a spacer (linker) arm i.e., a chemical moiety that extends or links other chemical groups, and preferably is a carbon chain containing from about 2 to about 12 carbon atoms, more preferably about 6 carbon atoms, containing a blocked amine group can be coupled during synthesis using conventional chemistry to the 5'-hydroxyl group of an oligonucleotide.
  • a primary amine is the preferred group for attachment to monofunctional or multifunctional reagents, and its attachment via a hexyl arm is preferred.
  • an oligonucleotide possessing a 5 » -terminal structure such as
  • n 2-12 inclusive, preferably 6;
  • X is -NH- or -NHC:0(CH 2 ) m NH-, preferably -NH-; wherein m is 2-12, inclusive;
  • Y is a 4,6-dichlorotriazine (preferred) or thiol (sulfhydryl) reactive moiety; and
  • A is an oligonucleotide, ranging from between about 9-50 bases, preferably between about 15-30 bases, with only the 5'-hydroxyl requiring modification for attachment.
  • an oligonucleotide can be modified at the 3'-end with a spacer arm containing a blocked amine group. This can be accomplished by conducting DNA synthesis on a solid support containing a conjugated ribonucleotide. After removal from the solid support, a DNA oligonucleotide is obtained that contains a single 3'-terminal ribonucleotide.
  • oligonucleotides are then activated with a monofunctional or multifunctional reagent.
  • Activated oligonucleotides refer in general to oligonucleotides that have been reacted with a chemical compound and rendered chemically active. As used herein, activatable refers to the potential to become chemically reactive.
  • Multifunctional reagents include, but are not limited to, homotrifunctional, heterotrifunctional, homobifunctional, and heterobifunctional reagents.
  • Activated oligonucleotides may be linked to polymer-coated solid supports according to the following chemistries.
  • oligonucleotide there are two modes by which the oligonucleotide can be covalently attached to the polymer at this point.
  • An amine-tailed oligonucleotide can be activated with a monofunctional or multifunctional reagent, for example cyanuric chloride whereby an alkylamino dichlorotriazine is formed, which is then reactive toward the amine-containing polymer.
  • the polymer on the surface of the bead can be activated with a reagent, preferably the homotrifunctional reagent cyanuric chloride, which is then reactive toward the amine-tailed or amine-derived oligonucleotide.
  • a reagent preferably the homotrifunctional reagent cyanuric chloride
  • cyanuric chloride a homotrifunctional reagent is preferred, other reagents can be used.
  • N-succinimidyl-4-(iodoacetamido)-benzoate (SIAB) is a heterobifunctional reagent, and disuccinimidyl suberate is a homobifunctional reagent.
  • the heterobifunctional reagent l-ethyl-3-(dimethylaminopropyl)- carbodiimide can be used.
  • Other similar monofunctional and multifunctional (heteromultifunctional and homomultifunctional) reagents are included for use in processes of the present invention.
  • the chemistries employed in the present invention result in the selective activation of an amino group on an oligonucleotide, without modification of any of the purine and pyrimidine bases of the oligonucleotide, as demonstrated in Example 1 below.
  • the placement of the amine-containing polymer on the bead surface and the covalent immobilization of an activated capture oligonucleotide onto such a surface increases the rate or extent of capture of target nucleic acid 5 to 25-fold compared with diamine compounds, for example, hexanediamine. Additionally, the nonspecific binding of biological material on the surface of the bead is substantially reduced.
  • the preferred chemistry employs cyanuric chloride (i.e., 2,4,6-trichlorotriazine) .
  • cyanuric chloride i.e., 2,4,6-trichlorotriazine
  • the chemistry of the cyanuric chloride reaction is as follows.
  • R is an oligonucleotide and preferably is of the structure depicted for a 5'-amino hexyl oligonucleotide.
  • Oligonucleotides possessing a 5'- or 3'-tethered (via a hexyl arm) nucleophilic amine moiety (or internal aminoalkyl groups substituted on pyrimidine or purine bases) are reacted with an excess, preferably from about 50 to about 200-fold, more preferably 125-fold, of recrystallized cyanuric chloride at, preferably, 15-50°C, more preferably at 19-25°C, in a part organic solvent for a preferred time period from about 30 minutes to about 6 hours, more preferably from about 1 to about 2 hours.
  • cyanuric chloride derivatives include 2-methoxy-4,6- dichlorotriazine (synthesized according to Schaeffer et al (J. Am. Chem. Soc. 73: 2990-2989 (1951)), 2,4,6-tribromotriazine, 2,4,6-triiodoatrazine, 2-monochloro-4,6-diiodotriazine, and 2- amino-4,6-dichlorotriazine.
  • the rate of reaction of these cyanuric chloride derivative reagents with amino-tailed oligonucleotides may be controlled as necessary, such as by performing reactions at lower temperatures.
  • 2-methoxy-4,6- dichlorotriazine is more reactive than cyanuric chloride and is prone to hydrolysis by water.
  • a similar compound, 2,4- dichlorotriazinyl - cellulose reacts directly with nucleic acids, presumably through the nucleophilic groups on the purine and pyrimidine bases (Hunger et al.. Anal. Biochem. 156:286-299 (1986) and Biagoni et al., Anal. Biochem.
  • organic solvents and acylating reagents are routinely used to block the aminogroups on purines and pyrimidines, and would, therefore, make specific activation of 5*- amino-tailed oligonucleotides difficult if not impossible in organic solvents.
  • amino-tailed oligonucleotides can specifically be activated in 0.1 M borate pH 8.3 with 2-methoxy-4,6- dichlorotriazine at 4°C.
  • the activated oligonucleotide is rapidly separated from the excess 2-methoxy-4,6- dichlorotriazine at 4°C on a Sephadex G50 straw column.
  • the 2- methoxy-4,6-dichlorotriazinyl - oligonucleotide is then immobilized to a polymer-coated solid phase, such as nylon beads coated with PEI, as described herein.
  • cyanuric chloride In addition to derivatives of cyanuric chloride, other compounds may substitute for cyanuric chloride to specifically activate amino-tailed oligonucleotides. These include, among others, 2,4-dinitrofluorobenzene, pyrimidines (e.g., 2,4-dichloropyrimidines) , 2,6-dichloro-3-nitropyridine, dichloropyridiazines (e.g., 3,6-dichloropyridiazine) , dichloropyrazines (e.g., 2,6-dichloropyrazine) , and trichloropyrimidines.
  • 2,4-dinitrofluorobenzene e.g., 2,4-dichloropyrimidines
  • 2,6-dichloro-3-nitropyridine 2,6-dichloro-3-nitropyridine
  • dichloropyridiazines e.g., 3,6-dichloropyridiazine
  • the 2-amino-4,6-dichlorotriazine may also be used to activate a hydroxyl-containing solid support to yield an activated solid support that will have the necessary activity to react specifically with an amino-tailed oligonucleotide as described herein.
  • hydroxyl- containing polymers such as cellulose can be activated by cyanuric chloride and then be coated with PEI polymer. Both of the more reactive triazinyl chlorides will react with the many amino groups available on the PEI polymer, and the PEI- cellulose is then reacted with activated oligonucleotide.
  • an oligonucleotide Once an oligonucleotide has been activated, such as with cyanuric chloride or derivatives or substitutes mentioned above or the like, it can be covalently linked to molecules that contain appropriately disposed r. cleophiles such as thiols, hydroxyls, or amines. More ;...
  • dichlorotriazine oligonucleotides can serve as electrophiles for covalent attachment to many compositions including, but not limited to: proteins, enzymes, antibod" s, lectins, antibodies and protein carriers used to raise ant dies; nucleophile- derived oligonucleotides, such as 5'- or 3'-aminohexyl-i- ⁇ iled oligonucleotides; nucleophile-containing polymers, sue-: .s poly(ethyleneimine) , polyallyla ine, and polyvinylamine .
  • Dichlorotriazine oligonucleotides are included in the present invention, as are processes for activating oligonucleotides with cyanuric chloride or derivatives thereof to form the corresponding activated oligonucleotides as discussed below.
  • the unreacted cyanuric chloride or derivatives thereof can be removed by exclusion chro atography or ultrafiltration, and the bead and derived oligonucleotide conjugated wherein they are mixed together and incubated at, preferably 20 to 50°C for 1 to 24 hours.
  • the residual (unreacted) amines on the bead surface can be blocked (capped) with an agent, such as succinic anhy d ride, preferably in
  • N-methyl pyrrolidone in the presence of an appropriate base such as sodium borate, to render the surface compatible (negatively charged) for nucleic acid hybridization.
  • an appropriate base such as sodium borate
  • Such blocking of amines occurs through an acylating reaction or reaction of amines with an activated ester, resulting in a nonactivatable moiety. It should be noted that the ability exists for the bead surface to chemically derived such that a positive, negative, or neutral charge can be placed on the bead.
  • 4,6-dichlorotriazine moieties can be replaced with thiol reactive substituents and, preferably, spacer arms are present.
  • the preferred spacer arms are derived from thiol reactive substituents linked to a 5'-tethered nucleophilic amine and are of the same formula as listed above with the exception that Y in the formula above is a thiol reactive moiety.
  • a preferred thiol reactive moiety has a reactive group of either an ⁇ halo-acyl or an a, ⁇ -unsaturated carbonyl.
  • the most preferred thiol reactive moieties are selected from the group consisting of haloacetamidobenzoyl and 4-(N-maleimidomethyl)- cyclohexane-1-carbonyl.
  • a polymeric structure as described above wherein the polymeric structure is derived with thiol (sulfhydryl) containing moieties can be used in the present invention.
  • the actual structure of the thiol containing moieties is non-critical as long as the thiol group or groups are available to react with thiol reactant moieties.
  • the thiol chemistries in this instance are replacements for cyanuric chloride chemistries.
  • the present invention also includes dipsticks having utility in nucleic acid hybridizations and comprising a nonporous solid support and a means for attaching a bead.
  • Nonporous solid supports are known in the art, and the present invention is concerned with attaching a bead to a dipstick.
  • An example of bead attachment is the presence of a perforation or perforations (or a depression or depressions) in the dipstick wherein beads can be attached.
  • perforations are employed and the beads are attached through a pressure fit with the circumference of the hole. Such a pressure fit can occur if, for example, the circumference of the perforation (or depression) is slightly less than the circumference of the bead so that the bead is pressed in place.
  • Preferred beads are as listed above, and may be covalently attached, either directly or through a spacer arm, to activated oligonucleotides of the same or different sequence per a given bead, as also described above.
  • the dipstick can contain more than one bead, preferably from about two to ten, each in their own hole, and more preferably, situated in a row along one edge of the dipstick.
  • Such a dipstick can function as an indicator card, wherein multiple beads covalently attached to oligonucleotides with different sequences or specificities can be closely aligned on a multisite dipstick, which can detect a multiplicity of pathogens in a single biological sample.
  • a particular bead may contain oligonucleotides with more than one nucleic acid sequence, for example, sequences from related organisms, or a bead may only contain oligonucleotides with a given nucleic acid sequence.
  • Orgar _ ⁇ s include bacteria and viruses as well as other microorga; as, and cell types include, for example, those involved in inherited diseases and metabolic disorders.
  • Many other detection applications will be apparent to one of ordinary skill in the art.
  • purported causative bacterial agents of periodontitis such as Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Bacteroides forsythus, Bacteroides intermedius, Eikenella corrodens, Fusobacterum nucleatum, and Wolinella recta, can be identified.
  • any member of a ligand pair can be attached to beads in the dipstick, and the dipstick can then be used to identify the corresponding ligand member.
  • antigens or antibodies could be attached to beads, as described above, in a dipstick and then their corresponding antibodies or antigens, respectively, could be identified.
  • biotin and streptavidin can be used.
  • the present invention also includes processes for nucleic acid detection wherein a composition comprising a polymer-coated bead, preferably having activatable amine groups, covalently attached to an activated oligonucleotide is contacted with a target nucleic acid under suitable conditions for hybridization and the hybridized product is detected.
  • Such processes can occur in a microtiter well, a flow-through column, and using a dipstick, as described above.
  • Target nucleic acid is usually a polynucleotide with an average length from about 20 to about 20,000 bases or nucleotides in length.
  • Suitable conditions for hybridization refer to stringent conditions wherein base-pairing mismatching does not occur and the hybridized product is perfectly base-paired.
  • Hybridization techniques are generally described in Hames, B.D., et al. (ed.), Nucleic Acid Hybridization, A Practical Approach, IRL Press, New York (1985) . As improvements are made in hybridization techniques, they can readily be applied to the present invention.
  • Sandwich assays can be preferably employed in the present processes wherein the target nucleic acid to be detected is either extracted or in the original sample and is sequestered (captured) on a solid support, such as beads, by hybridization (i.e., pairing of complementary bases) to capture oligonucleotide probes covalently immobilized on the surface of the support.
  • the captured nucleic acid is then hybridized to a signal oligonucleotide probe or, alternatively, this step can be performed simultaneously with the capture of the target by including the signal probe within, for example, the hybridization solution.
  • the signal probe can be, for example, labeled with biotin.
  • a reporter group as used herein is a group having a physical or chemical characteristic that can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity. Or, it may be provided by the ability of the reporter group to serve as a ligand recognition site. Any haptenic or antigenic compound can be used in combination with a suitably labeled antibody.
  • Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphotases, esterases, ureases, and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescers include luciferin, luminol, and oxetanediones. The above list is not complete, and the choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • the present invention concerns methods and compositions to increase the oligonucleotide loading capability of control pore glass (CPG) bead columns and the like in automated oligonucleotide synthesis.
  • CPG control pore glass
  • the CPG bead, "burnished" glass bead, silica gel, nylon bead, etc. is activated as described above, e.g., with 3- aminopropyltriethoxysilane, and the resulting aminopropyl - CPG, silica gel or glass bead is further reacted with cyanuric chloride or a derivative thereof described above.
  • the bead or gel is then reacted with polymer to coat the support as described above, e.g., with PEI.
  • the amines are then coupled with the appropriate chemistries to allow oligonucleotide synthesis.
  • the coated bead or CPG substantially amplifies oligonucleotide loading on the CPG surfaces, which is particularly important in the preparative scale synthesis of oligonucleotides, and is a convenient substitute for expensive CPG columns currently used in oligonucleotide synthesis.
  • chemistries may be used to activate the support, as described herein, as well as the subsequent activation used to conjugate the selected polymer to the support, depending on the functional groups involved.
  • the invention provides for the preparation of nylon coated magnetic particles.
  • Nylon coated magnetic particles are useful in hybridization assays, immunoassays, extractions, affinity chromatography, etc.
  • Nylon coated magnetic beads are particularly useful in the isolation of nucleic acids, including mRNA.
  • nylon 6 (100 mg) is dissolved in m-cresol (0.7 ml) and dichloromethane (1.6 ml) (see Verschoor et al., J. Immun. Meths. 127:43-49 (1990)) or other solvents. This solution is then added to a vigorously stirred (or sonicated) solution of ⁇ Fe 2 0 3 (magnetic iron oxide, obtained from Pfizer Minerals, Pigments and Metal Division, NY, NY; see also US 4,672,040, which is incorporated herein by reference) in either 20% dimethylformamide or ethanol or water.
  • ⁇ Fe 2 0 3 magnetic iron oxide, obtained from Pfizer Minerals, Pigments and Metal Division, NY, NY; see also US 4,672,040, which is incorporated herein by reference
  • the nylon may first be dissolved and mixed with polymer described above, preferably PEI polymer, and the solution co-precipitated onto the magnetic particles.
  • the nylon may be activated via alkylation and reacted with the polymer.
  • the nylon-polymer conjugate is then dissolved and precipitated onto the magnetic iron oxide particles, or the magnetic particles may be added during the polymerization reaction.
  • the present invention further provides means for convenient extraction and isolation of nucleic acids.
  • Centrifuge tubes, wells or the like are coated with a nylon- polymer coat, such as nylon-PEI as described above. This will yield a surface with multiple positive charges.
  • the sample to be extracted is added to the nylon-polymer coated tube and lysed and extracted using convention extraction methods, e.g., using the IsoQuick and the organic phase removed.
  • the aqueous phase is diluted, e.g., 10- to 20-fold, preferably about 15-fold, the nucleic acids in the sample adsorb to the positively charged surface of the coated tube.
  • the absorbed nucleic acids can then be washed, used to perform a PCR reaction in the same tube as generally described in U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference, or the nucleic acids can be eluted by a salt solution and used as desired.
  • nylon-polymer coated tubes having multiple positive charges can be prepared a variety of ways.
  • the nylon-polymer conjugate, where t ; nlymer is preferably PEI is dissolved and then precipitate o the surface of the tubes.
  • nylon micrc covalently covered with the polymer, prepared as i ed above are dissolved and precipitated onto the si of the tubes.
  • polymer-coated nylon disci . f and the like can be placed in microwells, centrifuge tuber nieth.
  • Example 1 describes the selective activation of aminohexyl-tailed oligonucleotides with cyanuric chloride.
  • Example 2 describes the comparison of nylon beads derived with various diamines and poly(ethyleneimine) in terms of the ability of the solid support to promote hybridization.
  • Example 3 describes the use of oligonucleotide derived nylon beads in a sandwi . ⁇ ⁇ ⁇ assay in which hybridization is detected using an insoluble colorimetric substrate.
  • Example 4 describes the use of oligonucleotide derived nylon beads in a sandwich assay in which target is detected in complex biological samples.
  • Example 5 describes the use of oligonucleotide derived nylon beads in a sandwich in which target is detected by chemiluminescence.
  • Example 6 describes the use of oligonucleotide derived nylon beads in a sandwich in which target is detected by a fluorescence-based signal system.
  • Example 7 describes the use of oligonucleotide derived nylon beads immobilized in a dipstick to form a multi-site detection panel or card.
  • SDS/FW is FW and 0.1% sodium dodecyl sulfate (SDS) .
  • HRP (horseradish peroxidase) substrate solution is 0.1 M sodium citrate pH 6.5, 0.2 M NaPhosphate, 0.5 mg/ml. 4-methoxy-l-naphthol, 0.02 mg/ml 3-methyl-2-benzo- thiazolinone hydrazone and 0.0135% hydrogen peroxide.
  • AP (alkaline phosphatase) substrate solution is l mM 5-bromo-4-chloroindoyl-3-phosphate, 1 mM nitroBlue tetrazolium, and 0.01% Tween 20 in TMNZ.
  • Lysis and hybridization solution is 3 M guanidinium thiocyanate, 2% N-lauroylsarcosine (sarcosyl) , 50 mM Tris pH 7.6, 25 mM EDTA.
  • the fluorescent substrate for alkaline phosphatase is
  • the chemiluminescent substrate for alkaline phosphatase was a pre-prepared cocktail from Lumigen, Inc. (Detroit, MI) .
  • Poly(ethyleneimine) was purchased from Polysciences (Warrington, PA) .
  • Burnished or unpolished nylon beads were purchased from Precision Ball Company (Chicago, IL) and The Hoover Group (Sault St. Marie, MI.)
  • Triethyloxonium tetrafluoroborate hexanediamine, phenylenediamine, succinic anhydride and N-methyl-pyrrolidinone (N-methyl-pyrrolidone, m-pyrol) were purchased from Aldrich Chemical (Milwaukee, IL) .
  • N-succinimidyl 4-(iodoacetamido)-benzoate (SIAB) and Tween 20 was purchased from Pierce (Rockford, IL) .
  • Guanidium isothioscyanate (GuSCN) was purchased form Kodak (Rochester, NY) .
  • the di- and triamines EDR-148, ED-400, ED-6000, and T-3000 were a gift from Texaco Chemical Company, (Houston, TX) .
  • Oligonucleotides complementary to regions conserved or hypervariable regions of the 16S-ribosomal RNA of either Actinobacillus actinomycete comitans (Aa) , Bacteroides gingivalis (Bg) , Bacteroides intermedius (Bi) , Eikenella corrodens (Ek) , Fusobacterium nucleatum (Fn) , or Wolinella recta (Wr) were synthesized using phosphoramidite chemistry on either an ABI 380B or a Milligen 7500 automated DNA synthesizer.
  • the oligonucleotides were prepared using the standard phosphoramidite chemistry supplied by the vendor or the H-phosphonate chemistry.
  • dA . dG, dC, and T phosphoramidites are commercially available in these forms, and synthetic nucleosides may readily be converted to the appropriate form.
  • Oligonucleotides were purified by adaptations of standard methods. Oligonucleotides with 5'-trityl groups were chromatographed on HPLC using a 12 ⁇ m, 300 A Rainin (Woburn, MA) Dyna ax C-8 4.2x250 mm reverse phase column using a gradient of 15% to 55% MeCN in 0.1 N Et 3 NH + 0Ac " , pH 7.0, over 20 min.
  • 25,000 3/32 inch diameter unpolished nylon beads were placed in a flask containing 1800 ml of 100% anhydrous n-methyl-pyrrolidinone and mixed for 5 minutes at ambient temperature. 200 ml of 1 molar triethyloxonium tetrafluoroborate in dichloromethane was added and the mixture was stirred for 30 minutes at ambient temperature. The beads were then decanted and washed quickly with 4, 500 ml changes of 100% n-methyl-pyrrolidinone.
  • the beads were then transferred to a solution consisting of 3% w/v 10,000 MW poly(ethyleneimine) , prepared from a 30% aqueous solution of poly(ethyleneimine) , in n-methyl-pyrrolidone and stirred for 12 to 24 hours at ambient temperature.
  • the beads were washed with 2000 ml n-methyl-pyrrolidone, 1000 ml SDS/FW and finally 10 x 2 liter stilled water.
  • the beads were then dried under a high vacuum for 4 to 5 hours without the use of heat.
  • the amine content of the beads was determined by reaction with picyrlsulfonic acid.
  • SIAB N-succinimidyl 4-(iodoacetamido)-benzoate
  • cyanuric chloride derived oligonucleotides poly(ethyleneimine) coated nylon beads described above were placed in a volume of 0.1 M sodium borate pH 8.3 equal to the volume of the beads at 4°C.
  • the purified cyanuric chloride derived oligonucleotide was then added to the beads, and the mixture was vigorously agitated at ambient temperature (19 to 23°C) for 60 minutes.
  • the beads were then washed twice with 0.1 M sodium borate pH 8.3.
  • Succinic anhydride was then added at a concentration of 10 mg/ml in 90% N-methyl-pyrrolidone, 10% 1 M sodium borate pH 8.3 with a volume three times that the volume of the beads. The reaction was allowed to proceed for 1 hour at ambient temperature.
  • the beads were then washed 3 times with 250 ml of 100% N-methyl-pyrrolidone, twice with distilled water, 5 times with 250 ml SDS/FW and then 4 times with 1 liter of distilled water. Beads were stored dry or in 25 mM EDTA. Radioactivity per bead was determined by liquid scintillation counting.
  • poly(ethyleneimine) coated nylon beads described above were placed in a volume of 0.1 M sodium borate pH 8.3 equal to the volume of the beads, and iminothiolane was added to a final volume of 5 mg/ml.
  • the beads were allowed to react for 1 hour at ambient temperature and then washed 10 times with 0.1 M sodium borate pH 8.3 and 10 mM EDTA at 4°C.
  • the thiolated poly(ethyleneimine) coated nylon beads described above were placed in a volume of 0.1 M sodium borate pH 8.3 and 25 mM EDTA equal to the volume of the beads at 4°C.
  • the purified iodoacetamidobenzoylated oligonucleotides were then added to the beads, and the mixture was vigorously agitated at ambient temperature (19 to 23°C) for 4 hours.
  • the beads were then washed twice with 0.1 M sodium borate and 25 mM EDTA and then incubated with a volume three times that of the beads with 10 mg/ml iodoacetamide in a 1:1 v/v ratio N-methyl-pyrrolidone and 0.1 M sodium borate pH 8.3. The reaction was allowed to proceed at ambient temperature for 1 hour.
  • the membrane was washed with 5 changes of the sodium borate buffer described above.
  • the introduced thiol groups were determined using 5,5-dithio-bis(2-nitrobenzoic acid).
  • the derived membrane was then cut into 0.28 cm 2 discs and washed once with 0.1 M sodium borate buffer.
  • IAB-oligonucleotide was prepared as described above and mixed with the membrane discs.
  • the solutions were incubated with either the derived nylon bead or the NytranTM (Scheicher & Schuell, Keene, NH) that had been covalently immobilized with 0.1 ⁇ g of respective oligonucleotide probe (capture probe) for 1 hour at ambient temperature with mild agitation.
  • the solid supports were then washed once with the lysis and hybridization solution, once with FW, and once with SDS/FW.
  • Streptavidin/HRP conjugate was added to a final concentration of 1 microgram/ml (based on streptavidin) in SDS/FW and incubated 10 to 15 minutes at ambient temperature with mild agitation.
  • the beads and filters were then washed three times with SDS/FW and then once with CAP buffer.
  • the quantity of target captured was determined by fluorescence quenching.
  • the beads were dried for 15 to 30 minutes at ambient temperature and then individually placed in a round bottom opaque white microtiter plate (Dynatek Laboratories, Chantilly, VA) .
  • the beads were then read using a fluorometer (Fluoroskan II, Flow Laboratories, McLean, VA) in which excitation was at 350 nanometers and emmision was at 456 namometers.
  • Example 1 describes the selective modification and activation of the tethered 5'-amine of oligonucleotides with cyanuric chloride. It is shown that the derivitization of the oligonucleotide occurs only at the tethered amine.
  • the sequence UP9A which either possessed a 5'-aminohexyl tail or did not possess a 5'-aminohexyl tail, was compared with respect to reactivity with cyanuric chloride.
  • the reaction was allowed to occur at 19°C for 30 minutes.
  • the reaction mixture was then analyzed by C-18 reverse phase HPLC utilizing a 5 to 45% acetonitrile gradient in TEA.
  • the chromatographs from the respective reaction mixtures and both types of starting oligonucleotide are shown in Fig. 3.
  • the non-tailed oligonucleotide sequence UP9A is shown in panel A and elutes off the column at 9.025 minutes whereas the amine-tailed oligonucleotide sequence UP9A elutes at 9.205 minutes (panel B) .
  • the chromatograph in panel C shows that the non-tailed oligonucleotide sequence UP9A does not react with cyanuric chloride, as the oligonucleotide continues to be eluted at 9.005 minutes.
  • the amine-tailed oligonucleotide sequence UP9A reacts almost to completion with cyanuric chloride resulting in a dichlorotriazine derivitive that is eluted at 11.6 minutes, almost 2.5 minutes later than the UP9A amine-tailed starting material.
  • the profiles indicate that only the UP9A sequence possessing the 5'-tethered amine reacted with cyanuric chloride demonstrating that cyanuric chloride reacted selectively with the amine and not with any of the sugars or bases present in the oligonucleotide.
  • Example 2 This example describes the derivation of nylon beads with several types of diamines and poly(ethyleneimine) , and then the subsequent attachment of 4,6-dichlorotriazine oligonucleotides. A comparison of the hybridization properties of the respective beads is also described.
  • the unreacted amines were then blocked with succinnic anhydride as described above and tested in a hybridization assay as described above.
  • the phenylenediamine beads showed no ability to capture target nucleic acid whereas the Jeffamines EDR-148, T-3000, and hexanediamine type beads captured nucleic acid target but at a rate about 25-fold less than that of the poly(ethyleneimine) beads as determined by the fluorescence quenching assay described above.
  • the results are summarized in the table below.
  • Example 3 compares the solid supports formed from NytranTM (Scheicher & Schuell, Keene, NH) membranes and nylon beads in a sandwich assay format in which a target nucleic acid sequence is sequestered and then detected using a colorimetric assay format.
  • 5-fold serial dilutions of the lysates were made using diluents containing the biotinylated signal oligonucleotides and 1 x 10 8 total cells of Aa, Bi, Ek, Fn, and Wr.
  • the solutions were then incubated for 30 minutes at ambient temperature with NytranTM (Scheicher & Schuell, Keene, NH) discs or 2 nylon beads that had covalently immobilized 0.1 ⁇ g of Bgl specific oligonucleotide probe (capture probe) .
  • the solid supports were washed with SDS/FW at ambient temperature and then incubated with 10 ng/ml of streptavidin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for 5 minutes at ambient temperature.
  • SA/HRP streptavidin/horseradish peroxidase
  • the solid supports were then washed with SDS/FW, FW, and then the presence of peroxidase was determined by incubating the filter with the HRP substrate solution described above to form an insoluble product.
  • Example 4 compares the solid supports formed from
  • NytranTM (Scheicher & Schuell, Keene, NH)membranes and nylon beads in a sandwich assay format in which a target nucleic acid sequence is sequestered and then detected in a complex biological sample containing whole blood.
  • 3 M GnSCN lysis solution was used to lyse 1 x 10 8 cells of Bacteroides gingivalis (Bg) spiked into a plaque sample containing visible quantities of blood (approximately 25 microliters packed cell volume) in 250 microliter volume at 19°C and then split into two equal volumes. The lysate was then heated to 65°C for 5 minutes.
  • a biotinylated 24-mer oligonucleotide probe complementary to conserved regions of bacterial 16s rRNA (signal probe) was added to a final concentration of 100 nanograms per ml.
  • the solid supports were then washed with SDS/FW at ambient temperature and incubated with 10 ng/ml of streptavidin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for 5 minutes at ambient temperature.
  • SA/HRP streptavidin/horseradish peroxidase
  • the solid supports were then washed with SDS/FW, FW, and then the presence of peroxidase was determined by incubating the filter with the HRP substrate solution described above to form an insoluble product.
  • Example 5 compares the solid supports formed from NytranTM (Scheicher & Schuell, Keene, NH) membranes and nylon beads in a sandwich assay format in which a target nucleic acid sequence is sequestered and then detected using a chemiluminescence assay format.
  • the solid supports were washed with SDS/FW at ambient temperature following by washing with 0.5% Tween 20, 1 mM MgC12, 0.01 M Tris-HCl pH 8.0 (APB) and then incubated with 0.4 ug/ l of streptavidin/alkaline phosphatase (SA/AP) conjugate in APB for 5 minutes at ambient temperature.
  • the solid supports were washed 5 times with APB, TMNZ, and then the presence of alkaline phosphatase was determined by incubating either the NytranTM (Scheicher & Schuell, Keene, NH) filters or the nylon beads with 200 microliters of Lumigen (from Lu igen, Inc. , Detroit, MI) in 5mm x 40 mm polypropylene tubes. The results are shown in the table below. Chemiluminescent Signal
  • the results indicate that, in the 30 minute hybridization, 3 x 10 4 cells were detected using the nylon beads as solid supports whereas only 1 x 10 8 cells were detected using the NytranTM (Scheicher & Schuell, Keene, NH) solid supports.
  • This approximately 10,000-fold difference in the lower level of detection of the target was due to the severe background of nonspecific binding of the alkaline phosphatase to the NytranTM (Scheicher & Schuell, Keene, NH) filters.
  • the nylon beads therefore, allowed the sensitive detection of Bg 16s rRNA using a chemiluminescence based signal system.
  • Example 6 demonstrates the nylon solid supports in a sandwich assay format in which a target nucleic acid sequence is sequestered and then detected using a fluorescence-based assay format.
  • 5-fold serial dilutions of the lysates were made using diluents in 3 M GuSCN lysing and hybridization solution containing the biotinylated signal oligonucleotides and 1 x 10 8 total cells of Aa, Bi, Ek, Fn, and Wr.
  • the solutions were then incubated for 30 minutes at ambient temperature with 2 black nylon beads prepared by The Hoover Group (Sault St. Marie, MI) that had covalently immobilized 0.1 ⁇ g of Bgl specific oligonucleotide probe (capture probe) .
  • the solid supports were washed with SDS/FW at ambient temperature following by washing with 0.5% Tween 20, 1 mM MgC12, 0.01 M Tris-HCl pH 8.0 (APB) and then incubated with 0.4 ug/ml of streptavidin/alkaline phosphatase (SA/AP) conjugate in APB for 5 minutes at ambient temperature.
  • the solid supports were then washed 5 times with APB, TMNZ, and then the presence of alkaline phosphatase was determined by incubating the nylon beads with 150 microliters of 0.5 mM 4-methyl-umbelliferyl phosphate (4-hydroxymethyl coumarin) in black microtiter well strips (Dynatek,
  • Example 7 describes the use of 3/32 inch nylon beads immobilized in a nonporous, plastic card to form a multi-panel detection composition described herein as a dipstick.
  • the dipstick (or indicator card) so formed possesses multiple and distinct sites that allow the specific detection of multiple pathogens in a single sample.
  • the specific detection of Bg and Ek is demonstrated.
  • a set of six identical dipsticks were prepared, in which the general configuration of the -lipstick is shown in Figs. 1 and 2.
  • the five beads, each possessing a different capture oligonucleotide, were placed in the dipstick from left to right in the following order: l: PA005 (positive control)
  • dipsticks were processed sequentially through the following solutions with constant agitation at 2 hertz: 10 minutes in the hybridization solution, 2 minutes in SDS/FW, 5 minutes in SA/HRP conjugate, 2 minutes in SDS/FW, 2 minutes in CAP buffer, and then developed 10 minutes in 4MN substrate solution.
  • the results are described in the following table.

Abstract

L'invention concerne des compositions et des procédés d'immobilisation covalente d'un oligonucléotide sur un support solide revêtu d'un polymère ou sur une structure similaire. En particulier, le support revêtu de polymères tel qu'une perle, possède un grand nombre de fractions activables, de préférence des amines primaires et secondaires. Un oligonucléotide est activé avec un réactif monofonctionnel ou multifonctionnel, de préférence le réactif homotrifonctionnel, le chlorure cyanurique. Les oligonucléotides immobilisés de manière covalente obtenus sur le support servent de sondes d'acide nucléique, et des essais d'hybridation peuvent être menés à bien, dans lesquels des acides nucléiques cibles spécifiques sont détectés dans des échantillons biologiques complexes. Les perles ou structures similaires peuvent être utilisées libres en solution, par exemple dans un format de puits de microtitrage, dans un format à écoulement traversant, tel que dans une colonne; ou dans une bande d'épreuve par immersion. De plus, des oligonucléotides de dichlorotriazine et des procédés d'activation d'oligonucléotides par traitement avec du chlorure cyanurique et des dérivés font partie de l'invention.
PCT/US1993/006044 1992-06-25 1993-06-24 Supports solides pour dosages d'hybridation de l'acide nucleique WO1994000600A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45441/93A AU4544193A (en) 1992-06-25 1993-06-24 Solid supports for nucleic acid hybridization assays

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90793192A 1992-06-25 1992-06-25
US07/907,931 1992-06-25

Publications (1)

Publication Number Publication Date
WO1994000600A1 true WO1994000600A1 (fr) 1994-01-06

Family

ID=25424872

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/006044 WO1994000600A1 (fr) 1992-06-25 1993-06-24 Supports solides pour dosages d'hybridation de l'acide nucleique

Country Status (2)

Country Link
AU (1) AU4544193A (fr)
WO (1) WO1994000600A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011912A2 (fr) * 1993-10-29 1995-05-04 Beckman Instruments, Inc. Synthese de biopolymeres utilisant des polymeres organiques actives en surface
EP0696046A2 (fr) 1994-08-04 1996-02-07 Patent-Treuhand-Gesellschaft für elektrische Glühlampen mbH Lampe à décharge à haute pression à culottage d'un seul cÔté
WO1997014815A1 (fr) * 1995-10-18 1997-04-24 Darwin Molecular Corp. Procedes pour preparer des supports solides destines a l'hybridation et pour reduire un fond non specifique
WO1997035031A1 (fr) * 1996-03-19 1997-09-25 Bio Merieux Detection d'une sequence nucleotidique avec amplification de signal
WO1999005308A2 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Appareil et procedes pour realiser une dispersion matricielle d'une solution sur un support solide
WO1999004896A1 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Alignements de biomolecules reposant sur une base de polyethylenimine
WO2000056790A1 (fr) * 1999-03-22 2000-09-28 Amersham Pharmacia Biotech Ab Groupe amino contenant des matrices support, leur utilisation et leur obtention
US6287762B1 (en) 1994-12-16 2001-09-11 Rhone-Poulenc Rorer S.A. Purification of a triple helix formation with an immobilized oligonucleotide
US6444422B2 (en) 1997-07-22 2002-09-03 Qiagen Genomics, Inc. Computer method and system for correlating data
US6558907B2 (en) 2001-05-16 2003-05-06 Corning Incorporated Methods and compositions for arraying nucleic acids onto a solid support
EP1537911A2 (fr) * 2003-11-07 2005-06-08 Hewlett-Packard Development Company, L.P. Medium pour séparations chromatographiques
US7038026B2 (en) 2000-05-26 2006-05-02 Centelion Purification of a triple heli formation with an immobilized oligonucleotide
AU2008201460B2 (en) * 1999-03-22 2010-02-04 Ge Healthcare Bio-Sciences Ab Amino group containing support matrices, their use and manufacture
US7790472B2 (en) 2001-09-24 2010-09-07 Austrian Research Centers Gmbh Fixed support for immobilizing biomolecules
US8431336B2 (en) 2000-07-07 2013-04-30 Diagnostics For The Real World, Ltd. Binding interactions in dipstick assays

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4721573A (en) * 1986-03-06 1988-01-26 J. T. Baker Chemical Company Use of sulfonic derivatives of acylated polyethyleneimine bonded phase silica products
WO1988001302A1 (fr) * 1986-08-11 1988-02-25 Siska Diagnostics, Inc. Procedes et compositions d'analyse a l'aide de sondes d'acide nucleique
US4806631A (en) * 1985-09-30 1989-02-21 Miles Inc. Immobilization of nucleic acids on solvolyzed nylon supports
US4828978A (en) * 1987-09-18 1989-05-09 Eastman Kodak Company Agglutination reagent and method of preparing same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4806631A (en) * 1985-09-30 1989-02-21 Miles Inc. Immobilization of nucleic acids on solvolyzed nylon supports
US4721573A (en) * 1986-03-06 1988-01-26 J. T. Baker Chemical Company Use of sulfonic derivatives of acylated polyethyleneimine bonded phase silica products
WO1988001302A1 (fr) * 1986-08-11 1988-02-25 Siska Diagnostics, Inc. Procedes et compositions d'analyse a l'aide de sondes d'acide nucleique
US4828978A (en) * 1987-09-18 1989-05-09 Eastman Kodak Company Agglutination reagent and method of preparing same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Volume 133, issued 1983, T.G. LAWSON et al., "Separation of Synthetic Oligonucleotides on Columns of Microparticulate Silica Coated with Crosslinked Polyethylene Imine", pages 85-91. *
ANALYTICAL BIOCHEMISTRY, Volume 156, issued 1986, H.-D. HUNGER et al., "CCA Paper: A New Two-Dimensional Cyanuric Chloride-Activated Matrix for Universal Application in Molecular Biology", pages 286-299. *
ANALYTICAL BIOCHEMISTRY, Volume 89, issued 1978, S. BIAGIONI et al., "A New Method for the Preparation of DNA-Cellulose", pages 616-619. *
ANALYTICAL CHEMISTRY, Volume 169, issued 1988, J.A. MATTHEWS et al., "Analytical Strategies for the Use of DNA Probes", pages 1-25. *

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011912A3 (fr) * 1993-10-29 1995-06-08 Beckman Instruments Inc Synthese de biopolymeres utilisant des polymeres organiques actives en surface
WO1995011912A2 (fr) * 1993-10-29 1995-05-04 Beckman Instruments, Inc. Synthese de biopolymeres utilisant des polymeres organiques actives en surface
EP0696046A2 (fr) 1994-08-04 1996-02-07 Patent-Treuhand-Gesellschaft für elektrische Glühlampen mbH Lampe à décharge à haute pression à culottage d'un seul cÔté
US8017744B2 (en) 1994-12-16 2011-09-13 Centelion Purification of a triple helix formation with an immobilized oligonucleotide
US6287762B1 (en) 1994-12-16 2001-09-11 Rhone-Poulenc Rorer S.A. Purification of a triple helix formation with an immobilized oligonucleotide
US6319672B1 (en) 1994-12-16 2001-11-20 Aventis Pharma S.A. Purification of a triple helix formation with an immobilized oligonucleotide
US8399636B2 (en) 1994-12-16 2013-03-19 Centelion Purification of a triple helix formation with an immobilized obligonucleotide
WO1997014815A1 (fr) * 1995-10-18 1997-04-24 Darwin Molecular Corp. Procedes pour preparer des supports solides destines a l'hybridation et pour reduire un fond non specifique
US5994065A (en) * 1995-10-18 1999-11-30 Rapigene, Inc. Methods for preparing solid supports for hybridization and reducing non-specific background
AU723889B2 (en) * 1995-10-18 2000-09-07 Qiagen Genomics, Inc. Methods for preparing solid supports for hybridization and reducing non-specific background
US7622566B2 (en) 1995-11-08 2009-11-24 Centelion Purification of a triple helix formation with an immobilized oligonucleotide
FR2746413A1 (fr) * 1996-03-19 1997-09-26 Bio Merieux Detection d'une sequence nucleotidique avec amplification de signal
US6033853A (en) * 1996-03-19 2000-03-07 Bio Merieux Nucleotide sequence detection with signal amplification
WO1997035031A1 (fr) * 1996-03-19 1997-09-25 Bio Merieux Detection d'une sequence nucleotidique avec amplification de signal
WO1999005308A3 (fr) * 1997-07-22 1999-05-27 Rapigene Inc Appareil et procedes pour realiser une dispersion matricielle d'une solution sur un support solide
AU735546B2 (en) * 1997-07-22 2001-07-12 Qiagen Genomics, Inc. Polyethylenimine-based biomolecule arrays
US6150103A (en) * 1997-07-22 2000-11-21 Qiagen Genomics, Inc. Polyethylenimine-based biomolecule arrays
WO1999005308A2 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Appareil et procedes pour realiser une dispersion matricielle d'une solution sur un support solide
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
US6444422B2 (en) 1997-07-22 2002-09-03 Qiagen Genomics, Inc. Computer method and system for correlating data
WO1999004896A1 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Alignements de biomolecules reposant sur une base de polyethylenimine
EP1433795A3 (fr) * 1999-03-22 2004-12-01 Amersham Biosciences AB Matrices de support contenant des groupes amino, leur utilisation et fabrication
EP1433795A2 (fr) * 1999-03-22 2004-06-30 Amersham Biosciences AB Matrices de support contenant des groupes amino, leur utilisation et fabrication
AU773644B2 (en) * 1999-03-22 2004-05-27 Ge Healthcare Bio-Sciences Ab Amino group containing support matrices, their use and manufacture
WO2000056790A1 (fr) * 1999-03-22 2000-09-28 Amersham Pharmacia Biotech Ab Groupe amino contenant des matrices support, leur utilisation et leur obtention
AU2008201460B2 (en) * 1999-03-22 2010-02-04 Ge Healthcare Bio-Sciences Ab Amino group containing support matrices, their use and manufacture
US6335438B1 (en) 1999-03-22 2002-01-01 Geir Fonnum Method for the manufacture of amino group containing support matrices, support matrices prepared by the method, and use of the support matrices
US7038026B2 (en) 2000-05-26 2006-05-02 Centelion Purification of a triple heli formation with an immobilized oligonucleotide
US8431336B2 (en) 2000-07-07 2013-04-30 Diagnostics For The Real World, Ltd. Binding interactions in dipstick assays
US6558907B2 (en) 2001-05-16 2003-05-06 Corning Incorporated Methods and compositions for arraying nucleic acids onto a solid support
US7790472B2 (en) 2001-09-24 2010-09-07 Austrian Research Centers Gmbh Fixed support for immobilizing biomolecules
EP1537911A2 (fr) * 2003-11-07 2005-06-08 Hewlett-Packard Development Company, L.P. Medium pour séparations chromatographiques
EP1537911A3 (fr) * 2003-11-07 2005-11-09 Hewlett-Packard Development Company, L.P. Médium pour séparations chromatographiques

Also Published As

Publication number Publication date
AU4544193A (en) 1994-01-24

Similar Documents

Publication Publication Date Title
US5667976A (en) Solid supports for nucleic acid hybridization assays
US5514785A (en) Solid supports for nucleic acid hybridization assays
Ness et al. A versatile solid support system for oligodeoxynucleotide probe-based hybridization assays
EP0770142B1 (fr) Procede pour augmenter la sensibilite de la detection d'hybrides entre un acide nucleique cible et une sonde
EP0128332B1 (fr) Méthode d'essai employant des séquences de polynucléotides
US4898951A (en) Compounds used as intermediates in the preparations of non-radioactive biological probes
EP0221308B1 (fr) Immobilisation d'acides nucléiques sur les support du nylon dérivé
EP0435470B1 (fr) Procédé de fixation d'acide nucléique sur une surface solide pour l'utilisation dans des tests d'hybridation d'acides nucléiques
EP0504321B1 (fr) Capture amelioree d'acide nucleique cible faisant appel a des oligonucleotides attaches de fa on covalente a des polymeres
WO1994000600A1 (fr) Supports solides pour dosages d'hybridation de l'acide nucleique
US5723344A (en) Device for the capture of target molecules, and capturing process using the device
EP0131830A1 (fr) Cordons d'acides nucléiques marqués et produits d'addition pour leur préparation
EP0703296B1 (fr) Détermination de polynucléotides par substitution d'un brin sur une sonde de capture
WO1997014815A1 (fr) Procedes pour preparer des supports solides destines a l'hybridation et pour reduire un fond non specifique
US5747256A (en) Homogeneous DNA probe titration assay
Yehle et al. A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA: RNA
WO1991018116A1 (fr) Procedes et compositions de reduction d'intensite de la fluorescence
EP0172153B1 (fr) Sonde-hybridation polynucléotidiques
EP0155854A2 (fr) Matériel de test biologique non-radioactif
US6306657B1 (en) Polynucleotide probe and kit for amplifying signal detection in hybridization assays
JP2651317B2 (ja) 核酸の検出法
CA1341365C (fr) Composition a base de sequences de polynucleotides visant a detecter un analyte
EP0401313B1 (fr) Conjugue macromoleculaire

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU MG MN MW NL NO NZ PL PT RO RU SD SE SK UA VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA