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WO1990001564A1 - Procedes d'analyse a cibles multiples par hybridation d'acides nucleiques - Google Patents

Procedes d'analyse a cibles multiples par hybridation d'acides nucleiques Download PDF

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Publication number
WO1990001564A1
WO1990001564A1 PCT/US1989/003378 US8903378W WO9001564A1 WO 1990001564 A1 WO1990001564 A1 WO 1990001564A1 US 8903378 W US8903378 W US 8903378W WO 9001564 A1 WO9001564 A1 WO 9001564A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
probes
dipstick
hybridization
target
Prior art date
Application number
PCT/US1989/003378
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English (en)
Inventor
Trevor H. Adams
Dennis E. Schwartz
Nicolaas M. J. Vermeulen
Charles R. Petrie
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Microprobe Corporation
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Filing date
Publication date
Application filed by Microprobe Corporation filed Critical Microprobe Corporation
Publication of WO1990001564A1 publication Critical patent/WO1990001564A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • This invention provides for technical improvements for conducting nucleic acid hybridization assays.
  • the improvements are designed to provide for a multiple target mode of conducting hybridization assays wherein a multiplicity of different nucleic acid probes for the site-specific capture of target nucleic acids are conjugated to specific locations upon a single dipstick or device.
  • the use of a dipstick has substantial mechanical advantages over prior art formats for nucleic acid hybridizations.
  • the dipsticks are composed of a handle, a nonporous support coated with a solid surface having at least one discrete region of nucleic acids covalently bound thereto.
  • the improvements also provide means for covalently attaching nucleic acids to solid supports, improved hybridization buffers, improved support surfaces, methods for reducing nonspecific background and methods for quantifying assay results.
  • the nucleic acid hybridizations described herein require the immobilization of a capture nucleic acid to a solid support surface.
  • Methods for the immobilization of nucleic acid to solid supports are known. Bischoff, R. et al., Introduction of 5'-Terminal Functional Groups into Synthetic Oligonucleotides for Selective Immobilization. Anal. Biochem. 164:336-344 (1987); Wolf, S.F. et al., Rapid Hybridization Kinetics of DNA Attached to Submicron Latex Particles, Nuc. Acids Res. 15:2911-2926 (1987); and J. N. Kremsky et al., Immobilization of DNA via Oligonucleotides Containing an Aldehyde or Carboxylic Acid Group at the 5' Terminus. Nuc. Acids Res. 15:2891-2910 (1987).
  • EP 200,381 A method for maintaining discrete regions of nucleic acid in a solid matrix for nucleic acid hybridization assays was disclosed in EP 200,381.
  • the invention of EP 200,381 involves the linking of nuclic acid to microspheres which are then entrapped in a porous matrix.
  • alkylammonium salts to influence the reannealing of nucleic acids. Orosz, J.M. and Wetmur, J.G., DNA Melting Temperatures and Renaturation Rates in Concentrated Alkylammonium Salt Solutions, Biopolymers 16:1183-1199 (1977); Chang, Chiang-Tung, et al., Effects of Microscopic and Macroscopic Viscosity on the Rate of Renaturation of DNA, Biopolymers, 13:1847-1858 (1974); EP 228,075 (page 3); and Wood et al., PNAS, 82:1585-1588 (1985).
  • Figure 1 is a multiple target assay system.
  • This invention provides for a dipstick for nucleic acid hybridization assays comprising: (a) a handle connected to a solid non-porous support coated with a solid surface having a porosity which does not effect the diffusion of the free nucleic acids and comprising at least one and preferably a multiplicity of discrete regions having nucleic acid probes covalently bound thereto. It is preferred that the thickness of the dipstick be between about .6 mm and about 10 mm, but more preferably between 1 and 2 mm.
  • the dipstick is generally longer than it is wide.
  • the portion of the dipstick immersed into test solutions is about 5 to about 10 times longer than it is wide.
  • the discrete regions of nucleic acid are generally uniformly spaced apart and the regions may be either a rectangular or circular pattern on the solid surface.
  • the nonporous solid support can be of the same material as the handle and can be any size or shape. It is preferably elongated and adapted for dipping into small volumes of liquid.
  • the solid support could be plastic, metal, or paper, and is preferably unchanged when exposed to hybridization media.
  • nonporous it is meant that nucleic acid in the sample solution does not become entrapped below the surface of the support.
  • a solid support having extremely small pores would also fall within this functional definition as it would be effectively nonporous.
  • the solid surface is preferably selected from the following group: polystyrene/latex; polystyrene; immobilized latex beads; carboxyl modified latex microspheres; carboxyl modified glass; and carboxyl modified teflon.
  • the surface may optionally be of the same material as the solid support.
  • the dipstick's solid surface preferably avoids a porosity which interferes with the diffusion rate of the free nucleic acids such that porosity is not a rate limiting factor in the nucleic acid hybridization assay.
  • the avoidance of a rate limiting porosity can be achieved by selecting large or small pores. By “small,” the porosity can be nonexistent (nonporous) or of such diameter that the surface is effectively nonporous.
  • the preferred size is an average pore diameter in excess of about 100 microns.
  • the surface charge of the dipstick is preferably neutral or positive when exposed to a neutral pH (about 6.0 to about 8.0).
  • the handle is an extension of the solid support to provide a means for transferring the dipstick from one solution to another solution without touching the surface having nucleic acid bound thereto.
  • the handle can be in the shape of a "t" or circular to maximize its surface area for grasping.
  • the dipstick of this invention preferably has nucleic acid covalently bound to it through spacer arms.
  • the preferred spacer arms are derived from thiol reactive substituents linked to a tethered nucleophilic amine on the 5' ends of the nucleic acid probes and are of the formula:
  • X is -NH- or -NHC:O(CH 2 ) m NH-
  • Y is a thiol reactive moiety
  • m is 2-12 inclusive
  • n is 2-12 inclusive. It is particularly preferred that n is six and X is -NH-.
  • a preferred thiol reactive moiety has a reactive group of either an ⁇ halo-acyl or an a, ß-unsaturated carbonyl.
  • the most preferred thiol-reactive moieties are selected from the group comprising haloacetamidobenzoyl and 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl.
  • This invention also provides for a dipstick as described above wherein the solid surface is derivatized with sulfhydryl containing moieties.
  • the chemical structure of the sulfhydryl containing moieties is non-critical so long as the sulfhydryl group or groups are available to react with thiol reactant moieties. It is preferred that the sulfhydryl containing moieties are polymeric compounds having a multiplicity of sulfhydryl groups.
  • the sulfhydryl groups are particularly useful when proteins are conjugated thereto.
  • the preferred proteins include thiolated bovine serum albumin; casein; and liquid gelatin.
  • This invention also provides for a dipstick as described above wherein the nucleic acid probes are complementary to regions of RNA found within ribosomes including both the 16S and 23S RNA.
  • the nucleic acid probes may be complementary to either hypervariable or conserved regions of the ribosomal RNA.
  • This invention also provides methods for assaying the presence of target nucleic acids, the method comprising contacting a hybridization medium containing target nucleic acids with a dipstick comprising a solid surface having a multiplicity of discrete regions with different nucleic acid probes covalently bound thereto.
  • a dipstick comprising a solid surface having a multiplicity of discrete regions with different nucleic acid probes covalently bound thereto.
  • different it is meant that the nucleic acid probes do not have identical nucleic acid sequences and will preferentially bind to different target nucleic acid.
  • This method is preferably conducted using the solid surfaces described above for the dipstick and is most preferably conducted using carboxyl modified latex microspheres.
  • the surface for conducting this method preferably does not inhibit the diffusion of nucleic acid. Most preferably, the surface has a porosity of the size ranges described above for the dipstick.
  • the method disclosed herein is preferably conducted using nucleic acid probes that are covalently bound to the solid surface through spacer arms.
  • the spacer arms are as described above for the dipstick.
  • the disclosed method has proteins conjugated to the solid surface through sulfhydryl bonding.
  • the preferred proteins are as described for the dipstick.
  • This method is preferably useful when the nucleic acid probes are complementary to sequences of nucleic acid indicative of pathogenic state in mammals. More preferred are nucleic acid probes which are complementary to sequences of RNA found within ribosomes. The preferred ribosomal RNA and the preferred regions of the RNA are as previously described for the dipstick. There is a particular preference for using the hypervariable sequences of the 16S rRNA derived from bacteria found in the human mouth. There is also application for this method to detect virus using nucleic acid probes that are complementary to sequences of viral nucleic acid such as probes which are complementary to sequences of human papilloma virus DNA.
  • This method can be conducted in two modes.
  • the method, described above can involve the use of a hybridization medium having both the target nucleic acid and the detectable nucleic acid present.
  • the detectable nucleic acid is complementary to sequences of the target nucleic acid that are different from the sequences to which the immobilized probe nucleic acid are complementary.
  • the hybridization of the target nucleic acid and the detectable nucleic acid are achieved in separate steps.
  • detectable nucleic acid is mixed with undetectable nucleic acid that bind in competition with each other onto the target nucleic acid.
  • the preferred size for the immobilized or capture nucleic acid is about between 12 and 100 nucleotide bases.
  • the above method may take place in a hybridization buffer comprising an ammonium salt selected from the group consisting of trialkylammonium salt and tetraalkyl-ammonium salt wherein the alkyl groups are the same or different and are comprised of between 1 and 3 carbon atoms inclusive.
  • the anionic portions of the salts are selected from the group comprising acetate, iodide, perchlorate, thiocyanate, chloride and bromide.
  • the preferred trialkylammonium salt is triethylammonium chloride.
  • the method and the dipstick can be surface modified by covalently binding a surface-modifying moiety through a covalent bond selected from the group consisting of disulfide linkage and thiol ether.
  • the surface-modifying moiety can be hydrophilic, hydrophobic, ionic or metallic. By hydrophobic it is meant that the dielectric constant for the relevant moiety is below 30, preferably between 1 and 30, and by hydrophilic it is meant that the dielectric constant is above 30, preferably between 30 and 80.
  • Metallic surface-modifying moieties include copper, gold, iron, chrome, silver and aluminum.
  • the preferred surface-modifying moiety is of the formula:
  • the method described above is preferably conducted in a multiple target mode wherein the reactive surface comprises a solid surface comprising a multiplicity of discrete regions each having different pathogen specific nucleic acid probes covalently bound thereto.
  • the solid surface is preferably selected from the solid surfaces described above for the dipstick.
  • the means for attaching the nucleic acid probes are preferably as previously described for the dipstick.
  • the solid support include surface-modifying moieties bound to surface through a covalent bond selected from the group consisting of disulfide linkage and thiol ether. These surface-modifying moieties are as previously described.
  • a method for decreasing the sensitivity of a nucleic acid hybridization assay by a known amount comprising adding a predetermined ratio of unlabeled polynucleotide signal probes to labeled polynucleotide signal probes, permitting the signal probes to bind to their complementary sequences, and detecting hybridization of the labeled signal probe.
  • Sandwich assays are a preferred format for reduction of sensitivity.
  • Bacterial nucleic acid is the preferred target. Kits having containers of signal probes with varying ratios of label to unlabeled probes are also described.
  • nucleic acid polynucleotide and oligonucleotide are interchangeable except where a specific size range is indicated by language context.
  • This invention discloses a nucleic acid hybridization assay format which uses a dipstick.
  • the dipstick format has mechanical advantages over other formats presently being used. It permits ready agitation without additional mechanical devices.
  • the handle permits easy transfer from the various solutions used in hybridizations.
  • the dipstick offers a more mechanically stable support than previously available technology.
  • the dipstick permits mechanical devices to rapidly read the results, such as by insertion of the dipstick in a reflectometer or similar device.
  • the dipstick permits extremely small amounts of sample solution (less than 500 ⁇ l) to be tested.
  • This invention also relates to means for and methods of detecting multiple and differing nucleic acid targets simultaneously in a single hybridization assay.
  • the detection of pathogenic microorganisms in biological specimens using nucleic acid hybridization assays is becoming increasingly widespread. Such assays are relatively simple in theory requiring only the release of intact nucleic acid from the specimen which is then hybridized with detectable nucleic acid probes specific for the pathogenic organism under question. By detecting specific hybridization binding between the probes and the extracted nucleic acids, the presence of the microorganisms is established.
  • the biological samples of interest include virtually any type of specimen which may contain microorganisms of interest.
  • Exemplary biological samples include fecal samples, blood, sputum, saliva, urine, semen, plaque samples, tissue samples, and the like.
  • the methods of the present invention will not differ regardless of whether exogenous or endogenous nucleic acid is being detected and hence the simultaneous detection of viral, bacterial or host nucleic acid is made possible.
  • Pathogenic viruses and microorganisms which may be detected in fecal or gastric samples, or as contaminants of foods, beverages or water, or the like, and include viruses, such as adenovirus, rotavirus and the like; bacteria, such as Salmonella spp. (S. typhimurium, S. typhi, S. paratyphi A, etc.), Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, S sonnei, etc.), Campylobacter spp. (C. jejuni, C. coli, C.
  • viruses such as adenovirus, rotavirus and the like
  • bacteria such as Salmonella spp. (S. typhimurium, S. typhi, S. paratyphi A, etc.), Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, S sonnei, etc.), Campylobacter
  • Clostridium difficile Clostridium difficile, Escherichia coli, Yersinia enterolitica, and the like; fungi, such as Candida albicans, and the like; and protozoans, such as Giardia lamblia, Entamoeba histolytica, Microsporidium spp., and the like.
  • Pathogenic viruses and microorganisms infecting the lung, bronchial and upper respiratory areas which may be detected in saliva, sputum or respiratory lavage samples include viruses, such as adenovirus, respiratory syncytial virus, human papillovavirus, human immunodeficiency virus, human T-cell lymphotrophic viruses, cytomegalovirus, hepatitis A and B virus, epstein-barr virus, and the like; bacteria, such as Streptococcus pyogenes (Group A beta-hemolytic streptococci), Haemophilus influenzae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Neisseria meningitidis, Mycobacterium tuberculosis, Spirochaetales, such as Treponema spp., and the like; fungi, such as Candida albicans, Histoplasma capsulatum and the like;
  • Pathogenic viruses and microorganisms which may be detected in blood (cells, serum and /or plasma), include: viruses, such as Human Immunodeficiency Virus, Hepatitis A and B Virus, Human T-Cell Lymphotrophic Viruses, and the like; bacteria, such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumonie, and the like; and, protozoans, such as Toxoplasma gondii, and the like.
  • viruses such as Human Immunodeficiency Virus, Hepatitis A and B Virus, Human T-Cell Lymphotrophic Viruses, and the like
  • bacteria such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumonie, and the like
  • protozoans such as Toxoplasma gondii, and the like.
  • Pathogenic microorganisms which may be detected in plaque samples include Actinobacillus actinomycetemcomitans, Bacteroides intermedius, Eikenella corrodens, Wolinella recta, Fusobacterium nucleatum, Bacteroides gingivalis, Bacteroides forsythus, and the like.
  • nucleic acid hybridization technology to produce a nucleic acid probe assay (e.g., using a single dipstick) which can detect at least one and preferably multiple pathogens simultaneously is a substantial improvement over the presently available assays which are capable of detecting only a single pathogen at a time.
  • dipsticks described herein will typically involve sandwich assays. Such assays utilize a covalently immobilized "capture” nucleic acid and labelled nucleic acid in solution. The clinical sample will provide the "target” nucleic acid. The "capture” nucleic acid and labelled nucleic acid probe hybridize with the "target” nucleic acid to form a “sandwich” hybridization complex.
  • This invention relates to nucleic acid hybridization assays which allow for the detection of multiple target nucleic acids (i.e., pathogens) on a single dipstick.
  • Accepted means for conducting such assays in a single target mode are known and general overviews of the technology can be had from a review of Nucleic Acid Hybridization: A practical Approach, Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1985; Hybridization of Nucleic acids Immobilized on Solid Supports, Meinkoth, J. and Wahl, G, Analytical Biochemistry, Vol. 238, 267-284, 1984 and U.S. Patent No. 4,358,535 which are incorporated herein by reference.
  • Target polynucleotides can be obtained from a variety of biological sources, depending upon the particular target one desires to detect.
  • Sources include all manner of biological materials which may harbor polynucleotide targets.
  • Polynucleotide targets can be: endogenous nonpathogenic nucleic acid sequences; mutations of the normal wild-type population, regardless of whether they are phenotypically expressed; or nucleic acid sequences arising from the presence of pathogens, such as viruses, bacteria, mycoplasmas, protozoa, rickettsia or fungi.
  • pathogens such as viruses, bacteria, mycoplasmas, protozoa, rickettsia or fungi.
  • Clinical samples of microbes, cells, tissue and the like can be obtained with standard techniques, such as lavage, scraping or biopsy.
  • the samples are typically dispersed in a buffer, which provides a biologically compatible solution.
  • a typical dispersal buffer solution would be 150mM NaCl, 20mM Tris-HCl (pH 7.5), 10mM EDTA, 10mM EGTA, or 150mM NaCl, 20mM NaPO 4 (pH7.5), 10mM EDTA, 10mM EGTA.
  • Samples may require exposure to a lysing solution in order to free target nucleic acid. Lysing buffers are known in the art. EP 199,439; Potts, T.V. and Berry, Em. Internat. J. Sys. Bacter., 33:765-771 (1983); Bonta, Y., et al., J. Dent.
  • these buffers are between pH 7.0 and 8.0, containing both chelating agents and surfactants.
  • a lysing solution is a buffered solution of detergent and a divalent metal chelator or a buffered chaotrophic salt solution containing a detergent, a reducing agent, and a divalent metal chelator.
  • Heat denaturing is optional as is the use of N-acetyl-muramidase (lysozyme).
  • Probes are either DNA or RNA oligonucleotides or polynucleotides, containing naturally occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine. Probes typically have sufficient complementarity with their target polynucleotides such that stable and specific binding occurs between target and probe.
  • the degree of homology required for formation of a stable hybridization complex (duplex) varies with the stringency of the hybridization medium and/or wash medium.
  • Polynucleotide or oligonucleotide probes for use in this invention can be obtained from the entire sequence or portions thereof of an organism's genome, from messenger RNA, or from cDNA obtained by reverse transcription of messenger RNA. After isolation of genomic DNA or cDNA fragments, the fragments are typically inserted into a replication vector, such as lambda phage, pBR322, M13, or vectors containing the SP6 or T7 promoter and cloned as a library in a bacterial host. Following appropriate screening procedures, a recombinant vector with the desired probe insert is isolated and labelled as described below. The vector is then grown in a suitable host. The probe and its vector are purified from the host cells by cell lysis and nucleic acid extraction. Following isolation, the probe can be purified away from the vector by digestion with selected restriction enzymes and sequenced. Further isolation of the probe can be achieved by using gel electrophoresis or high pressure liquid chromatography.
  • a replication vector such as lambda phage
  • DNA probes are preferably chemically synthesized using commercially available methods and equipment.
  • the solid phase phosphoramidite method can be used to produce short probes of between 15 and 50 bases and have a molecular weight of less than 16,000 daltons. These are referred to herein as "short probes.” (Caruthers, et al., Cold Spring Harbour Symp. Quant. Biol., 47:411-418, 1982, and Adams, et al., J. Am. Chem. SOC, 105:661, 1983).
  • nucleotide sequence When synthesizing a probe for a specific target, the choice of nucleotide sequence will determine the specificity of the test. For example, by comparing DNA sequences from several virus isolates, one can select a sequence for virus detection that is either type specific or genus specific. Comparisons of DNA regions and sequences can be achieved using commercially available computer programs.
  • Probes may be labelled by any one of several methods typically used to detect the presence of hybrid polynucleotides. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or
  • 32 P labelled probes or the like The choice of radioactive isotope depends on research preferences due to ease of synthesis, varying stability, and half lives of the selected isotopes.
  • Other labels include ligands which bind to labelled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope.
  • the radioactive nucleotides can be incorporated into probes by several means such as by nick translation of double-stranded probes; by copying single-stranded M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP; by transcribing cDNA from RNA templates using reverse transcriptase in the presence of radioactive dNTP; by transcribing RNA from vectors containing SP6 promoters or T7 promoters using SP6 or T7 RNA polymerase in the presence of radioactive rNTP; by tailing the 3'ends of probes with radioactive nucleotides using terminal transferase; or by phosphorylation of the 5' ends of probes using [gamma 32P]-ATP and polynucleotide kinase
  • Non-radioactive probes are often labelled by indirect means.
  • a ligand molecule is covalently bound to the probe.
  • the ligand then binds to an anti-ligand molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labelled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
  • Probes can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore ("labeled signal probes").
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g.. luminol.
  • hybridization solutions comprising from about 20 to 60% volume, preferably 30%, of a polar organic solvent.
  • a common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HCl, PIPES or HEPES, about 0.05 to 0.5% detergent, such as sodium dodecylsulfate, and between 1-10 mM EDTA, 0.01 to 5% ficoll (about 300-500 kilodaltons), 0.1 to 5% polyvinylpyrrolidone (about 250-500 kdal), and 0.01 to 10% bovine serum albumin.
  • the amount of labelled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichiometric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.
  • degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex.
  • the degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20° to 80oC, usually 25o to 75oC. For probes of 15-50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65oC. With routine experimentation, one can define conditions which permit satisfactory hybridization at room temperature.
  • the stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of 20% to 50%.
  • the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar reagents (e.g., sodium chloride, buffers, organic solvents and detergent), as provided in the hybridization solution.
  • reagents e.g., sodium chloride, buffers, organic solvents and detergent
  • the time period for which the support is maintained in the wash solutions may vary from minutes to several hours or more.
  • the probe may be conjugated directly with the label.
  • the label is radioactive
  • the dipstick with associated hybridization complex substrate is exposed to X-ray film.
  • the label is fluorescent
  • the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector (Physical Biochemistry, Freifelder, D., W.H. Freeman & Co., 1982, pp. 537-542).
  • the label is an enzyme
  • the sample is detected by incubation with an appropriate substrate for the enzyme.
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemiluminescence.
  • the preferred label for dipstick assays generates a colored precipitate to indicate a positive reading.
  • alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly colored and insoluble formazans.
  • Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the multiple target assay system described uses the sandwich assay format which is well known in the art of immunoassays and DNA probe assays.
  • the first description of a sandwich format for immunoassays was described in 1968 by Miles et al., Nature 219:186-189, while the first description of this format for the detection of nucleic acids was described by Dunn et al., Cell, 15:511-526 (1978).
  • a sandwich assay using DNA probes for the detection of single pathogens in clinical samples was described by Ranki et al., Gene, 21:77-85, 1983; Curr. Top. Microbiol. Immunol., 104:307- 318 (1983), and patents were issued to the same group in 1982 (U.S.
  • Patent No. 4,486,539) and 1986 U.S. Patent No. 4,563,419). These patents, however, were limited to a one-step assay for the detection of a single pathogen within a sample. No description was given of the potential for the detection of multiple pathogens within a single test sample using DNA/RNA probes in a sandwich type of format.
  • the first DNA probe subsequently is immobilized to a solid support and, when conducting a clinical assay, is used to capture a target nucleic acid from a complex cell lysate via hybridization to form a specific duplex.
  • the second DNA probe also hybridized with the target nucleic acid to form a specific duplex.
  • This second DNA probe is labeled and can be detected directly through a probe-linked label such as an enzyme, or indirectly through a biotin:labeled-streptavidin system.
  • the amount of the target nucleic acid (i.e., the pathogen) present in the clinical sample is indicated by the amount of labeled, second DNA probe which is contained within the hybridization complex.
  • the sandwich format can be used to develop assays which detect multiple targets simultaneously.
  • a principle feature of this assay is that discrimination of multiple nucleic acid targets (DNA and/or RNA) is achieved by site specific sequestering on a solid surface of the target nucleic acid itself.
  • Probe 1 An example of a multiple target assay for each of the 16S ribosomal RNA (rRNA) of three microbes is shown in Figure 1.
  • Probe 1, probe 3, and probe 5 are designed to hybridize only with the 16S rRNA of microbe A, microbe B and microbe C, respectively.
  • these probes are immobilized at three specific areas on a dipstick.
  • the sample is mixed with an appropriate hybridization reagent and the dipstick is immersed in the resulting solution.
  • Hybridization immediately proceeds between the dipstick-immobilized probes 1, 3 and 5 and the 16S rRNAs released from microbes A, B and C, respectively.
  • each 16S rRNA is sequestered precisely at the area on the dipstick which contains it immobilized and complementary probe. Once immobilized, each target 16S rRNA can be detected specifically with signal-generating probes 2, 4 and 6 which also are complementary, respectively, to the 16S rRNAs of microbes A, B and C.
  • the sample is mixed directly in a solution with an unlabeled DNA probe immobilized on a solid surface (e.g., a dipstick), a labeled solution-free DNA probe, and protease(s), hydrolases, detergent(s) and /or chaotropic agent(s) to rapidly lyse the cells and release target DNA or RNA.
  • Hybridization starts immediately after cell disruption, and the target DNA or RNA is simultaneously sequestered at the solid surface (by hybridizing to the immobilized first DNA probe) and labeled with signal (by hybridizing to the labeled second DNA probe).
  • the unbound labeled probe is washed away, and the amount of target DNA or RNA is quantified by the amount of bound (hybridized) labeled DNA probe.
  • the lysis buffer may limit the selection of a detectable label.
  • the use of proteases or chaotropic agents in the lysing step may preclude the use of enzymes as labels unless appropriate inhibitors are added after lysis.
  • the second DNA probe can be labelled with biotin. After hybridization and removal of proteases and/or chaotropic agents by a washing step, the hybridized biotin-labeled DNA probe can be detected with avidin conjugated with a signal generating system as described below for the two-step assay.
  • the two-step assay also can be run in an alternative mode - - after cell lysis, the target nucleic acid can be hybridized first with the signal-generating probe, and second with the immobilized probe.
  • any target probe as well as any signal-generating probes can be, but does not have to be, highly specific.
  • the flexibility arises because specificity of the assay for a particular microbe can be achieved in different ways as shown in the Table below. TABLE 1
  • Probe Signal Probe a) One/Two step Specific Specific* b) One/Two step Specific Non-Specific* c) One/Two step Partially Specific Specific* d) One/Two step Non-Specific Specific - - - - - -
  • nucleic acids having significant variations in their respective G:C and A:U or A:T ratios The influence upon hybridization due to differing compositions is most readily viewed between nucleic acids having the same length.
  • the G:C hydrogen bonding is more stable under typical salt conditions than the A:U or A:T bonding. Therefore nucleic acids having significantly different compositions will have significantly different ability to stably hybridize under similar hybridization conditions.
  • the most preferred salt is tetraethylammonium chloride which works best at 30-40oC in concentrations of between 2 and 3 moles per liter most preferably about 2.4 moles per liter.
  • the preferred nucleic acid chain length is about 15-50 nucleotides most preferably about 24 nucleotides.
  • test system can be made less sensitive by mixing the labeled signal probe (biotinylated) with unlabeled signal probe (not biotinylated) of the same sequence. During hybridization the unlabeled probe will compete with the labeled probe for hybridization with the target nucleic acid. Once hybridized, this probe will not produce signal and will thus make the test assay less sensitive.
  • E. coli bacteria is a common contaminant of water and foodstuffs; however, the presence of this microbe is only a health hazard when present above acceptable levels. Using these methods, one can readily desensitize an assay to reflect positive when the E._ coli concentration exceeds acceptable levels.
  • E._ coli concentration exceeds acceptable levels.
  • several microbes are associated with bacterial vaginosis which is a vaginal infection curable by treatment with the proper antibiotics. In the disease condition these organisms are typiclally present at different abnormally high levels.
  • a diagnostic assay based upon visualization of color can be constructed using either the single or multiple target modes, preferably the multiple target mode.
  • cutoff limits such that a positive test (eg., a detectable or specific shade of color on the dipstick) for a disease state will be indicated only when the concentration of a microbe exceeds a certain value.
  • This type of label dilution can be used to desensitize virtually any assay and can be individually and independently applied to any of the nucleic acid targets undergoing detection in the multiple target test.
  • label dilution will work for any type of label.
  • biotinylated probes or enzyme conjugated probes can be diluted in this fashion.
  • a stoichiometric dilution may not give exactly the desired result.
  • the rate of hybridization of the bulky enzyme-conjugated probe will be less than that of the smaller, unlabeled probe.
  • the solid support surface for the preferred multiple target mode of this invention is modified with sulfhydryl groups for subsequent reaction with the thiol- reactive oligonucleotide described below in section II part B.
  • the preferred method is to modify the surface with disulfide derivatives containing nucleophilic amines which participate in conjugation, and then to reduce the disulfide groups to monothiols by treatment with, for example, dithiothreitol, in order for the thiol reactive portion of the activated oligonucleotide to covalently bind to the surface.
  • Membranes polystyrene beads, teflon, polystyrene/latex beads, latex beads or any solid support possessing an activated carboxylate, sufonate or phosphate are suitable for use as solid surface substratum.
  • Porous membranes possessing pre-activated surfaces may be obtained commercially (eg. Pall
  • the following chemical moieties can be attached to the solid support surface: any aminoalkyl or aryl disulfides (for coupling of the oligonucleotides), any mercaptans (for surface modification), any amino thiol alcohols (for coupling of oligonucleotides), amino aryl disulfides (for coupling of the oligonucleotides), any NH2-R12-S-S-R13-SH where R12 and R13 are the same or different.
  • R 12 and R 13 are noncritical moieties that are not to be viewed as a limitation of this invention. They are typically organic radicals that vary in size, charge and polarity in accordance with the user's needs and objectives.
  • R 12 and R 13 are preferably as defined in the Summary of the Invention above.
  • the diamino substituted disulfide can be used to react with either the preactivated membranes or the carboxylated solid surfaces.
  • a direct substitution reaction occur according to reaction conditions described by the manufacturer.
  • the carboxyl group is first activated and then derivatized by reaction with the disubstituted aminodisulfide.
  • a reactive sulfhydryl group is generated by reacting the membrane with a reducing agent such as beta-mercaptoethanol, dithioerythritol, or dithiothreitol at 0.0001 to 0.001 moles/cm 2 in buffered neutral or basic aqueous solution for 30 to 60 minutes at 20o to 30oC. Unreacted material is removed by repeated washings in the presence of a detergent.
  • a reducing agent such as beta-mercaptoethanol, dithioerythritol, or dithiothreitol
  • filtration manifolds include those commercially available from Bio-Rad Laboratories (Richmond, CA) which is referred to as the Bio-Dot SF apparatus or the Minifold 11 apparatus which is available from Schleicher & Schuell (Keene, NH).
  • Bio-Rad Laboratories Richmond, CA
  • Minifold 11 apparatus which is available from Schleicher & Schuell (Keene, NH).
  • dots" or "slots” are formed on the membrane surface which contain only one oligonucleotide.
  • the membranes are then cut mechanically such that any configuration of the dipstick may be obtained.
  • the membrane may then be immobilized onto a plastic, glass or ceramic support with the use of appropriate adhesives.
  • the activated beads are exposed to 0.01 to 1 molar solution of an aminoalkyldisulfide or an aminoalkylmercaptan preferably cystamine or cysteamine (2-aminoethanethiol) in a non-amine containing buffer such as sodium borate, sodium phosphate or sodium carbonate at about pH 7 to 9 and allowed to react for 1 to 24 hours.
  • an aminoalkyldisulfide or an aminoalkylmercaptan preferably cystamine or cysteamine (2-aminoethanethiol
  • a non-amine containing buffer such as sodium borate, sodium phosphate or sodium carbonate
  • the disulfide of the conjugated cystamine is reduced to a sulfhydryl using a reducing agent such as 0.01 to 0.1 M dithiothreitol or dithioerythritol If cysteamine was employed in the last step, the beads do not require a reduction step.
  • Support surfaces described above without activatable carboxylate groups can also be activated. These supports can be any shape or size.
  • the surfaces are first coated with a solution of latex polymer which contains carboxyl groups (Carboxylate-Modified Tube Coating (CML), Seradyne, Inc. Indianapolis, IN). Following drying, the carboxyl groups on such coated surfaces can be activated, and then conjugated with modified oligonucleotides as explained below in section IV part C.
  • CML coating is a preferred mode of producing a multiple target dipstick.
  • the CML Tube Coating is diluted with alcohol such as 50% ethanol to make the desired dilution to optimize the properties of the CML Tube Coating for application to a desired surface.
  • CML Tube Coating After application to the solid surface, excess CML Tube Coating is removed and the CML Tube Coating is allowed to dry without the use of heat.
  • the carboxyl groups on the CML Coating are activated using any appropriate chemical activator such as 1-ethyl-3-(3-diethylaminopropyl)carbodiimide (EDC), and then reacted with an aminoalkyldisulfide or an amino alkylmercaptan, preferably cystamine or cysteamine followed by reduction in the case of the disulfide conjugate as described above for the beads.
  • EDC 1-ethyl-3-(3-diethylaminopropyl)carbodiimide
  • the CML Coating is applied to distinct regions of the plastic surface and then specific activated oligonucleotides are applied to the appropriate regions of CML coating.
  • the preferred method is to prepare unique plastic sheets of one specific oligonucleotide immobilized on the CML Coating on the solid support. The sheets are then cut and processed. A multiple target dipstick is prepared by attaching onto the appropriate dipstick device the individual plastic oligo-containing pieces. ii. Plastic, Glass. Ceramic Frits or Magnetic Frits or Beads
  • the frits are coated with CML and are processed in bulk with one specific oligonucleotide as described above.
  • the individual frits possessing the desired specific oligonucleotides are assembled in to the appropriate dipstick device giving rise to a multiple target dipstick.
  • CML Coating is applied and processed as described above and to each well a different or the same type oligonucleotide sequence may be covalently immobilized. This permits the formation of a device in which 96, 48, 24, or 8 different specific oligonucleotides may be immobilized and used in hybridization assays.
  • the solid surfaces now contain a sulfhydryl group ready to be activated with a thiol reactive oligonucleotide as described below in Section IV, Part C.
  • the preferred capture nucleic acids for use in this invention are synthetic oligonucleotides of between 20 and 100 bases.
  • a linker arm containing a blocked amine group can be coupled using conventional chemistry to the 5'-hydroxyl group of an oligonucleotide.
  • the activated oligonucleotides used as starting materials for this invention can be derived through several methods.
  • the reagents for the attachment of primary linker arms terminating in a primary amine are commercially available.
  • a primary amine is the preferred group for attachment to the heterobifunctional reagent, and its attachment via a hexyl arm is preferred.
  • Starting materials suitable for use in this invention are described in PCT U.S. 86/01290; Nucl.
  • an oligonucleotide possessing a 5'-terminal structure such as
  • X is an NH or NHC:O(CH 2 ) n NH
  • Y is a thiol reactive moiety
  • A is a poly or oligonucleotide
  • n and m are between 2 and 12, inclusive.
  • the oligonucleotide can range between about 9 and 50 bases, preferably between about 15-30 bases, with only the 5'-hydroxyl requiring modification for attachment.
  • the preferred thiol reactive moieties are an alpha-halo-acyl or an alpha- or beta-unsaturated carbonyl or alpha- or beta-unsaturated lactones.
  • thiol reactive moieties are alpha-haloacetamidobenzoyl and 4-(N-maleimidomethyl) cyclohexane-1-carbonyl.
  • Preferred halogens are iodine and bromine.
  • the thiol-reactive oligonucleotides are referred to as activated oligonucleotides.
  • an oligonucleotide can be modified at its 3 'end with a linker 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 which contains a single 3'-terminal ribonucleotide.
  • This can be modified with a linker arm containing a nucleophilic amine by, for example, 1) oxidizing the ribonucleotide cis-glycol with periodate, 2) treating oligonucleotide so modified with, for example, butane diamine to form a Schiff base, and 3) treating with sodium borohydride or cyanoborohydride to form a stable reduced Schiff base derivative in which one of the amines is left free for subsequent conjugation.
  • DNA bases can also be modified to become thiol reactive before or after preparation in a DNA synthesizer.
  • natural nucleic acids DNA and RNA
  • molecularly cloned nucleic acids as well as synthetic nucleic acids, can be modified.
  • cytosine a more nucleophilic amine is linked to the 4-position of cytosine by a variety of chemistries. The amine can be added by treatment with hydrazine to generate N-4-aminocytosine (Sverdlov, E.D., et al., FEBS Letters, 62, p. 212. Feb. 1976). This reaction is catalyzed by bisulfite.
  • an amine can be added to the 4-position by bisulfite catalyzed transamination reactions where a diaminoalkane is added to the 4-position (Shapiro and Weisgras, Biochemical and Biophysical Research Communications, 40:839, 1970).
  • Bi-functional semicarbazide can also be used to add nucleophilic amines to the 4-position of cytosine (Hayatsu and Ukita, Biochemical and Biochemical Research Communications, 14:198, 1964).
  • the base with the free amino group in the oligo is then made thiol reactive through the reaction of the nucleophilic amine and a NHS ester of a carboxylic acid derivative substituted with a alpha- or beta-unsaturated carbonyl, or alpha-halocarbonyl groups or the like.
  • nucleic acid bases with nucleophilic amines on other positions can be used for reactions with NHS esters of a carboxylic acid derivative substituted with a alpha- or beta-unsaturated carbonyl, or alpha-halo ⁇ acyls.
  • NHS esters of a carboxylic acid derivative substituted with a alpha- or beta-unsaturated carbonyl, or alpha-halo ⁇ acyls For instance, 5-[N-(7-aminohexyl)-1-acrylamido]-2'-deoxyuridine 5 '-triphosphate (Calbiochem, La Jolla, CA) can be added to the 5' end of oligonucleotides in reactions with terminal deoxnucleotidyl transferase ("Molecular Cloning, A Laboratory Manual," T. Maniatis, et al. eds., Cold Spring Harbor Laboratory, p. 148, 1982).
  • oligonucleotide bearing the tethered amine is covalently bound to a reagent having two different types of functional groups.
  • These bifunctional reagents typically have an N-hydroxysuccinimidyl (NHS) ester, which is amine reactive as the first functional group, and a thiol reactive group, as the second functional group.
  • NHS ester acylates the free amine of the 5' end of the oligonucleotide.
  • the acylating conditions are well known and highly selective.
  • the exocyclic amines on adenine and cytosine bases are not nucleophilic and do not readily react with the NHS ester.
  • the thiol reactive functional group is available for conjugation with surfaces containing free sulfhydryl groups.
  • An oligonucleotide with a tethered nucleophilic amine linked to its 5' end can be derivatized with such thiol-reactive groups by relatively simple chemistry.
  • a reagent such as SIAB is added to the oligonucleotide in aqueous solutions at a 10 to 100 fold molar excess.
  • the pH is buffered between about 6 and 9 and the temperature is between about 20° and 30oC. After a reaction period of about 30 to 90 minutes the excess unreacted reagent is removed by exclusion gel filtration.
  • the preferred thiol reactive moiety is an alpha-halo-acyl compound or an alpha- or beta-unsaturated carbonyl compound.
  • the thiol reactive group may include: maleimides, pyridyl sulfides, and active halogens, alpha- or beta-unsaturated lactones and the like.
  • the active halogens are typically alpha-haloacyl.
  • Useful halogens include chlorine, bromine iodine, and fluorine with iodine and bromine being preferred.
  • Reagents useful for this invention can be purchased from Pierce Chemical Co., Rockford, IL. Examples include:
  • the thiolated surface of section IV, part A is reacted with the activated oligonucleotide, using a 5 to 100-fold excess of activated oligonucleotide over the maximum oligonucleotide binding capacity of the solid support.
  • This method gives 5 to 100 micrograms of oligonucleotide (for a 24-mer) per cm 2 for a Pall Immunodyne membrane or 0.05 to 1 microgram per cm 2 for undiluted Seragen CML Tube Coating.
  • the reaction occurs in aqueous buffer between ph 5.5 and 9.5 (with the pH range of 7-8 being preferred) and is allowed to proceed from 1 to 16 hours at about 19o to 24 oC. Volumes of the reaction are held to a minimum.
  • the conjugate is separated from free oligonucleotide by repeated washings in buffers containing ionic detergents such as sodium dodecyl sulfate, sodium laurylsulfate or hexadecyltrimethylammonium bromide (CTAB) or nonionic detergents such as Tween 20, with a final wash in water.
  • ionic detergents such as sodium dodecyl sulfate, sodium laurylsulfate or hexadecyltrimethylammonium bromide (CTAB) or nonionic detergents such as Tween 20, with a final wash in water.
  • CTAB hexadecyltrimethylammonium bromide
  • Tween 20 hex
  • Oligonucleotides modified with thiol-reactive groups can be conjugated to the derivatized solid support by a variety of means. These include slow filtration in the case of membranes, immersion in a solution of functionalized oligo when plastics frits are used or overlaying the activated oligo solution on the activated surface of a flat plastic, glass or ceramic sheet.
  • nucleic acid hybridization assays of all types. These assays have numerous applications in the medical and biological sciences as well as in numerous industrial settings. A general review of these procedures can be found in Nucleic Acid Hybridization, A Practical Approach, Eds. Hames, B.D. and Higgins, S.J., IRL Press, Washington D.C. (1985); Hybridization of Nucleic Acids Immobilized on Solid Supports, Meinkoth, J. and Wahl, G., Anal. Biochem., 238:267-284 (1984); and U.S. Patent No. 4,358,535.
  • the thiolated surface on it's own possesses very low background levels of non-specific binding of nucleic acid due to the negative charge of the surface at solution pH values of 7.2 or greater.
  • the amount of capture oligonucleotide conjugated to a surface can be increased in the following way.
  • a polymer, possessing appropriate side groups which may be activated chemically to form thiols, may be conjugated to an active surface through a terminal or internal primary amine. After immobilization, the polymer can be treated to produce reactive thiols and these can be reacted with thiol-reactive capture oligonucleotides. — Using this type of procedure higher levels of immobilized capture oligonucleotides can be obtained.
  • Poly-S-CBZ-L-cysteine is a preferred polymer. Reaction conditions are as described above for aminoalkyl mercaptans and aminoalkyldisulfides.
  • D. Postmodification of the Surface of the Solid Support After conjugation of the surface of the solid support with a capture DNA probe, there is a large excess of free sulfhydryl groups. The ratio of these remaining sulfhydryl groups to covalently coupled capture oligonucleotide can range from 10:1 to 1,000,000:1. The presence of these excess thiol groups permits a second modification of the surface of the solid support by the selective chemical coupling of R-SH groups, where R is any chemical functionality. This chemical coupling can be either irreversible (e.g., by reaction with a maliemide derivative) or reversible (e.g., by formation of a disulfide bond).
  • the ability to conduct a second modification of the surface of the solid support is important for achieving a variety of desired surface properties on the solid support.
  • the following are illustrative of some possible examples:
  • the thiolated surface provides the chemical means for irreversible modification in the second conjugation step by treatment modification of the residual thiols with the appropriate reagent to provide covalent bonds. Such conjugations can modify the charge, hydrophobicity, hydrophilicity, color, reflectance, transmittance, porosity, frictional coefficient, porosity, conductivity and heat capacity of the surface.
  • the thiolated surface also provides the means whereby proteins, nucleic acids, antibodies, antigens or other macromolecules of interest may be irreversibly coupled to the solid support. Such modified surfaces will control, in part, the rate of hybridization of target nucleic acid to the capture probe on the solid surface support and will be important in reducing the level of nonspecific background arising during the detection of the hybridized labelled probe.
  • the thiolated surface also provides the chemical means for reversible modification in the second conjugation step by the formation of disulfide bonds between the surface and a desired reagent (e.g., formation of surface-S-S-R bonds). Such conjugations can modify transmittance, frictional coefficient, porosity, conductivity and heat capacity of the surface.
  • the thiolated surface also provides the means whereby specific proteins, nucleic acids, antibodies, antigens or other macromolecules of interest may be reversibly coupled to the solid support. These types of moieties may serve as receptors, ligands, or substrates in the signal development processes.
  • the reversibly modified surface described directly above in Section 2 can be further modulated after sequestering of the target nucleic acid via the formation of the hybridization complex with the immobilized capture probe and the labeled probe.
  • the solid surface is "chemically cleansed” by selective reduction of only those groups coupled by disulfide bonds to the surface. This will have important consequences in the reduction of undesired background typically arising in DNA probe assays.
  • the dipstick can be used in a sandwich assay to capture target nucleic acid and hybridized labeled probe.
  • any labeled probe which has bound non-specifically to the protein on the dipstick can be removed by treating with a reducing agent such as dithiothreitol. During such treatment, the disulfide bond between the protein and the dipstick is cleaved and the protein is released into the solution with its non-specifically associated labeled DNA probe.
  • this type of “chemical cleansing” (disulfide reduction to remove specific conjugated groups), in addition to removing background signal, can also be used to alter the charge, hydrophilicity, hydrophobicity, color, reflectance, transmittance, friction coefficient, porosity, conductivity and heat capacity of the surface of the solid support.
  • the following serves to illustrate more specifically the reversible "chemical cleansing" described directly above.
  • the surface is placed in a neutral or basic buffer (if aqueous conditions are required), or organic solvent containing 0.0001-0.1 M thiolated bovine serum albumin (BSA) with 0.001-0.1 M iodosobenzoate, and mixed for 10 to 60 minutes at 15 to 50oC.
  • BSA bovine serum albumin
  • the iodosobenzoate promotes an oxidation reaction which produces a disulfide bond between the dipstick and the BSA.
  • Excess reagents are removed by alcoholic washes followed by aqueous washes containing ionic detergents. Surfaces modified in this way contain irreversibly conjugated capture probe and reversibly conjugated BSA-protein.
  • all disulfides on the surface may be cleaved by treatment with dithiothreitol, beta-mercaptoethanol or the like. This highly specific cleavage removes much of the labeled DNA probes which is non-specifically bound to the dipstick surface. Such treatment allows the development of DNA probe assays which have high signal to noise ratios, and hence are extremely sensitive.
  • This cleansing is particularly advantageous where the target nucleic acid to be detected is present within a complex mixture of biological materials.
  • complex mixtures which are expected in both clinical and industrial settings include sputum, saliva, blood, urine, fecal material, foodstuffs, water supplies, and the like.
  • the surface may be made hydrophilic by the introduction of a hydrophilic functionality such as thioglucose.
  • the surface may be made a different color by the introduction of a colored functionality or the reduction of compounds known as tetrazolium salts or other compounds which maintain a color upon reduction.
  • the reflectance of the surface may be modified by the introduction of, for example, metal containing complexes such as Fe, Cu, Ag or the like via sulfhydryl group reaction.
  • the porosity of the surface of the solid support may be modulated by the introduction of the large polymeric molecules such as polycysteine.
  • the solid supports used to construct the dipstick described herein can be polystyrene, polypropylene, polycarbonate sheet plastic or plastics, or glass and ceramics in any conformation or shape amenable to the desired dipstick format.
  • Examples include porous polypropylene frits or porous glass, and nonporous material such as flat polystyrene sheets, microtiter plates and wells, glass tubes and the like.
  • the rate of hybridization of nucleic acids in solution to a complementary nucleic acid immobilized on a solid support is normally a three step process depending on the nature of the solid support. If the solid support is a filter membrane such as those commercially available from Pall or Millipore, the rate limiting steps leading to hybridization consist of the following:
  • the rate of hybridization of nucleic acids in solution to a solid support is substantially limited by external and internal diffusional control. Therefore, to maximize the efficiency and rate of hybridization, several key parameters of the sandwich assay system have been defined.
  • concentrations of the nucleic acids should be kept as high as possible.
  • the volumes should be minimized to the fullest extent.
  • the distances should be minimized between the surface and the target nucleic acid in solution.
  • polymeric forms of capture nucleotides will provide good kinetics for hybridizing to target probes.
  • the capturing polymers are then sequestered on the dipstick.
  • the preferred polymer is polyethyleneamine having capturing oligonucleotides covalently bound thereto.
  • porosity of the solid support should be at or of a size that does not impose internal diffusional control on the rate of reaction.
  • Preferred surfaces should have an average porosity that are in excess of 100 microns. It should be noted that the upper and lower ranges of the preferred porosity range will vary in accordance with the length of the oligonucleotides and target nucleic acids in solution.
  • Capture probe was covalently conjugated to a CML surface (carboxy modified latex) coated within a polyethylene frit or to a Pall Immunodyne membrane. In both experiments, the probe was immobilized at the same concentration per unit of surface area, yet the probe on the CML surface hybridized 18-24 times faster than the probe conjugated to the Pall membrane surface.
  • Example 1 Derivatization (Thiolation) of the Pall Immunodyne Membrane and Other Solid Supports.
  • the beads were then washed extensively with PBS containing 0.1% Tween and then reduced with 0.1 M DTT for 30 minutes at 22oC and again extensively washed with PBS/Tween.
  • the DTNB assay (described above) indicated 5 X 10- 11 moles of cystamine were bound (and reduced) to the beads.
  • Cystamine binding to the Carbox-Modified Tube Coating (CML, Seragen Diagnostics, Inc. Indianapolis, IN) was similarly demonstrated after painting polyethylene frits (0.28 cm squared) with a 1 to 5 dilution of the stock CML paint, drying and processing the frits as described for the polystyrene/latex beads. Approximately 2.5 X 10 -10 moles of cystamine were bound per frit.
  • This example involves the direct conjugation of an oligonucleotide to a solid support involves conjugation via heterobifunctional reagents.
  • the first step is to modify a synthetic probe with a linker arm reagent attached to its terminal 5'-hydroxyl group.
  • An aminohexyl linker arm with a terminal amino group is attached to the 5'-hydroxyl of the synthetic oligonucleotide via a phosphodiester linkage on an automated DNA synthesizer during the last step in the synthesis.
  • the reagent used for the introduction is 6- (methoxytritylamino)hexyl 2-cyanoethyl N,N-disopropylphosoramidite, prepared from 6-aminohexanol in a manner similar to the synthesis of the 3- (methoxytritylamino)propyl m e t h y l N , N -diisopropylphosphoramidite, as described by Connolly, Nucleic Acids Research., 15:3131 (1987).
  • the linker arm was attached to the probe, and the deprotected probe purified in a method similar to the methods described in the Connolly reference.
  • the synthetic probe chosen for this study was a DNA sequence complementary to a hypervariable region of the 16S RNA of Bacteriodes gingivitis.
  • the probe was designated Bg5B6N and has the following sequence: 5'XCCGATGCTTATTCTTACGGTACAT 3' where X denotes the hexyl arm linker.
  • SIAB-oligonucleotide is prepared by adding 0.1 to 2.0 grams of Sulfo-SIAB to 50 micrograms of the oligonucleotide described above in 300 microliters of Na Borate buffered aqueous solution at pH 8.3 and reacting for 1 hour in the dark at ambient temperature. The excess reagent is removed by desalting (size exclusion) on a G-25 Sephadex column in PBS or PBS containing 3M NaCl.
  • Example 3 Conjugation of the Activated Oligonucleotide to the Solid Support.
  • the activated oligonucleotide can be bound to the solid support by two methods: (a) immersion if polystyrene/latex beads, CML coated surfaces or porous membranes are employed or (b) filtration if porous membrane is used.
  • oligonucleotide binding was monitored by 32 P radioactivity as the oligonucleotide was labelled at the 3' end with 32 P-cordycepin phosphate. 0.12 to 1.2 ⁇ g of Bg5B6n was applied in 1 to 10 microliter volumes by filtration to 2-aminoethanethiol derivatized Pall
  • Example 5 Modification of the Solid Support Via Oxidation of the Surface Sulfhydryl Groups.
  • the Pall membrane was derivatized with 2-aminoethanethiol, conjugated with SIAB-Bg5B6N (as described above) and then the remaining free sulfhydryl groups capped as follows:
  • the 0.28 cm squared membrane discs were placed in either 0.01 M triphenylmethyl mercaptan (TMM) or 0.01 M dimethylaminoethanethiol (DMAE) in 10% ethanol containing also 0.01 M iodosobenzoate.
  • TMM triphenylmethyl mercaptan
  • DMAE dimethylaminoethanethiol
  • the reaction temperature was immediately brought to 90 oC and then cooled to 22oC during a 15 minute incubation period.
  • the filters were washed with PBS and then an aqueous buffered solution containing SDS.
  • a DNTB assay indicated the degree of capping: TABLE 2
  • TMM hydrophobic triphenylmethyl mercaptan
  • DMEA dimethylaminoethanethiol
  • Example 6A The Multiple Target Dipstick for Detecting Peridontal Bacteria.
  • the steps for the binding of capture oligonucleotides to the multiple target dipstick are identical to the steps described above for Examples 2 and 3 above with the physical separation of the four different capture nucleotides being applied to four of the painted regions of the dipstick. Unreacted oligonucleotide is then removed by washing the dipstick in a wash solution of hybridization buffer containing 0.01 M Tris-HCl pH 8.0, 5 mM EDTA, 0.09 M NaCl and 0.5% SDS.
  • the dipsticks can be stored dry and should be washed with distilled water prior to drying. The dipstick is stable for long period when stored dry or when stored at 4oC in appropriate buffers.
  • Curette scrapings of bacterial plaque from human teeth are either cultured or placed directly into 1 ml of 20 mM Tris-HCL, 150 mM NaCl, 10 mM EDTA, 10 mM EGTA.
  • the sample can be frozen until use.
  • 100 ng of an unrelated carrier 24 mer oligonucleotide is added to the sample if the bacterial count is low.
  • 500 ul of sucrose lysis buffer is added comprising: 75% sterile sucrose, 10 mM EDTA, 10 mM EGTA, and 50 mM Tris-HCl at pH 8.0. The sample is vortexed briefly.
  • the bacterial nucleic acids (.01-10 ng/ml) are then placed into a hybridization solution containing 30% formamide, 0.09 M NaCl, 0.01 M Tris-HCl, 5 mM EDTA, 0.1% SDS, 1X Denhardt's solution at pH 8.0.
  • Oligonucleotides, complementary to the conserved regions of the bacterial rRNA: CACGA(G/A) CTGACGACA ( G/A ) CCATGC and TACGGNTACCTTGTTACGAC and conjugated to horse radish peroxidase are added to a concentration of .01-.2 ⁇ g/ml. Conjugation can be achieved using known techniques.
  • Example 6B Demonstration of a Multiple Target Dipstick Using Four Specific Oligonucleotides Covalently Immobilized on Pall Membrane for the Detection of Specific Bacteria in Patient Plaque Samples.
  • the dipsticks were then placed in 1.0 ml sonicated solutions containing 3 M GnSCN, 50 mM EDTA, 50 mM Trip pH 8.0, 2% Sarcosyl and 1 x 10 8 cultured bacterial cells described above (or a mixture in any possible permutation of the cell types described above), or, species typed plaque samples possessing from 100 to 1 x 10 8 cells of the type described above in addition to other oral flora.
  • the hybridization solution was then brought to 100 ng/ml of 5'-terminal linked biotinylated oligonucleotide (18 to 20 nucleotides in length possessing a universal sequence, four different oligonucleotides per hybridization).
  • the dipsticks were incubated with constant agitation at 19 oC for 4 hours.
  • the dipsticks were then washed 3 times with filter wash solution (0.09 M NaCl, 50 mM Tris pH 8.0, 50 mM EDTA) and then 3 times with filter wash solution containing 0.5% SDS.
  • the filters were then probed with 5 mg/ml of Streptavidin/horseradish peroxidase conjugate in filter wash containing 0.5% SDS. The incubation was for 1 hour at 19 oC.
  • the dipsticks were then washed at ambient temperature 3 times with SDS/filter wash and three times with filter was without SDS.
  • the filters were developed as described in Example 7 below.
  • One 24-nucleotide species specific sequence from the hypervariable region of each of the 16s rRNAs from Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Eikenella corrodens, and Bacteroides intermedius is synthesized possessing a 5'-terminal primary amine hexyl-linker as described in Example 6.
  • Each oligonucleotide is covalently immobilized on a Pall membrane as described in Examples 1-5 above using a Minifold 11 slot blot apparatus (Schleicher & Schuell, Keene, NH) .
  • each type of the 24-mer oligonucleotide is immobilized per slot and each slot is approximately 1 cm in length separated by a distance of 3 cm.
  • the Pall membrane is cut such that each strip of membrane possesses the four different oligonucleotide regions. Each strip is then used in a separate hybridization assay.
  • the dipsticks are then placed in 1.5 ml solutions containing 30% formamide, 0.09 M NaCl, 0.01 M Tris, pH8.0, 5 mM EDTA, 0.1% SDS, 1X Denhardt's solution and 0.2 ug of a 48-mer synthetic oligonucleotide complementary to the 24 nucleotide sequence immobilized on the membrane as well as a 24 nucleotide length oligonucleotide which has been covalently conjugated to horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the filter membranes were developed in 0.1 M Na Citrate pH 5.5, 0.5 mg/ml 4-methoxy-l-naphthol, 0.02 mg/ml 3-methyl-2-benzo-thiazolinone hydrazone and 0.0135% hydrogen peroxide.
  • a multiple target dipstick for different types of HPV's can be prepared as described in Example 6 from three different S'-aminohexyloligonucleotides listed below: TABLE 4
  • the dipsticks immobilized with different combinations of the three capture oligonucleotides are placed into a hybridization solution containing a mixture of HPV6, 16 and 18 plasmid DNA's (0.01-10 ng/ml), 30% formamide, 0,09M NaCl, 0.01M Tris-HCl, 5mM EDTA, 0.1% SDS, IX Denhardt's solution at pH 8.0 at 42oC for 1-24 hours and the unhybridized nucleic acids are removed by repeated washing with hybridization media minus formamide and SDS.
  • polypropylene frits possessing an average pore size of 125 microns were obtained from the Porex Corporation.
  • the frits were coated with CML and derivatized with capture oligonucleotide as described in Example 1.
  • the rate of hybridization of the immobilized capture probe on the frit to complementary nucleic acid in solution was then determined and compared to the rate obtained with the Pall membrane. As shown in Table 2, the rate of hybridization was increased approximately 25-fold when the polypropylene or polyethylene frit was used in place of the membrane as the solid support.
  • a solid support which possesses no porosity was also examined (a flat polyethylene sheet) as a solid support and was derivatized with a capture oligo as described above using CML coating.
  • the rate of hybridization was determined to occur approximately 50 times faster than when the membrane support was employed (Table 2) .
  • Capture probe immobilized to polyethylene beads which were used in solution hybridization were taken as the control for rate comparison.
  • the beads in solution were determined to possess a reaction T1/2 of about 2 minutes as described in Table 2.
  • Poly-S-CBZ-L-cysteine was obtained from Sigma Chemical Company (St. Louis, MO) and dissolved at a concentration of 20 mg/ml in dimethyl formimide and 50 microliter volumes were applied to 0.28 cm squared discs of Pall membrane and incubated at ambient temperature for 15 minutes. The membrane discs were then incubated in 5 ml of concentrated ammonium hydroxide for 30 minutes at 20 degrees C. to de-block the CBZ groups and generate the free sulfhydryl groups.
  • a DTNB assay indicated the presence of the sulfhydryl groups on the polymer covalently attached to the surface of the membrane.
  • Activated oligonucleotide (SIAB-oligo) was then bound to the derivatized Pall membrane by immersion as described in Example 1. Radioactivity monitoring of the membrane after the conjugation indicated that the filter possessed approximately a 10-fold increase in capture oligo concentration over the membrane that was derivatized with cysteine. Thus, the use of a homomeric polymer of cysteine allowed a significant increase in capture oligo concentration on the solid support.
  • Example 11 Demonstration of Reduction of Nonspecific Background by Chemical Reduction of the Solid Support Surface.
  • Pall membrane discs were prepared as described in Example 5 and were either left uncapped or capped to the 100% level with either 2-aminoethanethiol or thiocholestrol.
  • the three types of filters were then incubated in duplicate with 50 ng/ml of a 4 to 5 million molecular weight Streptavidin/horseradish peroxidase conjugate in filter wash (0.09 M NaCl, 50 mM Tris pH 8.0, 50 mM EDTA) for 1 hour at 10oC.
  • SDS or detergent or a blocking agent such as BSA this leads to a very high level of nonspecific binding of the conjugate to the surface of the solid support.
  • the filters were then washed 3 times with filter wash containing 0.5% SDS.
  • Example 12 The Equalization of Melting Characteristics of Probes with Different GC Content in TEABR.
  • the filters were probed with either P-32 labelled Bg5B or Bg8B in 2.8 to 3.4 M TEABR solutions (in 0.1 M increments) containing 50 mM Tris pH 8.0, 2 mM EDTA, 0.1% SDS for 30 minutes at 29oC.
  • the filters were then washed once with their respective TEABR solution at 29oC and then with three non-stringent washes containing the buffer described above with 0.9 M NaCl replacing the TEABR.
  • the filters were then monitored for radioactivity. Similar extents of hybridization were observed only for the TEABR concentration of 3.1 M indicating the melting temperatures were equalized leading to comparable rates and extents of hybridization of Bg5B and Bg8B.
  • HPV18A3B 3' CCT GTC GTG CTC GGTTGCAGCACG 5 '

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Abstract

L'invention concerne des améliorations techniques relatives à la conduite d'analyses d'hybridation d'acides nucléiques. Les améliorations permettent un mode de conduite d'analyses d'hybridation à cibles multiples dans lequel une multiplicité de sondes d'acide nucléique différentes, permettant la capture d'acides nucléiques cibles spécifique à des sites, sont conjugués à des emplacements spécifiques sur un seul dispositif ou bâtonnet d'immersion. L'emploi d'un bâtonnet d'immersion présente des avantages mécaniques substantiels par rapport aux dispositifs antérieurs pour l'hybridation d'acides nucléiques. Les bâtonnets d'immersion se composent d'un manche, d'un support non poreux recouvert d'une surface solide comportant au moins une région d'acides nucléiques qui y sont liés de manière covalente. Les améliorations concernent également des moyens de fixation covalente d'acides nucléiques à des supports solides, des tampons d'hybridation améliorés, des surfaces de support améliorées, des procédés de réduction de fond non spécifique ainsi que des procédés de quantification des résultats d'analyse.
PCT/US1989/003378 1988-08-09 1989-08-07 Procedes d'analyse a cibles multiples par hybridation d'acides nucleiques WO1990001564A1 (fr)

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