WO1989007606A1 - Acide nucleique d'investigation ayant en partie un seul brin et en partie deux brins, et son procede de production - Google Patents

Acide nucleique d'investigation ayant en partie un seul brin et en partie deux brins, et son procede de production Download PDF

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Publication number
WO1989007606A1
WO1989007606A1 PCT/EP1989/000122 EP8900122W WO8907606A1 WO 1989007606 A1 WO1989007606 A1 WO 1989007606A1 EP 8900122 W EP8900122 W EP 8900122W WO 8907606 A1 WO8907606 A1 WO 8907606A1
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nucleic acid
acid probe
probe according
metal ions
stranded
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PCT/EP1989/000122
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German (de)
English (en)
Inventor
Günter VALET
Andreas Oser
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Max-Planck-Gesellschaft Zur Förderung Der Wissensc
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Publication of WO1989007606A1 publication Critical patent/WO1989007606A1/fr

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    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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

Definitions

  • the invention relates in part to single-stranded and in part double-stranded nucleic acid probes which are complementary to a nucleic acid to be detected in their single-stranded region and contain a Guer cross-linking compound covalently bound to their double-stranded region, a process for their preparation and a method for the detection of nucleic acids by hybridization with such a nucleic acid probe to form a hybrid double strand.
  • radioactive labeled DNA probes include either direct labeling of the DNA samples by fluorochromes or enzymes or indirect labeling after attaching a group to which another labeled group is in turn bound after hybridization has taken place.
  • all of these methods have disadvantages.
  • radiolabelled DNA probes there is the problem of the instability of the radioactive compounds and the signals which become weaker over time, as well as the problem of safety when dealing with radioactive compounds.
  • fluorochro-labeled DNA probes are not sensitive enough.
  • Enzymes can be inactivated by stringent hybridization conditions and, due to an immune reaction, detectable groups attached to the hybridizing sequences. sequences are known, hinder the hybridization reaction. Indirect detection systems can also cause a high background due to unspecific bindings of the second partner of the immune reaction (antibodies or avidin / streptavidin in the case of biotin).
  • a partly single-stranded and partly double-stranded nucleic acid probe which, in its double-stranded region, contains a crosslinking compound covalently bound and is characterized in that a chelating agent for metal ions is bound to the nucleic acid probe via a functional group of the crosslinking compound is.
  • the nucleic acid probe in its single-stranded region is complementary to a nucleic acid to be detected.
  • This preferred nucleic acid probe according to the invention binds with its single-stranded region to the nucleic acid to be detected and, after the formation of a hybrid double strand of nucleic acid probe and nucleic acid to be detected, enables the detection of the hybrid double strand via the binding of fluorogenic metal ions to the chelating agent via the crosslinking agent Connection is bound to the nucleic acid probe.
  • the single-stranded region of the nucleic acid probe contains a homopolymer DNA tail, via which the probe preferred according to the invention has any nucleic acid sequence which is complementary to a nucleic acid to be detected and which has a homopolymer tail complementary to the homopolymer tail of the probe. can be coupled.
  • the cross-linking compound is preferably provided with an amino, thiol, hydroxyl or carboxy group as a functional group.
  • This functional group is expediently located on a side arm of the cross-linking connection.
  • Preferred as such a cross-linking compound is an intercalating compound which is embedded in the double-stranded area of the nucleic acid probe and is then covalently connected to the nucleic acid probe.
  • Preferred are psoralens, phenanthridium diazides, mitomycin C derivatives and carcinophilin A.
  • compounds of the general formula I are especially:
  • Y. "and Y-, H, alkyl or preferably represent methyl and Y. is a side arm which carries the functional group.
  • Y. is preferred here:
  • phenanthridium diazides are, above all, compounds of the general formula II
  • S represents a side arm which carries a functional group.
  • the structure is preferred, where S represents a side arm which carries a functional group.
  • R 1 , R ", m and n have the meanings defined for the general formula I of psoralens.
  • the synthesis of such a preferred phenanthridium azide is possible by known methods, for example by alkylation of the ring nitrogen atom. -? -
  • S represents a side arm that carries a functional group.
  • R ', R ", m and n correspond to those mentioned for the formula I and II.
  • a side arm S or Y with the meanings explained above is preferably coupled to the 19-C or 19a-C-carboxy group or to the 20-C or 20a-C-OH group of the molecule.
  • DTPA diethylenetriaminepentaacetate
  • EDTA ethylene - diamine tetraacetate
  • other polyaminocarboxylates are particularly suitable as chelating agents for metal ions.
  • These metal chelators must carry activated groups in order to be able to bind them to functional groups, for example a) intramolecular cyclic anhydrides of metal chelators, such as cyclic anhydride of DTPA (caDTPA), b) isothiocyanate groups as in 1- (p-isocyanatophenyl) EDTA , c) azo groups as in 1- (p-diazophenyl) EDTA, d) azido groups as in 4-azido-2-nitrophenyl-EDTA, e) N-hydroxysuccinimide groups or f) N-maleimido groups.
  • a) intramolecular cyclic anhydrides of metal chelators such as cyclic anhydride of DTPA (caDT
  • Preferred chelating agents are caDTPA, the mixed anhydride of DTPA and isobutylcarboxycarboxylic acid, 1- (p-isothiocyanatophenyl) -DTPA, 1- (p-isothiocyanatophenyl) -EDTA, 1- (p-diazophenyl) -EDTA or a phenylazide - derivative used by DTPA or EDTA.
  • chelating agents for metal ions are not only understood to mean a compound as mentioned above, but also a cryptate, that is to say a macropolycyclic ligand for metal ions.
  • the cryptate consists of O, o (-bipyridine or 1,10-phenantroline units.
  • a cryptate can already contain fluorescent metal ions.
  • Fluoroscent metal ions which are preferably used are ions of the rare earth metals.
  • the ions Eu 3+, Tb3-1- or / and Sm3 + are particularly preferred.
  • the nucleic acid probe additionally contains a linker molecule between the cross-linking compound and the chelating agent.
  • This linker molecule is bound to the functional group of the cross-linking compound and to the chelating agent.
  • the linker molecule is therefore preferably a bifunctional crosslinking reagent.
  • the linker molecule is particularly preferably a heterobifunctional crosslinking reagent, such as, for example, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester, the N-maleimido group being thiol group-specific and the N-hydroxysuccinimide ester being amino groups -is specific.
  • the nucleic acid probe contains between the cross-linking compound or optionally the linker molecule and the chelating agent additionally a macromolecular carrier molecule with at least one functional amino or thiol group.
  • This carrier molecule can contain any number of functional groups which are used for binding chelate ligands and for coupling to the nucleic acid sample via the crosslinking compound or the linker molecule.
  • Suitable carrier molecules are homopolymeric or heteropolymeric polypeptides and other chemical and biological macromolecules with functional groups.
  • the carrier molecule is preferably a homopolymeric or heteropolymeric polypeptide.
  • the carrier molecule is particularly preferably polylysine or polycysteine.
  • Another object of the invention is a method for producing a nucleic acid according to the invention which is complementary to a nucleic acid to be detected, which is characterized in that a DNA sequence complementary to a nucleic acid sequence to be detected is inserted into a double-stranded plasmid, with a restriction endonuclease in the region of Cuts inserts and thereby linearizes the plasmid, selectively degrades only one strand of the linearized double strand in the region of the insert with an exonuclease, covalently couples to the remaining double strand a cross-linking compound which contains at least one functional group, and binds a chelating agent to the functional groups.
  • plasmids which contain only a few restriction sites and thus permit cleavage in the insert without the plasmid sequence also being cut thereby would, for example the plasmid pBR322.
  • a DNA strand from the 5 'end for example with exonuclease III on the opposite sides of the linearized plasmid, or the strand from the 3' end with L-exonuclease, is degraded.
  • the cross-linking compound which is preferably a compound intercalating between a DNA double strand
  • the DNA is then effected.
  • a psoralen or a phenantridium diazide is preferably used for this and the covalent binding to the nucleic acid probe is effected by irradiation with UV light.
  • mitomycin C or carcinophilin A is used for this and the covalent binding to the nucleic acid probe is effected by acidifying the reaction solution.
  • the cross-linking compound preferably contains an amino, thiol, hydroxy or as a functional group - 3 -
  • the chelating agent which contains a group activated for reaction with a functional group, is then attached to this functional group.
  • This chelating agent is preferably caDPTA, the mixed anhydride of DTPA and isobutylcarboxylic acid, 1- (p-isolothio ⁇ yanatophenyl) DTPA, 1- (p-isothio- ⁇ yanatophenyl) EDTA, 1- (p-diazophenyl) EDTA or a phenyl azide - derivative of DTPA or EDTA.
  • a chelating agent in the sense of the invention is also understood to mean a cryptate.
  • a cryptate composed of 0 ⁇ , ⁇ / - bipyridine or 1,10-phenantroline units is preferably used here. It is also preferred here to use cryptates which already contain fluorogenic metal ions, the fluorogenic metal ions in turn preferably being ions of the rare earth metals. Cryptates are particularly preferred
  • a linker molecule is additionally introduced between the crosslinking compound and the chelating agent.
  • This linker molecule is reacted with the functional or activated groups of the cross-linking compound and the chelating agent.
  • a linker molecule which is a bifunctional crosslinking reagent.
  • a heterobifunctional crosslinking reagent that is to say a crosslinking reagent which contains two different functional groups, for example 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester, is particularly preferably used as the linker molecule.
  • the linker molecule and the chelating agent additionally include a macromolecular carrier molecule with at least one functional amino or thiol group.
  • a macromolecular carrier molecule with at least one functional amino or thiol group.
  • chelating molecules are increasingly made available for fluorogenic metal ions, as a result of which the detection signal to be obtained later is amplified.
  • a homopolymeric or heteropolymeric polypeptide particularly preferably polylysine or polystyrene, is used as the carrier molecule.
  • Another object of the invention is another method for producing a nucleic acid probe which contains a sequence complementary to a nucleic acid to be detected, in which a nucleic acid probe according to claim 3 with a single-stranded DNA or RNA, the sequence required for detection hybridization and a homopolymer Single strand, which is complementary to the nucleic acid probe according to claim 3, contains hybridized.
  • the invention further relates to a method for the detection of nucleic acids by hybridization with a nucleic acid probe to form a hybrid double strand, which is characterized in that a nucleic acid probe according to the invention which contains the sequence complementary to a nucleic acid to be detected is used after hybridization, a solution of a fluorogenic metal ion is added and, after removing the metal ion solution and washing, the hybrid double strand formed is exposed to the fluorescence of the I - ⁇ ⁇ -
  • Another object of the invention is a method for the detection of nucleic acids by hybridization with a nucleic acid probe to form a hybrid double strand, in which one uses a nucleic acid probe according to claim 3 to 15, this either with a single-stranded DNA or RNA which the a nucleic acid complementary sequence to be detected and a homopolymeric nucleic acid tail, which is complementary to the homopolymeric single strand of the nucleic acid probe according to claim 3, and simultaneously or thereafter hybridizes with the nucleic acid probe to be detected, adds a solution of a fluorogenic metal ion after hybridization and after removing the metal ion solution and Washing the bound metal ions is extracted by an amplifier solution and the hybrid double strand formed is detected via the fluorescence of the metal ions in the amplifier solution.
  • Rare earth ions are preferably used as the fluorogenic metal ions. Are particularly preferred
  • the hybridization is carried out with a nucleic acid to be detected, which is bound on a support, for example on a filter, gel, microtiter plate, histological section, cell, the fluorogenic metal ions are added after hybridization has taken place and after washing to remove excess uncomplexed metal ions, the metal ions are detached from the complex by an intensifying solution and their fluorescence in the amplified solution is determined.
  • a non-ionic detergent 0.1 - 0.05%, v / v, e.g.
  • Triton Triton
  • a "synergistic base” (10 - 100 ⁇ M, e.g. 50 ⁇ M tri-n-octylphosphine oxide) and an energy Dissolve donor (10 ⁇ M - 100 ⁇ M of a ⁇ -diketone, eg 15 ⁇ M 2-naphthoyltrifluoroacetone) in an aqueous buffer solution with a pH between 2.5 and 4.
  • a preferred strengthening solution contains 0.1 M acetate phthalate buffer, pH 3.2, 0.1% Triton X-100 (v / v), 15 ⁇ M 2-naphthoyltrifluoroacetone and 50 ⁇ M tri-n-octylphosphine oxide.
  • nucleic acid probe labeled with the chelate ligands cannot already be used in a metal-bound manner since the metal ions would dissociate from the chelate under these conditions . Consequently, the metal ions are only added after the hybridization reaction has taken place. An exception to this are, however, mild hybridization conditions in which a nucleic acid probe pre-labeled with metal ions can also be used.
  • the determination of the fluorescence of the metal ions after removal from the labeled hybrid double strand in solution is above all an advantage if, for example, the method according to the invention is used in the context of clinical and medical applications, since the nucleic acids to be detected can often not be used in this way clean form used, but there are still contaminants from accompanying biological material, such as cells, tissues, etc., present. This can result in a disturbance in the measurement, but this can be caused by removing the fluorescent -. 3 -
  • Another advantage that results from the removal of the metal ions from the marked hybrid double strand is that, with small amounts of metal ions bound to the hybrid strand, the solution of the ions can still be concentrated and a more precise measurement can thereby be achieved.
  • the metal ions are expediently added even in the form of a complex.
  • the complex formation constant of this complex should also be relatively high (log K at least 5), but significantly lower than that of the metal ion compared to the chelating agent bound to the probe. This prevents non-specific binding of the metal ions to other binding sites, such as phosphate groups of the nucleic acids or binding sites on the filter, etc.
  • Another object of the invention is a method for the detection of nucleic acids by hybridization with a nucleic acid probe to form a hybrid double strand, which is characterized in that a nucleic acid probe according to the invention is used which contains a cryptate as chelating agent which already contains a fluorogenic metal ion.
  • a nucleic acid probe according to the invention which contains a cryptate as chelating agent which already contains a fluorogenic metal ion.
  • the fluorescence of the metal ions can also be determined directly on the hybrid double strand after hybridization has taken place. It will ⁇ ) H
  • a nucleic acid probe and a method for the detection of nucleic acids are thus provided which make it possible to detect nucleic acids with high specificity without using radioactive compounds or enzymatically or immunologically labeled probes.
  • the use of a carrier molecule to which several chelating agents can be bound which results in an amplification of the signal from fluorogenic metal ions, results in a high sensitivity of the method according to the invention.
  • Figure 1 shows schematically the synthesis of psoralen SH
  • Fig. 2 shows the elution profile of a
  • Psoralen-SH was produced according to the method of Saffran et al, Proc.Natl.Acad.Sci.üSA 79, 4594-4598 (1982). The individual reaction steps of the synthesis are shown in Figure 1. All reactions were carried out in the dark. la) Preparation of 4'-chloromethyl-4,5 ', 8-trimethyl-psoralen (2):
  • PSH (5) was freshly made for each labeling reaction. 24 ⁇ l of a 1.7 mM stock solution of compound 4 in ethanol, 40 ⁇ l 100 mM dithioerythitol (DTE) and 16 ⁇ l H 2 O were mixed in an Eppendorf vessel and incubated for one hour at room temperature in the dark . - / -
  • the reaction was stopped by adding 2.5 ul 100 mM EDTA.
  • the DNA was precipitated with ethanol, washed with 70% ethanol and resuspended in 36 ⁇ l H_0.
  • exonuclease III 90 U
  • 10xExo-III buffer 660 mM Tris-HCl, 6.6 mM MgCl 2 , 10 mM mercaptoethanol, pH 7.6 was added and the solution at 37 ° C. for 30 minutes long incubated to make the plasmid partially single-stranded.
  • the reaction was stopped by adding 5 ul 100 mM EDTA.
  • pKKHBs34 The partially single-stranded pKKHBs34 (hereinafter referred to as pKKHBs34 ') was separated from the enzyme, nucleotides and buffer via a NENSORB-20 nucleic acid cleaning cartridge in accordance with the manufacturer's instructions.
  • Psoralen molecules intercalate into double-stranded DNA and undergo photocycloaddition with DNA thymidine residues when irradiated with long-wave UV light (Cimino et al, Ann.Rev.Bochem. 5_4, 1151-1193 (1985)).
  • Double strands are easily formed in TA / AT sequences, single strand DNA remains unmodified.
  • the amount of PSH covalently bound to the DNA was determined by reacting the SH groups with the thiol-specific fluorescent dye, 7-di-ethylamino-3- (4'-maleimidophenyl) -4-methylcoumarin ( CPM).
  • CPM 7-di-ethylamino-3- (4'-maleimidophenyl) -4-methylcoumarin
  • the fluorescence of CPM is significantly increased when it is covalently bound to thiol groups.
  • SH-labeled pKKHBs34 * (7.5 ⁇ g) was in 50 ⁇ l 50 mM phosphate buffer (pH 7.8) containing 200 ⁇ M CPM and 1 mM EDTA, dissolved.
  • the DTPA content in PLL was determined by equilibrium dialysis.
  • DTPA-PLL conjugate (20 ⁇ g) was incubated for two hours with 500 ⁇ M EuCl 3 , 500 ⁇ M EDTA in 200 ⁇ l 50 mM sodium citrate buffer (pH 6.0) and then equilibrated against 5 mM sodium acetate, 10 mM NaCl ( pH 5.5) dialyzed.
  • the europium and thus the DTPA content was determined by time-delayed fluorometry Right.
  • DTP -bound PLL (100 ⁇ g, 6 nmol) in 100 ⁇ l 100 mM KHCO-. (pH 8.2) was cooled on ice.
  • the heterobifunctional cross-linking agent, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid N-hydroxy-succinimide ester (10 ⁇ g, 30 nmol, from a 1 mg / ml solution in dry dimethylformamide) became slow added with shaking.
  • the solution was shaken for a further 30 minutes at room temperature and, after adjusting the pH to 6.5 with 0.6 N HCl, SH-labeled pKKHBs34 '(7.5 ⁇ g) was incubated overnight at room temperature.
  • the resulting conjugate was isolated by DEAE chromatography (Fig. 2).
  • pKKHBs34 as target DNA in various amounts and pBR322 and calf thymus DNA as controls were immobilized on nitrocellulose filter disks (BA 85 NC filter, 4-5 mm diameter, Schleicher and Schuell) and denatured using standard methods (Anderson and Young, in Harnes BD and Higgins (ed.), Nucleic Acid. Hybridization ; A Practical Approach, IRL Press, Oxford, Washington, DC, 73-111 (1985)).
  • Chelation of the hybrid-bound DTPA with European ions was achieved by incubating the filters from Example 6 in 100 ⁇ M EuCl 3 , 100 ⁇ M EDTA, 1 ⁇ SSC (pH 7.0, 200 ⁇ l per microtiter plate) for two hours at room temperature.
  • the filters were washed at least six times in 2x SSC and finally for 15 minutes in 1 ml "booster solution" (0.1 M acetate phthalate buffer, pH 3.2, 0.1% Triton X-100 (v / v) , 15 ⁇ M 2-naphthoyltrifluoroacetone, 50 ⁇ M trin-octylpho'sphin ⁇ oxide) were shaken in polystyrene vessels to remove DTPA-bound Eu 3+. The supernatant was decanted and the fluorescence was determined in an "Arcus 1230" time-delayed fluorometer (LKB-Wallac, Turku, Finland). Excitation and emission were 340 and 613 nm, respectively. Delay and count times were both set to 400 ⁇ s (Fig. 3).

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Abstract

Un acide nucléique d'investigation ayant en partie un seul brin et en partie deux brins et qui contient un composé de réticulation transversale lié par covalence à son domaine à deux brins, présente un chélateur d'ions métalliques liés à l'acide nucléique d'investigation par un groupe fonctionnel du composé de réticulation transversale. Cet acide nucléique convient au dépistage d'acides nucléiques par hybridation menant à la formation d'un double brin hybride. Une fois l'hybridation achevée, on ajoute des ions métalliques fluorogènes, puis on extrait les ions métalliques liés au moyen d'une solution de renforcement, après avoir éliminé la solution d'ions métalliques et avoir lavé l'acide nucléique hybridé; le double brin ainsi formé par hybridation est dépisté grâce à la fluorescence des ions métalliques dans la solution de renforcement.
PCT/EP1989/000122 1988-02-11 1989-02-09 Acide nucleique d'investigation ayant en partie un seul brin et en partie deux brins, et son procede de production WO1989007606A1 (fr)

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DE3804243A DE3804243A1 (de) 1988-02-11 1988-02-11 Teils einzel- und teils doppelstraengige nukleinsaeuresonde und verfahren zu ihrer herstellung
DEP3804243.6 1988-02-11

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP0822261A1 (fr) * 1996-07-29 1998-02-04 Academia Sinica Méthode rapide pour la détection de la susceptibilité microbienne

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Publication number Priority date Publication date Assignee Title
US5399719A (en) 1993-06-28 1995-03-21 Steritech, Inc. Compounds for the photodecontamination of pathogens in blood
EP1105539A2 (fr) 1998-08-21 2001-06-13 Naxcor Dosages utilisant des acides nucleiques reticulables immobilises
US6303799B1 (en) 1998-11-10 2001-10-16 Naxcor Polynucleotide crosslinking agents

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EP0131830A1 (fr) * 1983-07-14 1985-01-23 Molecular Diagnostics, Inc. Cordons d'acides nucléiques marqués et produits d'addition pour leur préparation
EP0156287A2 (fr) * 1984-03-21 1985-10-02 Cetus Corporation Procédé pour marquer des acides nucléiques en utilisant des dérivés du psoralen
EP0212951A2 (fr) * 1985-08-15 1987-03-04 Amoco Corporation Acides nucléiques marqués
WO1987002708A1 (fr) * 1985-10-24 1987-05-07 Siska Diagnostics, Inc. Sondes d'acide nucleique marquees par des composes chelates de lanthanides
WO1987003622A1 (fr) * 1985-12-13 1987-06-18 Princeton University Analyse d'hybridisation amplifiee
EP0235726A2 (fr) * 1986-03-05 1987-09-09 Miles Inc. Détection rapide des séquences d'acides nucléique dans un échantillon par marquage de l'échantillon
EP0237833A2 (fr) * 1986-03-05 1987-09-23 Miles Inc. Test d'hybridation en phase liquide pour détecter des séquences polynucléotidiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0131830A1 (fr) * 1983-07-14 1985-01-23 Molecular Diagnostics, Inc. Cordons d'acides nucléiques marqués et produits d'addition pour leur préparation
EP0156287A2 (fr) * 1984-03-21 1985-10-02 Cetus Corporation Procédé pour marquer des acides nucléiques en utilisant des dérivés du psoralen
EP0212951A2 (fr) * 1985-08-15 1987-03-04 Amoco Corporation Acides nucléiques marqués
WO1987002708A1 (fr) * 1985-10-24 1987-05-07 Siska Diagnostics, Inc. Sondes d'acide nucleique marquees par des composes chelates de lanthanides
WO1987003622A1 (fr) * 1985-12-13 1987-06-18 Princeton University Analyse d'hybridisation amplifiee
EP0235726A2 (fr) * 1986-03-05 1987-09-09 Miles Inc. Détection rapide des séquences d'acides nucléique dans un échantillon par marquage de l'échantillon
EP0237833A2 (fr) * 1986-03-05 1987-09-23 Miles Inc. Test d'hybridation en phase liquide pour détecter des séquences polynucléotidiques

Non-Patent Citations (1)

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Title
Nucleic Acids Research, Band 16, Nr. 3, 11. Februar 1988, IRL Press Ltd, (Oxford, GB), A. Oser et al.: "Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence", Seiten 1181-1196 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0822261A1 (fr) * 1996-07-29 1998-02-04 Academia Sinica Méthode rapide pour la détection de la susceptibilité microbienne
US5789173A (en) * 1996-07-29 1998-08-04 Academia Sinica Methods for rapid antimicrobial susceptibility testing

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