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WO1991017264A1 - Analyse de sequences specifiques d'acide nucleique - Google Patents

Analyse de sequences specifiques d'acide nucleique Download PDF

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Publication number
WO1991017264A1
WO1991017264A1 PCT/GB1991/000696 GB9100696W WO9117264A1 WO 1991017264 A1 WO1991017264 A1 WO 1991017264A1 GB 9100696 W GB9100696 W GB 9100696W WO 9117264 A1 WO9117264 A1 WO 9117264A1
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WO
WIPO (PCT)
Prior art keywords
probe
target
nucleic acid
acid sequence
exonuclease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1991/000696
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English (en)
Inventor
Clive Graham Copley
Christopher Boot
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Imperial Chemical Industries Ltd
Original Assignee
Imperial Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial Chemical Industries Ltd filed Critical Imperial Chemical Industries Ltd
Publication of WO1991017264A1 publication Critical patent/WO1991017264A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6823—Release of bound markers

Definitions

  • This invention relates to an assay method for the detection of specific nucleic acid sequences, to an optionally labelled oligonucleotide and to kits comprising components suitable for use in the method.
  • probes comprising nucleic acid sequences which are complementary to the specific nucleic acid sequences to be detected, i.e. together with the nucleic acid sequences to be detected the probe sequences will form sections of double stranded DNA.
  • the complementary nucleic acid sequences of the probes will hybridise with the corresponding nucleic acid sequences specific to the bacteria or other materials whose presence is to be detected and the occurrence of hybridisation is detected by a suitable means, frequently autoradiography or a fluorescence technique.
  • a probe comprising a nucleic acid sequence complementary to a sequence specific to Salmonella strains could be used and the hybridisation of the probe sequence with the target sequence in the Salmonella strains could be detected by a suitable means.
  • a significant disadvantage of many assay methods presently employed is that the detection systems which they use lead to a requirement for immobilisation of hybridised DNA. Methods which enabled the assays to be performed in solution would represent a considerable improvement. Additionally, several existing assay methods dependent upon the very sensitive polymerase chain reaction (PCR) technique are often complicated, requiring many cycles comprising several temperature changes over an extended period of time.
  • PCR polymerase chain reaction
  • nucleolytic agent it is not essential for the nucleolytic agent to be added to the medium before the target has been contacted with the probe, but for convenience it is preferred.
  • the nucleolytic agent is preferably an exonuclease but other agents, chemical or enzymatic agents, which degrade the hybridised probe faster than the target can be envisaged.
  • the invention will be described in terms of an assay method using an exonuclease as nucleolytic agent, that is to say using an exonucleolytic agent.
  • the exonucleolytic agent used in the assay method of the invention may be any exonuclease which requires double stranded nucleic acids for activity and degrades the hybridised probe faster than the target to which it is hybridised.
  • a preferred exonuclease is lambda exonuclease, a readily available exonuclease, but other exonucleases, including 5' to 3' and 3' to 5' exonucleases, are also suitable.
  • Lambda exonuclease is a 5' to 3' exonuclease requiring a 5' phosphate group and double stranded nucleic acids for activity.
  • oligonucleotides with 5' phosphate groups
  • Other suitable exonuclease enzymes may require other terminal groups for example 3 1 phosphate groups or hydroxyl groups for activity.
  • a preferred assay method of the invention is for detecting the presence or absence of a target nucleic acid sequence in a medium which comprises the steps of:
  • degraded probe The method used for detecting the presence or absence of degraded probe will depend upon the nature of the probe, and suitable methods will be apparent to persons skilled in the art. For example where the probe is labelled by a fluorophore or chemiluminescent label then degraded probe may be detected by observing its fluorescence or luminescence; similarly, where the probe is labelled using a radioactive label degraded probe can be detected using known techniques such as autoradiography.
  • a cycling effect occurs which enables a small amount of a target nucleic acid sequence to produce a relatively large quantity of degraded probe oligonucleotide sequences. This produces an amplification effect which is an important advantage of the method of the invention.
  • step (1) a target comprising the target nucleic acid sequence to be detected is shown in the presence of probe oligonucleotides having 5' phosphate groups and comprising complementary probe nucleic acid sequences, one of which probe oligonucleotides has hybridised with the target sequence.
  • step (2) a lambda exonuclease attacks the probe component of the double stranded DNA formed by the hybridised target and probe, degrading the hybridised probe but leaving the target intact.
  • step (3) the degraded probe is released from the target and a further undegraded probe oligonucleotide hybridises with the target sequence, and the cycle is repeated.
  • the various fragments of the degraded probe are detected by a means available during operation of the method.
  • a urogenital swab or a blood or urine sample or other biological sample can be taken, subjected to suitable further treatment, for instance by heating, and then made up into a suitable medium for treatment by the assay method.
  • the assay method is then carried out to produce an effect which can be detected by suitable means.
  • suitable temperature e.g. temperature
  • a suitable temperature is 37°C and a suitable pH is 9.
  • Any suitable means may be used to detect the various fragments of the degraded labelled probe produced in the assay method of the invention.
  • the probe preferably comprises 10 to 100 bases, more preferably 15 to 50, especilly 20 to 30 bases.
  • bases refers to the bases found in nucleic acids, such as ⁇ . A, T, G and C.
  • a label should be at or near the 3' terminus whilst when the enzyme degrades in the 3' to 5' direction a label should be at or near the 5' terminus.
  • the probe is labelled in such a way that it is detectable, for example using any of the labels used in biochemistry, such as by a chemiluminscer or fluorophore, a radioactive label, or other detectable chemical substance such as an active substrate, co-factor or enzyme donor peptide for a second enzyme system.
  • the radioactive label preferably contains a radioactive isotope of a carbon, nitrogen, sulphur, iodine or
  • phosphorus atom particularly P, which may form part of a nucleotide.
  • An example of a chei ⁇ iluminescser is luciferin, and fluorescein is an example of a fluorophore.
  • Suitable means for detecting the degradation of probe oligonucleotides in the assay method include separation of the products on polyacrylamide gels and preferably either: (a) directly detecting degradation by the incorporation of a fluorophore on a suitable terminal nucleotide (3' in the case of lambda exonuclease) and measuring the change in fluorescence polarisation as the probe containing the fluorophore is degraded from large to small [for example, as described in our European Patent Application EP 0382433]; or (b) designing the probe oligonucleotide such that degradation results in release of an active substrate, co-factor or enzyme donor peptide for a second enzyme system: such as the release of NAD followed by a standard oxidation-reduction reaction, or the release of a peptide derived from beta-galactosidase which combines with an apoenzyme to give the holoenzyme capable of generating a signal from appropriate substrate [for example as described in Clin. Che
  • the specific sequence In the case of a double stranded target the specific sequence must be made available for hybridisation to the probe oligonucleotide, this can be achieved by conventional methods such as denaturation.
  • additional oligonucleotides either side of the oligonucleotide probe known in the art as “blocking oligonucleotides” can alleviate the potential problem of target which is normally double stranded re-annealing.
  • blocking oligonucleotides can alleviate the potential problem of target which is normally double stranded re-annealing.
  • the use of multiple probe oligonucleotides can also serve to enhance the sensitivity of the assay method as well as (if arranged consecutively) preventing re-annealing of a double stranded target.
  • the assay method of the invention has the advantage that it does not require the use of a large number of temperature changes over an extended period of time. According to a further aspect of the invention there is provided a kit for detecting the presence or absence of a target nucleic acid sequence which comprises:
  • a probe comprising a nucleic acid sequence complementary to a region of the target nucleic acid sequence
  • nuclease which requires double stranded nucleic acids for activity and which degrades the probe, when hybridised with the target, faster than said target.
  • the kit also contains an appropriate pH buffer, or a protocol for carrying out the assay method of the invention or both.
  • the precise nature of the pH buffer depends upon the properties of the nuclease, and will be selected such that it is capable of buffering a solution to a pH at which the nuclease is active.
  • a kit containing a nuclease requiring a pH of 9 for optional activity would preferably contain a pH buffer which maintain a pH of 8 - 10, especially pH 8.5 to 9.5, more especially around pH 9.
  • the preferred probe and nuclease are as hereinbefore described in relation to the assay method of the invention.
  • Figure 1A is an autoradiograph showing the effects of varying lambda exonuclease concentrations (measured in Units) on varying quantities of oligonucleotide 1377.
  • Figure IB is an autoradiograph showing the effect of varying lambda exonuclease concentrations on varying quantities of oligonucleotide 1125 (as defined hereinafter).
  • Figure 2 is an autoradiograph showing the effect of lambda exonuclease on single stranded oligonucleotide probe in the presence of varying amounts of its target complementary strand.
  • Figure 3 illustrates the amplification effect provided by the preferred assay method of the present invention. This Figure is hereinbefore discussed in more detail.
  • Formula 1 illustrates the binding of labelled oligonucleotide 1377 on the positive strand of M13 mpl9.
  • 32 encircled star is a label, for example P-dd ATP or a fluorophore.
  • Oligonucleotide 1125 and 1377 had 5' chemically attached phosphate groups and had been purified by application to a Mono Q column.
  • Oligonucleotide 1125 is a Vectorette universal primer used in Chemical Genetics' UK Patent Application No.8818020.3.
  • Oligonucleotide 1377 is complementary to the positive strand of M13 mpl9 DNA and has been specifically designed for use in the exonuclease cycling assay (see Formula 1).
  • oligonucleotide 0.05 pmoles of labelled oligonucleotide was added to either 0.45 pmoles, 4.95 pmoles or 49.95 pmoles of 3' blocked oligonucleotide.
  • Lambda exonuclease was added at concentrations of 10U, 1U, 0.1U, 0.01U or 0.001U. Digestion reactions and visualization of the products were carried out as described above.
  • M13 mpl9 single-stranded DNA (positive strand) was diluted to 2.5 pmole, 0.8 pmole, 280 fmole, 30 fmole, 10 fmole, 3.3 fmole, 1.1 fmole and 380 amole.
  • 0.05 pmole of labelled 1377 and 4.95 pmole of 3' blocked 1377 were added to these along with 0.1U of lambda exonuclease. Digestion reactions and visualization of the products were carried out as described earlier.
  • Oligonucleotide 1125 is a PCR primer used in Chemical Genetics and was initially selected because it was available with a terminal 5' phosphate group. In the role for which it was designed it is preferred that 3' complementarity is not present, if the formation of primer dimers is to be avoided. However, in the assay described here 5' complementarity is to be avoided if non-specific degradation is not to be a problem. Examination of the structure of oligonucleotide 1125 reveals that the most stable configuration would contain 5' complementarity as shown in Formula (2): Formula (2)
  • the Tm of this structure is predicted to be 48°C.
  • Oligonucleotide 1377 was specifically designed from a 40 base sequence complementary to the positive strand of M13 mpl9 by examining the self-complementarity of all available 20mer oligonucleotides in this region. Examination of the sequence of oligonucleotide 1377 reveals the most stable self-complementary structures have 5' overhangs, as shown in Formulae (3) and (4), and thus are relatively immune from degradation by lambda exonuclease: Formula (3)
  • the Tm of these structures is predicted to be 24°C, that is 13°C lower than the reaction temperature, and thus a large proportion of this oligonucleotide would be in single stranded form.
  • Both oligonucleotides 1125 and 1377 were prepared with a 5' terminal phosphate group. This was achieved by conventional automated DNA synthesis on an ABI3 80B DNA synthesiser, using the reagent "phosphate-ON" (Clontech) . Before use the oliginucleotides were radiolabelled by the addition of radioactive ddAMP to their 3' termini.
  • Figure 1A shows the effects of varying lambda exonuclease concentrations (measured in Units) on varying quantities of oligonucleotide 1377.
  • Lanes 1 to 4 used 0.5 pmoles of oligonucleotide 1377
  • lanes 5 to 9 used 5pmoles of oligonucleotide 1377
  • lanes 10-14 used 50 pmoles of oligonucleotide 1377.
  • Lane X is not relevant.
  • Figure IB shows the effects of varying lambda exonuclease concentrations on varying quantities of oligonucleotide 1125. There were 10 Units used in lane 1; 1 Unit in lane 2; 0.01 Unit in lane 3; 0.01 Unit in lane 4; 0.001 Unit in lane 5; and no exonuclease in lane 6. All lanes used 5pmoles of oligonucleotide 1125.
  • Figure 2 shows the effect of lambda exonuclease on single-stranded oligonucleotide probe in the presence of varying amounts of its target complementary strand.
  • lanes 1 to 10 there was used 0.1 Units of lambda exonuclease, and in all lanes there were 5 pmoles of single-stranded probe oligonucleotide.
  • the concentrations of target sequence in lanes 1 to 11 were as follows: lane 1, 2.5 pmoles; lane 2 , 0.8 pmoles; lane 3, 280 fmoles; lane 4, blank; lane 5, 30 fmoles; lane 6, 10 fmoles; lane 7, 3 fmoles; lane 8, 1.1 fmoles; lane 9, 380 amoles; lane 10, none; lane 11, none and no exonuclease.

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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

Un procédé d'analyse dans lequel on détecte une séquence cible d'acide nucléique en utilisant une sonde marquée comprenant une séquence complémentaire d'acide nucléique dans des conditions causant l'hybridation de la cible et de la sonde et où une nucléase nécessitant un ADN à double brin pour son activité dégrade la sonde plus rapidement que la cible. Un effet cyclique conduit à la formation de quantités importantes de sonde dégradée, par rapport à l'importance de la cible, ce qui implique une amplification des produits de dégradation détectables. L'invention décrit également de nouveaux oligonucléotides et des 'kits' d'analyse.
PCT/GB1991/000696 1990-05-02 1991-05-01 Analyse de sequences specifiques d'acide nucleique Ceased WO1991017264A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909009903A GB9009903D0 (en) 1990-05-02 1990-05-02 Assay for specific nucleic acid sequences
GB9009903.7 1990-05-02

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WO1991017264A1 true WO1991017264A1 (fr) 1991-11-14

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2697851A1 (fr) * 1992-11-10 1994-05-13 Bio Merieux Système et procédé de détection d'une séquence d'acide nucléique selon une méthode d'amplification par restriction enzymatique sur phase solide.
EP0578138A3 (fr) * 1992-07-10 1994-10-26 Hitachi Ltd Méthode pour la détection de gènes.
EP0639647A3 (fr) * 1993-07-08 1995-07-12 Tanabe Seiyaku Co Procédé pour déterminer des séquences d'acides nucléiques.
WO1995014106A3 (fr) * 1993-11-17 1995-08-24 Id Biomedical Corp Detection cyclique par scission de sondes de sequences d'acides nucleiques
WO1996020287A3 (fr) * 1994-12-23 1996-09-06 Behringwerke Ag Detection d'acides nucleiques par formation d'un produit catalyse par une cible
WO1996040999A1 (fr) * 1995-06-07 1996-12-19 Behring Diagnostics Gmbh Detection d'acides nucleiques par la formation d'un produit dependant d'une matrice
WO1997019192A1 (fr) * 1995-11-20 1997-05-29 Trustees Of Boston University Procede et sonde servant a detecter une sequence cible d'acide nucleique
US5679510A (en) * 1993-04-27 1997-10-21 Hybridon, Inc. Quantitative detection of specific nucleic acid sequences using lambdoid bacteriophages linked by oligonucleotides to solid support
EP0711361A4 (fr) * 1993-06-04 1998-04-15 Third Wave Tech Inc Nucleases 5' derivees d'adn-polymerase d'adn thermostable
WO2000049179A1 (fr) * 1999-02-18 2000-08-24 Promega Corporation Procedes de determination de la presence de sequences cibles d'acides nucleiques et leurs applications
WO2000049182A3 (fr) * 1999-02-18 2001-04-05 Promega Corp Methodes analytiques et materiaux permettant de detecter les acides nucleiques
US6235480B1 (en) 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6270973B1 (en) 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6312902B1 (en) 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6335162B1 (en) 1998-03-13 2002-01-01 Promega Corporation Nucleic acid detection
US6391551B1 (en) 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
EP1064400A4 (fr) * 1998-03-13 2003-05-02 Promega Corp Detection d'acide nucleique
AU765428B2 (en) * 1998-03-13 2003-09-18 Promega Corporation Methods for determining the presence of nucleic acid target sequences and applications thereof
US6703211B1 (en) 1998-03-13 2004-03-09 Promega Corporation Cellular detection by providing high energy phosphate donor other than ADP to produce ATP
US7090975B2 (en) 1998-03-13 2006-08-15 Promega Corporation Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
EP2480689A4 (fr) * 2009-09-24 2013-05-15 Seegene Inc Détection de séquences d'acides nucléiques cibles au moyen de réactions exonucléolytiques cycliques
WO2023240564A1 (fr) * 2022-06-16 2023-12-21 深圳华大智造科技股份有限公司 Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques

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Publication number Priority date Publication date Assignee Title
EP0123513A1 (fr) * 1983-04-22 1984-10-31 AMERSHAM INTERNATIONAL plc Méthode pour détecter des mutations en ADN
EP0142299A2 (fr) * 1983-10-25 1985-05-22 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Méthode pour la mesure de polynucléotides et trousse de réactifs utilisée dans cette méthode
EP0200057A2 (fr) * 1985-05-02 1986-11-05 AlliedSignal Inc. Réactif diagnostique, trousse et procédé utilisant déplacement polynucléotidique, séparation, clivage enzymatique et détection d'adénosine-phosphate
US4725537A (en) * 1985-09-19 1988-02-16 Allied Corporation Assay, reagent and kit employing nucleic acid strand displacement and restriction endonuclease cleavage
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
WO1989009284A1 (fr) * 1988-03-24 1989-10-05 University Of Iowa Research Foundation Systemes d'hybridation catalytiques servant a detecter des sequences d'acide nucleique, fondes sur l'activite de ces sequences en tant que cofacteurs dans des reactions catalytiques ou une sonde d'acide nucleique complementaire marquee est soumise a une operation de clivage
WO1989010415A1 (fr) * 1988-04-29 1989-11-02 Meiogenics, Inc. Procedes de detection de sequences d'acide nucleique

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0123513A1 (fr) * 1983-04-22 1984-10-31 AMERSHAM INTERNATIONAL plc Méthode pour détecter des mutations en ADN
EP0142299A2 (fr) * 1983-10-25 1985-05-22 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Méthode pour la mesure de polynucléotides et trousse de réactifs utilisée dans cette méthode
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
EP0200057A2 (fr) * 1985-05-02 1986-11-05 AlliedSignal Inc. Réactif diagnostique, trousse et procédé utilisant déplacement polynucléotidique, séparation, clivage enzymatique et détection d'adénosine-phosphate
US4725537A (en) * 1985-09-19 1988-02-16 Allied Corporation Assay, reagent and kit employing nucleic acid strand displacement and restriction endonuclease cleavage
WO1989009284A1 (fr) * 1988-03-24 1989-10-05 University Of Iowa Research Foundation Systemes d'hybridation catalytiques servant a detecter des sequences d'acide nucleique, fondes sur l'activite de ces sequences en tant que cofacteurs dans des reactions catalytiques ou une sonde d'acide nucleique complementaire marquee est soumise a une operation de clivage
WO1989010415A1 (fr) * 1988-04-29 1989-11-02 Meiogenics, Inc. Procedes de detection de sequences d'acide nucleique

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578138A3 (fr) * 1992-07-10 1994-10-26 Hitachi Ltd Méthode pour la détection de gènes.
FR2697851A1 (fr) * 1992-11-10 1994-05-13 Bio Merieux Système et procédé de détection d'une séquence d'acide nucléique selon une méthode d'amplification par restriction enzymatique sur phase solide.
US5679510A (en) * 1993-04-27 1997-10-21 Hybridon, Inc. Quantitative detection of specific nucleic acid sequences using lambdoid bacteriophages linked by oligonucleotides to solid support
EP1505160A3 (fr) * 1993-06-04 2005-05-04 Third Wave Technologies, Inc. Nucléases 5' dérivées d'ADN-polymérase thermostable
EP0711361A4 (fr) * 1993-06-04 1998-04-15 Third Wave Tech Inc Nucleases 5' derivees d'adn-polymerase d'adn thermostable
EP0639647A3 (fr) * 1993-07-08 1995-07-12 Tanabe Seiyaku Co Procédé pour déterminer des séquences d'acides nucléiques.
WO1995014106A3 (fr) * 1993-11-17 1995-08-24 Id Biomedical Corp Detection cyclique par scission de sondes de sequences d'acides nucleiques
US5660988A (en) * 1993-11-17 1997-08-26 Id Biomedical Corporation Cycling probe cleavage detection of nucleic acid sequences
US5792614A (en) * 1994-12-23 1998-08-11 Dade Behring Marburg Gmbh Detection of nucleic acids by target-catalyzed product formation
WO1996020287A3 (fr) * 1994-12-23 1996-09-06 Behringwerke Ag Detection d'acides nucleiques par formation d'un produit catalyse par une cible
US6368803B1 (en) * 1994-12-23 2002-04-09 Dade Behring Inc. Detection of nucleic acids by target-catalyzed formation
US6110677A (en) * 1994-12-23 2000-08-29 Dade Behring Marburg Gmbh Oligonucleotide modification, signal amplification, and nucleic acid detection by target-catalyzed product formation
US6121001A (en) * 1994-12-23 2000-09-19 Dade Behring Marburg Gmbh Detection of nucleic acids by target-catalyzed product formation
EP1260593A3 (fr) * 1994-12-23 2003-12-03 Dade Behring Inc. Détection d'acides nucléiques par formation d'un produit catalysé par une nucléase
US5882867A (en) * 1995-06-07 1999-03-16 Dade Behring Marburg Gmbh Detection of nucleic acids by formation of template-dependent product
WO1996040999A1 (fr) * 1995-06-07 1996-12-19 Behring Diagnostics Gmbh Detection d'acides nucleiques par la formation d'un produit dependant d'une matrice
WO1997019192A1 (fr) * 1995-11-20 1997-05-29 Trustees Of Boston University Procede et sonde servant a detecter une sequence cible d'acide nucleique
US6235480B1 (en) 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
AU765428B2 (en) * 1998-03-13 2003-09-18 Promega Corporation Methods for determining the presence of nucleic acid target sequences and applications thereof
US6270973B1 (en) 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6312902B1 (en) 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6335162B1 (en) 1998-03-13 2002-01-01 Promega Corporation Nucleic acid detection
US6268146B1 (en) 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6379898B2 (en) 1998-03-13 2002-04-30 John W. Shultz Nucleic acid detection
US6391551B1 (en) 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
EP1064400A4 (fr) * 1998-03-13 2003-05-02 Promega Corp Detection d'acide nucleique
US6270974B1 (en) 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6653078B2 (en) 1998-03-13 2003-11-25 Promega Corporation Multiplex method for nucleic acid detection
US7090975B2 (en) 1998-03-13 2006-08-15 Promega Corporation Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
US6703211B1 (en) 1998-03-13 2004-03-09 Promega Corporation Cellular detection by providing high energy phosphate donor other than ADP to produce ATP
US6730479B2 (en) 1998-03-13 2004-05-04 Promega Corporation Detection of nucleic acid hybrids
WO2000049179A1 (fr) * 1999-02-18 2000-08-24 Promega Corporation Procedes de determination de la presence de sequences cibles d'acides nucleiques et leurs applications
WO2000049182A3 (fr) * 1999-02-18 2001-04-05 Promega Corp Methodes analytiques et materiaux permettant de detecter les acides nucleiques
EP2480689A4 (fr) * 2009-09-24 2013-05-15 Seegene Inc Détection de séquences d'acides nucléiques cibles au moyen de réactions exonucléolytiques cycliques
WO2023240564A1 (fr) * 2022-06-16 2023-12-21 深圳华大智造科技股份有限公司 Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques

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