WO1995030025A1 - Detection ou titrage d'acides nucleiques cibles - Google Patents

Detection ou titrage d'acides nucleiques cibles Download PDF

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Publication number
WO1995030025A1
WO1995030025A1 PCT/GB1995/000971 GB9500971W WO9530025A1 WO 1995030025 A1 WO1995030025 A1 WO 1995030025A1 GB 9500971 W GB9500971 W GB 9500971W WO 9530025 A1 WO9530025 A1 WO 9530025A1
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Prior art keywords
primers
amplification
nucleic acid
target nucleic
labelled
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PCT/GB1995/000971
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English (en)
Inventor
Ørjan OLSVIK
Jack Andreassen
Original Assignee
Dynal As
Dzieglewska, Hanna, Eva
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Publication date
Application filed by Dynal As, Dzieglewska, Hanna, Eva filed Critical Dynal As
Priority to AU23155/95A priority Critical patent/AU2315595A/en
Publication of WO1995030025A1 publication Critical patent/WO1995030025A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • This invention relates to the detection or assay of target nucleic acids, for example to assist identification of nucleic acid variations of medical or forensic interest.
  • nucleic acid analysis has provided a valuable diagnostic or forensic tool by permitting identification of mutant or variant nucleic acids, eg. in diagnosis of disease related mutations or in tissue typing. It is possible to identify extremely small quantities of target nucleic acids, sometimes a single molecule, by initial selective amplification followed by appropriate labelling to permit detection or quantification of the amplified material.
  • the polymerase chain reaction (PCR) has been used extensively in this way; other amplification systems include the Self-Sustained Sequence Replication (3SR) process, the Ligase Chain Reaction (LCR) , and the Nucleic Acid Sequence-based Amplification (NASBA) process.
  • 3SR Self-Sustained Sequence Replication
  • LCR Ligase Chain Reaction
  • NASBA Nucleic Acid Sequence-based Amplification
  • the present invention is based on the concept of carrying out the amplification by introducing the sample into a medium contained in a sealed container comprising all the components necessary for the procedure, including means for immobilising the amplified nucleic acid together with a labelled ligand so that withdrawal of the labelled ligand from solution by immobilisation provides an indication of the presence and/or quantity of target nucleic acid in the sample.
  • a method of detection or assay of a target nucleic acid in a sample wherein the sample is introduced into a medium containing one or more amplification enzymes and at least two amplification primers and subjected to at least one cycle of an amplification procedure such that any target nucleic, acid present is amplified, characterised in that at least one of said primers is immobilised or provided with means for immobilisation and another is labelled or provided with means for labelling whereby said primers are incorporated into amplified target nucleic acid which is thereby immobilised, if necessary by reaction with a solid phase reactive with said means for immobilisation, and labelled, if necessary by addition of a label reactive with said means for labelling, and the immobilised labelled primers are thereby removed from solution in said medium, followed by examination of the medium to detect the presence or quantity of any label left in solution and hence determine the presence, absence or quantity of target nucleic acid in the sample.
  • RNA Ribonucleic acid
  • DNA Ribonucleic Acid
  • PNA Peptide Nucleic Acid
  • the method of the invention may be illustrated in its simplest form in relation to the Ligase Chain Reaction (LCR) .
  • LCR Ligase Chain Reaction
  • a point mutation in a target nucleic acid is detected by use of two primers which hybridise to one strand of the nucleic acid over consecutive sequences of DNA such that the point mutation lies at the terminus of one of the primers.
  • one primer has a terminal base overlying the point mutation and if that terminal base is complementary to the underlying base of the point mutation, it will be possible to ligate the two primers using a suitable ligase. If, however, the terminal base is not complementary to the point mutation, ligation will not take place.
  • the ligation reaction may be carried out in the presence of one or more nucleotides to fill the gap.
  • two further primers are also provided for the complementary strand and, again, will permit ligation if the base of the point mutation is appropriate and there is no mismatch.
  • the double stranded DNA so produced may be subjected to strand separation, for example by heating, and the separate single strands may then serve as templates for further ligation primers. If this procedure is repeated, exponential production of ligated DNA takes place and the relevant region of the target DNA is amplified. On the other hand, if there is a mis-match at the site of the point mutation, no amplification will take place. Standard amplification conditions, well known and described in the literature may be used, and this applies to all the amplification procedures.
  • the means for immobilisation attached to one primer may conveniently be an affinity binding partner such as biotin or a hapten such as digoxigenin which binds to a corresponding binding partner for example avidin, streptavidin or an anti-hapten on a solid phase.
  • a solid phase may be, for example, the walls of the vessel containing the medium or a dipstick or, more preferably particles which can readily be separated from the medium.
  • the solid phase may be provided with a "particulate" or porous surface.
  • Superparamagnetic particles such as Dynabeads (Dynal AS, Oslo, Norway) are especially suitable since they permit the normal kinetics of the reaction to proceed unimpaired while allowing rapid and simple removal from solution using a magnet .
  • Labels for use in the method of the invention will, in general, be those giving a direct visual signal such as fluorescent molecules eg. fluorescein isothiocyanate (FTC) or chemiluminescent molecules such as acridinium esters.
  • fluorescent molecules eg. fluorescein isothiocyanate (FTC) or chemiluminescent molecules such as acridinium esters.
  • FTC fluorescein isothiocyanate
  • chemiluminescent molecules such as acridinium esters.
  • suitable labels are commercially available, including fcr example the fluorescent Chro aTide ® labels, consisting of fluorophore- and hapten-labelled NTPs useful for generating labelled nucleic acids (Molecular Probes, Inc., Eugene, OR, USA) or bisbenzimide fluorochromes available from Calbiochem, La Jolla, CA, USA.
  • labels include tris (2, 2 ' -bipyridine) -ruthenium (II) chelate (TBR) and synthetic or naturally occurring dyes and pigments.
  • Particles may be used as labels eg. colloidal gold particles in the size range 3-20 nm.
  • the label molecules may be attached directly to the DNA by covalent bonds, for example via 5'-amino or 5 ' -SH groups.
  • the label molecules may be attached to DNA-binding proteins such as the lad repressor which binds to the lac operon; the DNA sequence of the lac operon may thus be attached as a 'handle' to one of the primers, for example by chemical synthesis of the primer and handle, and the labelled repressor protein added to label the primer or the amplified DNA incorporating the primer.
  • DNA-binding proteins such as the lad repressor which binds to the lac operon
  • the DNA sequence of the lac operon may thus be attached as a 'handle' to one of the primers, for example by chemical synthesis of the primer and handle, and the labelled repressor protein added to label the primer or the amplified DNA incorporating the primer.
  • the sample may be any sample containing the target nucleic acid. It may for example be a laboratory sample or a clinical, biological or environmental sample, eg. serum or other body fluid or blood-derived product, faeces, food or allied products, cell suspension, cell culture, tissue homogenate or cell lysate etc. It may be desirable to dilute the sample or to submit a more crude or complex sample to one or more purification steps, for example to remove potential contaminants or substances which may interfere in the assay, such as haemoglobin.
  • a laboratory sample or a clinical, biological or environmental sample eg. serum or other body fluid or blood-derived product, faeces, food or allied products, cell suspension, cell culture, tissue homogenate or cell lysate etc. It may be desirable to dilute the sample or to submit a more crude or complex sample to one or more purification steps, for example to remove potential contaminants or substances which may interfere in the assay, such as haemoglobin.
  • the components of the reaction mixture will be provided in a suitable vessel adapted to permit visual inspection or photometric assay of any labelled primer dissolved in the medium.
  • the vessel is preferably sealed with a membrane which may be punctured by a syringe to introduce the sample.
  • a simple lid or cover may serve to avoid contamination.
  • the components in dried form, for example lyophilised form, and for water to be added, for example by injection through the above membrane, to dissolve the components.
  • the use of magnetic particles for immobilisation makes it possible to keep these separate from the other components by magnetic attraction to a locus above the level of any fluid in the reaction vessel.
  • the primers to be immobilised may carry ligands such as biotin and remain in solution throughout the amplification reaction before being contacted with the magnetic beads (coated, for example, with streptavidin) to effect immobilisation and ultimate withdrawal from the medium.
  • the means for immobilisation may be linked to the primer via a region of nucleic acid which does not hybridise to the target.
  • This may be a standardised oligonucleotide, such as oligodA which may readily be hybridised to a complementary oligonucleotide sequence, such as oligodT or an equivalent PNA sequence, which is immobilised or carries means for immobilisation.
  • oligodA oligodA
  • complementary oligonucleotide sequence such as oligodT or an equivalent PNA sequence
  • such oligonucleotides may separate and only recombine when hybridising conditions are restored or even only when the reaction mixture is cooled to room temperature after amplification.
  • the complementary oligonucleotide carrying means for immobilisation is of such a length as to be contiguous with the adjacent primer, the ligation reaction will serve to ligate not only the two primers but also the additional complementary oligonucleotide.
  • a gap between the oligonucleotide and the primer may be filled by effecting ligation in the presence of appropriate nucleotides.
  • the labelled primer will hybridise to the strand of the target nucleic acid complementary to that to which the immobilised primer hybridises; in this way all the amplified nucleic acid in double stranded form will be both immobilised and labelled. Since, however, the objective is merely to immobilise labelled primer in order to indicate the presence of the target nucleic acid, it is also possible for the labelled primer to hybridise to the same strand as the immobilised primer, although two primers for the complementary strand are needed for exponential amplification.
  • solid phase and/or label is to be attached to the primers throughout the amplification reaction
  • attachment is preferably covalent, for example via 5'-SH groups on terminal bases of the primers which may be bound to maleimide groups on the solid phase or 5'-amino groups which may bind to activated carboxyl groups on the solid phase.
  • PCR In PCR, only two primers are used for each cycle of amplification, one for each strand of target DNA and thus, for use in the present system, one of these should be immobilised or possess means for immobilisation while the other should be labelled or carry means for labelling.
  • the polymerase used in PCR is usually heat stable; Taq polymerase is most commonly used.
  • PCR is commonly effected in two series of cycles using nested primers, that is a first series of cycles may be effected with two primers not adapted for immobilisation or labelling to provide an initial amplification while a second series of cycles is effected using at least one further primer which hybridises to the DNA strands between the first primers, thereby amplifying a shorter sequence of the target DNA but with increased sensitivity due to the use of the further primer(s) which must hybridise accurately to the amplified DNA.
  • the second immobilising primer In the present system, it is possible to perform a second series of cycles using a second immobilising primer and the same labelled primer; in order to prevent this second primer from being involved in the first series of PCR cycles, the second immobilising primer must be held above the level of the medium and only introduced after the first series of cycles has been completed. This can be achieved by immobilising the second primer on magnetic beads which can be held magnetically in the upper part of the reaction vessel until required or by coating the upper region of the vessel with means reactive with the second primer and inverting the vessel at the end of the reaction.
  • the 3SR method of amplification has the advantage over PCR that it is isothermal (37-42°C) .
  • Double stranded target DNA is transcribed to RNA copies of the separate strands using RNA polymerase and each RNA copy is caused to hybridise to a DNA primer having a 5 ' - polymerase binding site permitting a DNA strand to be synthesised by the action of reverse transcriptase,- the RNA strand in each duplex is then digested away using RNase to leave two DNA strands (thus avoiding the strand separation step used in PCR which is normally effected by heating) .
  • each separate DNA strand is then caused to hybridise to a primer which is identical to that used to hybridise with the RNA copy generating the complementary DNA strand and a second pair of DNA strands is synthesised by the action of reverse transcriptase.
  • a primer which is identical to that used to hybridise with the RNA copy generating the complementary DNA strand and a second pair of DNA strands is synthesised by the action of reverse transcriptase.
  • a single double stranded DNA sequence produces two double stranded sequences which may then be transcribed by reverse transcriptase to pairs of RNA copies to begin the cycle again and thus permit exponential amplification.
  • the primers are incorporated into the amplified DNA and thus, in accordance with the method of the invention, one primer may be immobilised or possess means for immobilisation and one may be labelled or possess means for labelling.
  • RNA polymerase In order to amplify target RNA, one may simply perform one 'limb' of the 3SR method described above, using the same primers, to convert the single stranded RNA to double stranded DNA which will then be transcribed by RNA polymerase as described above, to a pair of RNA copies to initiate amplification.
  • the amplification is preferably effected in a sealed vessel into which only the sample and possibly water, aqueous buffer, or other suitable medium, are introduced.
  • contamination is less serious after amplification is completed and components such as labelled ligand reactive with one of the primers incorporated into the amplified nucleic acid may be added at this stage. This enables the amplification procedure to be effected in the absence of the label which may be a relatively large molecule which might exert steric effects on the reaction if attached to a primer throughout.
  • primers are provided with means for subsequent attachment of solid phase and label respectively, these must naturally be different.
  • high affinity binding systems such as biotin and avidin or streptavidin are best for immobilisation.
  • One particularly suitable means of attaching a label is via a DNA binding protein as described above.
  • the solid phase and label are attached to the primers, however, they are preferably bound covalently in order to survive the heating stages of the PCR and LCR reactions .
  • the quantity of labelled reagent introduced into the system should, in general, be calculated to be slightly less than that expected to be removed by the intended number of amplification cycles if target nucleic acid is present. However, if the reaction is effected under standard conditions, it may be possible to quantify the amount of target nucleic acid in the sample by using a slight excess of label and determining the amount remaining after magnetic separation.
  • reaction quantities for an LCR reaction, for example 10 pmoles of the non-labelled amplification primer and 1 pmole of the labelled primer may be used.
  • detection primer for the above-mentioned amplification primer quantities, up to 0.1 pmole of the detection primer would suffice. Conditions and quantities may however be varied according to choice and the methods and systems being used or investigated. The important consideration is that all the labelled primer should hybridise to the target sequence, if it is present.
  • the components for the reaction may be provided in a vessel permitting light to pass through the medium in a region from which the immobilised amplified DNA has been withdrawn.
  • a cuvette of the type used in photometry or a microtitre well (or array of wells) may be suitable. If the illumination is along one axis of the vessel, the amplified DNA may be immobilised (magnetically or otherwise) to one of the walls parallel to that axis to leave a clear light path.
  • a vessel containing one or more amplification enzymes, a primer which is immobilised or carries means for immobilisation, a primer which is labelled or carries means for attachment of a label, any further primers which are required and any nucleotide bases which are required.
  • the vessel will preferably be sealed by a membrane permitting introduction of the sample and any water necessary by a syringe.
  • a vessel may, for example, be a tube or a microtitre well and may conveniently be transparent along at least one axis, or at one end.
  • the components will preferably be in dry form, eg. lyophilised form.
  • a supply of high purity water or other suitable medium may be provided for introduction into the reaction vessel conveniently in a solid disposable syringe when the vessel is sealed by a membrane.
  • a permanent magnet eg. a neodymium based magnet
  • the magnet may be used to draw the particles to an upper region of the vessel .
  • Fig. 1 shows the reaction scheme of the invention using the Ligase Chain Reaction (LCR) ;
  • Fig. 2 shows diagrammatically the procedure using LCR.
  • a transparent vessel is provided containing an aqueous buffer, pH 7.0, a thermostable ligase and oligonucleotide primers P 1 , P 2 , P 3 and P 4 as well as oligonucleotides P 5 attached to Dynabeads (Dynal AS, Oslo) via biotin/streptavidin binding and P 6 , attached to a fluorescent label.
  • P 5 hybridises to the 'handle' (cross-hatched) region of P 1 while P 6 hybridises to the 'handle' region of P 4 .
  • a sample containing target double stranded DNA is introduced and subjected to strand separation by heating to 95°C.
  • the oligonucleotides P x -P 4 hybridise to the target DNA and P 5 and P 6 hybridise to P 1 and P 4 .
  • the thermostable ligase then effects ligation to form double stranded DNA.
  • the double stranded DNA is subjected to strand separation, whereupon the newly ligated strands and the target DNA are available as templates for a further ligation reaction.
  • Fig. 2 shows diagrammatically the use of M28 magnetic beads (Dynal AS) to effect magnetic separation after LCR.
  • A the label (chromatophor) and M28 beads are distributed throughout the medium and a photometric reading shows the absorbance produced by the chromatophor.
  • B the M28 beads and chromatophor are joined by incorporation into amplified DNA and are removed by application of a magnet so that the photometer gives low or zero absorbance.
  • C there has been no amplification due to the absence of target DNA and the M28 beads are not bound to the chromatophor which thus remains distributed throughout the medium and gives a similar photometric reading to A.
  • a positive control tube would probably be run employing a known amount of target DNA and a negative control would be run using irrelevant DNA.
  • the gene encoding the heat-labile enterotoxin (LTh or LTlb) located on plasmid(s) in enterotoxigenic Escherichia coli strains was used as target DNA.
  • the target sequences were located in position (PI) 46 to 65 (and P3 rev) and (LCR P2) 65 to 84 (and LCR P4 rev) using the gene sequences and numbers published by Leong et al. (Infect Immun 48: 73-77, 1985) .
  • the positive control (LTlb pos) was E. coli strain ATCC 43886, and the negative control was E. coli strain ATCC 43922.
  • control reference assay was a PCR procedure described by Olsvik and Strokbine (pp 271-276 In Persing et al . , Diagnostic Molecular Microbiology, ISBN 1-55581-056-X, 1993) .
  • Test strains (6 positive and 6 negative enteric E. coli isolates) were taken from the laboratory collection of the Enteric Diseases Laboratory Section, Centers for Diseases Control and Prevention, Atlanta, GA.
  • Oligonucleotide LCR PI had an 18 oligonucleotide tail with sequences from the E. coli lac operon (Homes et al., J. Clin. Microbiol. 29: 2375-2379, 1991) , and P4 a 20 oligonucleotide tail from the E. coli SLT 1 operon, 215-234 (Olsvik and Strokbine supra, pp 271-276) .
  • a reverse oligonucleotide to the lac tail (P5 trap) was synthesized with a 5 ' -biotin molecule, and a reverse oligonucleotide to the SLT1 tail (P6 signal) was 5'- labelled with fluorescein.
  • test organisms one small colony of each were added directly to clear glass tubes and preheated for 2 minutes as described by Fields et al. (J. Clin. Microbiol. 30: 2117-2121, 1992) , to effect lysis and DNA liberation.
  • the LCR reaction was then carried out immediately in situ using the ligase, buffers and cycling conditions described by Cernesky et al. (J. Infect. Dis. 170: 1308-1311, 1994) , except that 100 micro gram Dynabeads, Streptavidin coated, was added prior to LCR amplification.
  • the tube was then placed in a magnetic rack to draw the magnetic beads to the side of the tube and the intensity of the fluorescein in the central volume of the tube was observed.

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Abstract

L'invention concerne un procédé de détection ou de titrage d'un acide nucléique cible présent dans un échantillon, où ce dernier est introduit dans un milieu, contenant une ou plusieurs enzymes d'amplification et au moins deux amorces d'amplification, soumis à un cycle au moins d'une procédure d'amplification pour amplifier tout acide nucléique cible présent. Ce procédé se caractérise en ce qu'une des amorces au moins est immmobilisée ou munie d'un dispositif d'immobilisation et une autre est marquée on munie d'un dispositif de marquage. Ces amorces sont incorporées dans un acide nucléique cible amplifié qui se trouve ainsi immobilisé, si nécessaire par réaction avec une phase solide pouvant réagir avec le dispositif d'immobilisation, et marqué, si nécessaire par addition d'un marqueur pouvant réagir avec ce dispositif de marquage. Les amorces immobilisées et marquées sont alors retirées de la solution présente dans ce milieu, puis on soumet ce dernier à un examen pour détecter la présence ou la quantité de tout marqueur subsistant dans cette solution, après quoi on détermine la présence, l'absence ou la quantité d'acide nucléique cible présent dans l'échantillon. Un autre aspect de l'invention concerne une cuve étanche contenant les composantes nécessaires pour ce procédé de titrage ou de détection.
PCT/GB1995/000971 1994-04-29 1995-04-28 Detection ou titrage d'acides nucleiques cibles WO1995030025A1 (fr)

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GB9408607A GB9408607D0 (en) 1994-04-29 1994-04-29 Assay

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007235A2 (fr) * 1995-08-14 1997-02-27 Abbott Laboratories Methode integrale d'amplification d'acide nucleique
US5912129A (en) * 1998-03-05 1999-06-15 Vinayagamoorthy; Thuraiayah Multi-zone polymerase/ligase chain reaction
GB2346145A (en) * 1998-10-22 2000-08-02 Stuart Harbron Detection of amplified products in nucleic acid assays

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EP0154505A2 (fr) * 1984-02-28 1985-09-11 Ortho Diagnostic Systems Inc. Diagnostic d'anomalies d'un gène par rapport à la carte d'enzyme de restriction utilisant l'hybridation en sandwich à cette fin
EP0237833A2 (fr) * 1986-03-05 1987-09-23 Miles Inc. Test d'hybridation en phase liquide pour détecter des séquences polynucléotidiques
EP0362042A1 (fr) * 1988-09-26 1990-04-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé d'analyse d'une séquence spécifique d'ADN ou d'ARN, réactifs et nécessaires pour sa mise en oeuvre
WO1990006042A2 (fr) * 1988-11-21 1990-06-14 Dynal As Detection et determination quantitative d'arn et d'adn
WO1990011369A1 (fr) * 1989-03-22 1990-10-04 Cemu Bioteknik Ab Diagnostic en phase solide de conditions medicales
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome
WO1992016659A1 (fr) * 1991-03-21 1992-10-01 Eastman Kodak Company Element et procede pour l'amplification et la detection de l'acide nucleique au moyen de sondes collees
WO1993010267A1 (fr) * 1991-11-15 1993-05-27 Igen, Inc. Dosages rapides pour produits d'amplification
WO1993015222A1 (fr) * 1992-01-29 1993-08-05 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede pour eviter les risques de contamination, notamment applicable aux techniques d'amplification de l'adn ou de l'arn
WO1994002634A1 (fr) * 1992-07-24 1994-02-03 University Of South Australia Procede d'amplification et de detection

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154505A2 (fr) * 1984-02-28 1985-09-11 Ortho Diagnostic Systems Inc. Diagnostic d'anomalies d'un gène par rapport à la carte d'enzyme de restriction utilisant l'hybridation en sandwich à cette fin
EP0237833A2 (fr) * 1986-03-05 1987-09-23 Miles Inc. Test d'hybridation en phase liquide pour détecter des séquences polynucléotidiques
EP0362042A1 (fr) * 1988-09-26 1990-04-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé d'analyse d'une séquence spécifique d'ADN ou d'ARN, réactifs et nécessaires pour sa mise en oeuvre
WO1990006042A2 (fr) * 1988-11-21 1990-06-14 Dynal As Detection et determination quantitative d'arn et d'adn
WO1990011369A1 (fr) * 1989-03-22 1990-10-04 Cemu Bioteknik Ab Diagnostic en phase solide de conditions medicales
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome
WO1992016659A1 (fr) * 1991-03-21 1992-10-01 Eastman Kodak Company Element et procede pour l'amplification et la detection de l'acide nucleique au moyen de sondes collees
WO1993010267A1 (fr) * 1991-11-15 1993-05-27 Igen, Inc. Dosages rapides pour produits d'amplification
WO1993015222A1 (fr) * 1992-01-29 1993-08-05 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede pour eviter les risques de contamination, notamment applicable aux techniques d'amplification de l'adn ou de l'arn
WO1994002634A1 (fr) * 1992-07-24 1994-02-03 University Of South Australia Procede d'amplification et de detection

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007235A2 (fr) * 1995-08-14 1997-02-27 Abbott Laboratories Methode integrale d'amplification d'acide nucleique
WO1997007235A3 (fr) * 1995-08-14 1997-03-20 Abbott Lab Methode integrale d'amplification d'acide nucleique
US5912129A (en) * 1998-03-05 1999-06-15 Vinayagamoorthy; Thuraiayah Multi-zone polymerase/ligase chain reaction
GB2346145A (en) * 1998-10-22 2000-08-02 Stuart Harbron Detection of amplified products in nucleic acid assays
GB2346145B (en) * 1998-10-22 2001-01-24 Stuart Harbron Detection of amplified products in nucleic acid assays

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