WO2002077273A1 - Gene typing method without dna extraction - Google Patents

Gene typing method without dna extraction Download PDF

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WO2002077273A1
WO2002077273A1 PCT/FR2002/001016 FR0201016W WO02077273A1 WO 2002077273 A1 WO2002077273 A1 WO 2002077273A1 FR 0201016 W FR0201016 W FR 0201016W WO 02077273 A1 WO02077273 A1 WO 02077273A1
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preparation
carried out
heating
genotypic analysis
minutes
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PCT/FR2002/001016
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French (fr)
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Philippe Amouyel
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Institut Pasteur De Lille
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Publication of WO2002077273A1 publication Critical patent/WO2002077273A1/en

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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a rapid genotyping method, without prior chemical extraction of DNA.
  • the genotyping of individuals consists of the analysis of their genotype, and notably includes the determination of polymorphisms.
  • SNP single nucleotide polymorphism
  • Genotyping studies are generally carried out on genomic DNA extracted, for example from blood leukocytes, by conventional methods of DNA extraction.
  • proteases In addition, they generally use proteases, the cost of which is not negligible.
  • the nucleic acid which serves to identify the genotype has not undergone chemical extraction.
  • chemical extraction is meant any step based on the use of the differential solubility of the molecules (nucleic acid and contaminants) between two immiscible phases, or on the use of the differential reactivity of these molecules with respect to a chemical agent.
  • extraction therefore includes the conventional precipitation of nucleic acids in the presence of a precipitation agent of ethanol or isopropanol type for example.
  • the present invention therefore relates to a method of genotyping a nucleic acid without chemical extraction step, comprising or consisting of: - bringing a cell preparation into the presence of a detergent agent, which allows the lysis of cells; heating the preparation obtained, then centrifuging it; the recovered supernatant, which comprises nucleic acids, being finally subjected to genotypic analysis.
  • the method of the invention does not require the addition of proteases, which further reduces DNA preparation time and lowers the overall cost of the method.
  • the method of the invention also does not require the use of stabilizers of the high molecular material type, such as proteins or gelatin, or also cellulose or polyethylene glycol, described for example in application EP 428 197.
  • stabilizers of the high molecular material type such as proteins or gelatin, or also cellulose or polyethylene glycol, described for example in application EP 428 197.
  • the method of the invention does not use polysaccharides either, as is the case for example with the method of application EP 620,282.
  • the cell preparation serving as starting material can for example be a blood sample total, or a preparation of blood leukocytes. It can also be tissues, such as tissue biopsies but also smears, in particular endo-buccal.
  • the detergent agents which can be used are well known to those skilled in the art. Examples include SDS (Sodium Dodecyl Sulfate) or sarcosyl.
  • the cell preparation can be incubated with Lauryl sarcosyl in red blood cell fyse buffer (7.7 mM Tris HCI, MgCI 2 , 6H 2 0 5mM, 10 mM NaCl, pH 7.6).
  • no other ingredient is added to this mixture to stabilize the nucleic acids or to degrade cell membranes or proteins.
  • the nucleic acid preparation is incubated at a temperature of about 70 to 100 ° C, preferably 85-95 ° C, more preferably 90 ° C.
  • An incubation time of the order of 30 minutes to 1 hour is suitable, for example around 40 minutes.
  • the preparation can be incubated at 90 ° C for 40 minutes.
  • centrifugation The conditions of the following centrifugation are adapted to precipitate the contaminants (such as cell debris) in the form of a pellet and leave the nucleic acid in the supernatant.
  • a centrifugation of the order of 5 to 15 minutes (preferably 10 minutes) between approximately 4000 and 7000g (preferably approximately 5600g) is particularly suitable.
  • the recovered supernatant which includes the nucleic acids, can be directly analyzed. These nucleic acids are essentially formed by genomic DNA.
  • Genotypic analysis is carried out according to methods known to those skilled in the art.
  • a determination of alleles can for example be carried out after amplification (preferably of the PCR type, chain amplification by a polymerase) of the DNA region in which there is a polymorphism of interest.
  • amplification preferably of the PCR type, chain amplification by a polymerase
  • the amplification products can then be separated in a matrix, for example by electrophoresis on an agarose gel, the polymorphism of interest being then easily detected according to standard techniques.
  • Genotypic analysis of SNPs by mass spectrometry can also be performed (Sauer et al, 2000). It is based on the primer extension method which generates products with a specific mass for each allele. The alleles are distinguished by their difference in mass, which is easy to interpret automatically by computer.
  • Genotypic analysis can also be carried out by implementing sequencing, preferably automatic.
  • the method of the invention is not only very fast, but is also very sensitive. It is therefore particularly suitable for any clinical application which would require the reliable and rapid determination of the genotype of a patient.
  • the method of the invention can for example be used to identify the D alleles of the ACE gene, in relation to a risk of restenosis after the installation of a stent. It can also be useful for determining mutations in tumor cells, by analyzes carried out on samples of tumor cells, during a surgical intervention. The determination of these mutations can then orient the surgeon's gesture, depending on the origin of the cancer thus identified.
  • the method of the invention also has an interesting practical and commercial aspect: inexpensive, it can be implemented on cell samples from subjects whose DNA does not need to be stored.
  • genotyping of alleles of the APOE gene for the purpose of prognosis of the risk of developing Alzheimer's disease (FR 2 716 894).
  • Genotyping for the ACE gene coding for the angiotensin converting enzyme was carried out on 345 patients who underwent coronary stent implantations, in the 12 to 24 hours preceding the analysis. 15 ⁇ l. whole blood taken from EDTA anticoagulant were mixed with 60 ⁇ l of 0.5% Lauryl sarcosyl in red blood cell lysis buffer (7.7 mM Tris HCI, MgCI 2 , 6H 2 O 5m, 10 mM NaC1 , pH 7.6).
  • the ACE fragment containing the desired polymorphism was amplified using a DNA thermocycler from Perkin-Elmer, using DNA polymerase from Thermus ' aquaticus (Amersham).
  • the ACE polymorphism was detected as described previously in the article by Tiret et al, 1992, except for the addition of DMSO to increase the amplification of the ACE I allele (Fogarty et al, 1994; Shanmugam et al , 1993).
  • the reaction products were analyzed by electrophoresis on an agarose gel for allelic identification.
  • the patients were also genotyped with a three-primer system (Hamon et al, 1995), for confirmation.
  • the amplification products were deposited on electrophoresis gels (NuSieve agarose at 4% FMC Bioproducts).
  • the rapid genotyping method made it possible to obtain results in only about 4 hours.
  • the rapid DD carrier detection test had a sensitivity of 100%, a specificity of 98.7%, a positive predictive value of 97.4% and a negative predictive value of 100%.
  • Patients with a DD genotype for CEA have a higher risk of restenosis after implantation of the stent. Quickly genotypes, these patients will be able to benefit from preventive therapy as soon as possible.

Abstract

The invention concerns a fast gene typing method without chemical DNA extraction, comprising contacting a cell preparation with a detergent agent, thereby enabling the cells to be lyzed, then heating the resulting preparation and centrifuging it, and finally subjecting the recovered supernatant to a genotypic analysis.

Description

Méthode de génotypage sans extraction de l'ADN Genotyping method without DNA extraction
L'invention a trait à une méthode de génotypage rapide, sans extraction chimique préalable de l'ADN. Le génotypage d'individus consiste en l'analyse de leur génotype, et comprend notamment la détermination de polymorphismes. Aujourd'hui de nombreux polymorphismes de l'ADN, répartis sur l'ensemble du génome, ont été identifiés. Ces mutations, fonctionnelles ou non, traduisant la diversité de chaque individu, sont recensées dans des banques de données accessibles, par exemple auprès du National Center for Biotechnology Information, et dans lesquelles les généticiens puisent pour localiser des gènes de maladies. Il s'agit le plus fréquemment de polymorphismes nucléotidiques (« single nucleotide polymorphisme », SNP). Ces analyses génotypiques peuvent être mises à profit pour évaluer le risque qu'un individu développe une maladie (Lathrop et al, 2000) ou pour adapter les protocoles thérapeutiques au profil génotypique d'un patient (Amouyel, 2000).The invention relates to a rapid genotyping method, without prior chemical extraction of DNA. The genotyping of individuals consists of the analysis of their genotype, and notably includes the determination of polymorphisms. Today, numerous DNA polymorphisms, distributed throughout the genome, have been identified. These mutations, functional or not, reflecting the diversity of each individual, are listed in accessible databases, for example from the National Center for Biotechnology Information, and from which geneticists draw to locate disease genes. They are most frequently nucleotide polymorphisms ("single nucleotide polymorphism", SNP). These genotypic analyzes can be used to assess the risk that an individual develops a disease (Lathrop et al, 2000) or to adapt therapeutic protocols to the genotypic profile of a patient (Amouyel, 2000).
Les études de génotypage sont généralement réalisées sur de l'ADN génomique extrait, par exemple à partir de leucocytes sanguins, par des procédés classiques d'extraction de l'ADN. L'article Amant et al, 1997, qui rapporte une analyse des polymorphismes du gène ACE (enzyme de conversion de l'angiotensine), fait ainsi référence à des protocoles de préparation de l'ADN en routine ( arcadet et al, 1987).Genotyping studies are generally carried out on genomic DNA extracted, for example from blood leukocytes, by conventional methods of DNA extraction. The article Amant et al, 1997, which reports on an analysis of the polymorphisms of the ACE gene (angiotensin converting enzyme), thus refers to protocols for routine DNA preparation (arcadet et al, 1987).
Ces procédés d'extraction des acides nucléiques ont toutefois l'inconvénient d'être relativement longs à réaliser (entre 24 et 48 heures généralement).These nucleic acid extraction methods, however, have the disadvantage of being relatively long to perform (generally between 24 and 48 hours).
En outre, ils mettent généralement en œuvre des protéases, dont le coût n'est pas négligeable.In addition, they generally use proteases, the cost of which is not negligible.
Or, il serait avantageux que des résultats de génotypage puissent être obtenus très rapidement, et à un coût raisonnable, par exemple à des fins de diagnostic ou pour adapter le traitement au patient dont le génotype a été identifié. Les auteurs de la présente invention ont maintenant mis au point une méthode de génotypage qui résout ce problème simplement. Conformément à l'invention, l'acide nucléique qui sert à l'identification du génotype n'a pas subi d'extraction chimique. En particulier aucune extraction au phénol, au chloroforme, à l'éther ou à l'isobutanol n'est réalisée. Par « extraction chimique », on entend toute étape basée sur l'utilisation de la solubilité différentielle des molécules (acide nucléique et contaminants) entre deux phases non miscibles, ou sur l'utilisation de la réactivité différentielle de ces molécules vis à vis d'un agent chimique. Au sens de l'invention, le terme « extraction » comprend donc la précipitation classique des acides nucléiques en présence d'un agent de précipitation de type éthanol ou isopropanol par exemple.However, it would be advantageous if genotyping results could be obtained very quickly, and at a reasonable cost, for example for diagnostic purposes or to adapt the treatment to the patient whose genotype has been identified. The authors of the present invention have now developed a genotyping method which solves this problem simply. In accordance with the invention, the nucleic acid which serves to identify the genotype has not undergone chemical extraction. In particular, no extraction with phenol, chloroform, ether or isobutanol is carried out. By “chemical extraction” is meant any step based on the use of the differential solubility of the molecules (nucleic acid and contaminants) between two immiscible phases, or on the use of the differential reactivity of these molecules with respect to a chemical agent. Within the meaning of the invention, the term “extraction” therefore includes the conventional precipitation of nucleic acids in the presence of a precipitation agent of ethanol or isopropanol type for example.
En fait, les auteurs de l'invention ont supprimé ces étapes usuelles d'extraction, et les ont remplacées par un simple chauffage suivi d'une centrifugation.In fact, the authors of the invention have eliminated these usual extraction steps, and replaced them by simple heating followed by centrifugation.
La présente invention a donc pour objet une méthode de génotypage d'un acide nucléique sans étape d'extraction chimique, comprenant ou consistant en : - la mise en présence d'une préparation cellulaire avec un agent détergent, qui permet la lyse des cellules ; le chauffage de la préparation obtenue, puis sa centrifugation ; le surnageant récupéré, qui comprend des acides nucléiques, étant enfin soumis à une analyse génotypique.The present invention therefore relates to a method of genotyping a nucleic acid without chemical extraction step, comprising or consisting of: - bringing a cell preparation into the presence of a detergent agent, which allows the lysis of cells; heating the preparation obtained, then centrifuging it; the recovered supernatant, which comprises nucleic acids, being finally subjected to genotypic analysis.
Aucune étape d'extraction chimique, ni de chromatographie ou autre traitement des préparations cellulaires n'est nécessaire. L'intérêt principal de la méthode de l'invention est donc son extrême simplicité.No chemical extraction, chromatography or other processing of the cell preparations is necessary. The main interest of the method of the invention is therefore its extreme simplicity.
De manière notable, et contrairement à la plupart des procédés classiques d'extraction, la méthode de l'invention ne nécessite pas d'ajout de protéases, ce qui permet de réduire encore le temps de préparation de l'ADN et diminue le coût global de la méthode.Significantly, and unlike most conventional extraction methods, the method of the invention does not require the addition of proteases, which further reduces DNA preparation time and lowers the overall cost of the method.
La méthode de l'invention ne nécessite également pas l'utilisation de stabilisateurs de type matériaux de haut poids moléculaire, tels que des protéines ou de la gélatine, ou encore de la cellulose ou du polyéthylène glycol, décrits par exemple dans la demande EP 428 197.The method of the invention also does not require the use of stabilizers of the high molecular material type, such as proteins or gelatin, or also cellulose or polyethylene glycol, described for example in application EP 428 197.
La méthode de l'invention ne met pas non plus en oeuvre de polysaccharides, comme c'est par exemple le cas de la méthode de la demande EP 620 282. La préparation cellulaire servant de matériel de départ peut être par exemple un échantillon de sang total, ou une préparation de leucocytes sanguins. Il peut également s'agir de tissus, tels que des biopsies tissulaires mais aussi des frottis, notamment endo-bucaux.The method of the invention does not use polysaccharides either, as is the case for example with the method of application EP 620,282. The cell preparation serving as starting material can for example be a blood sample total, or a preparation of blood leukocytes. It can also be tissues, such as tissue biopsies but also smears, in particular endo-buccal.
Les agents détergents utilisables sont bien connus de l'homme du métier. On peut par exemple citer le SDS (Sodium Dodecyl Sulfate) ou le sarcosyl. Par exemple, la préparation cellulaire peut être incubée avec du Lauryl sarcosyl dans du tampon de fyse de globuie rouge (Tris HCI 7,7mM, MgCI2, 6H20 5mM, NaCI 10 mM, pH 7,6). De préférence, on n'ajoute à ce mélange aucun autre ingrédient pour stabiliser les acides nucléiques ou dégrader les membranes cellulaires ou les protéines.The detergent agents which can be used are well known to those skilled in the art. Examples include SDS (Sodium Dodecyl Sulfate) or sarcosyl. For example, the cell preparation can be incubated with Lauryl sarcosyl in red blood cell fyse buffer (7.7 mM Tris HCI, MgCI 2 , 6H 2 0 5mM, 10 mM NaCl, pH 7.6). Preferably, no other ingredient is added to this mixture to stabilize the nucleic acids or to degrade cell membranes or proteins.
Conformément à la présente invention, la préparation d'acide nucléique est incubée à une température d'environ 70 à 100°C, de préférence 85-95°C, de préférence encore 90°C.In accordance with the present invention, the nucleic acid preparation is incubated at a temperature of about 70 to 100 ° C, preferably 85-95 ° C, more preferably 90 ° C.
Un temps d'incubation de l'ordre de 30 minutes à 1 heure convient, par exemple 40 minutes environ.An incubation time of the order of 30 minutes to 1 hour is suitable, for example around 40 minutes.
De manière avantageuse, on peut effectuer une incubation de la préparation à 90°C pendant 40 minutes.Advantageously, the preparation can be incubated at 90 ° C for 40 minutes.
Les conditions de la centrifugation qui suit sont adaptées pour précipiter les contaminants (tels que des débris cellulaires) sous la forme d'un culot et laisser l'acide nucléique dans le surnageant. Une centrifugation de l'ordre de 5 à 15 minutes (de préférence 10 minutes) entre approximativement 4000 et 7000g (de préférence 5600g environ ) convient particulièrement.The conditions of the following centrifugation are adapted to precipitate the contaminants (such as cell debris) in the form of a pellet and leave the nucleic acid in the supernatant. A centrifugation of the order of 5 to 15 minutes (preferably 10 minutes) between approximately 4000 and 7000g (preferably approximately 5600g) is particularly suitable.
Le surnageant récupéré, qui comprend les acides nucléiques, peut être directement analysé. Ces acides nucléiques sont essentiellement formés par de l'ADN génomique.The recovered supernatant, which includes the nucleic acids, can be directly analyzed. These nucleic acids are essentially formed by genomic DNA.
L'analyse génotypique est réalisée selon les méthodes connues de l'homme du métier.Genotypic analysis is carried out according to methods known to those skilled in the art.
Une détermination d'allèles peut être par exemple réalisée après amplification (de préférence du type PCR, amplification en chaîne par une polymérase) de la région d'ADN dans laquelle existe un polymorphisme d'intérêt. Pour cela, on peut créer dans une amorce de PCR un site de clivage pour une enzyme de restriction, n'existant pas chez les individus porteurs d'un des allèles. On peut également utiliser une technique d'hybridation utilisant des sondes oligonucléotidiques spécifiques des allèles. Les produits d'amplification peuvent ensuite être séparés dans une matrice, par exemple par électrophorèse sur un gel d'agarose, le polymorphisme d'intérêt étant ensuite aisément détecté selon les techniques standards.A determination of alleles can for example be carried out after amplification (preferably of the PCR type, chain amplification by a polymerase) of the DNA region in which there is a polymorphism of interest. For this, it is possible to create in a PCR primer a cleavage site for a restriction enzyme, which does not exist in individuals carrying one of the alleles. It is also possible to use a hybridization technique using allele-specific oligonucleotide probes. The amplification products can then be separated in a matrix, for example by electrophoresis on an agarose gel, the polymorphism of interest being then easily detected according to standard techniques.
Une analyse génotypique des SNP par spectrométrie de masse peut également être réalisée (Sauer et al, 2000). Elle repose sur la méthode d'extension d'amorces qui génère des produits ayant une masse spécifique pour chaque allèle. Les allèles se distinguent par leur différence de masse, facile à interpréter automatiquement par ordinateur.Genotypic analysis of SNPs by mass spectrometry can also be performed (Sauer et al, 2000). It is based on the primer extension method which generates products with a specific mass for each allele. The alleles are distinguished by their difference in mass, which is easy to interpret automatically by computer.
L'analyse génotypique peut également être réalisée en mettant en œuvre un séquençage, de préférence automatique. La méthode de l'invention s'avère non seulement très rapide, mais est aussi d'une grande sensibilité. Elle est donc particulièrement adaptée pour toute application clinique qui nécessiterait la détermination fiable et rapide du génotype d'un patient. La méthode de l'invention peut par exemple être mise à profit pour identifier les allèles D du gène ACE, en relation avec un risque de resténose après la pose d'une endoprothèse vasculaire. Elle peut également être utile pour déterminer des mutations dans des cellules tumorales, par des analyses réalisées sur des prélèvements de cellules tumorales, au cours d'une intervention chirurgicale. La détermination de ces mutations peut alors orienter le geste du chirurgien, selon l'origine du cancer ainsi identifiée.Genotypic analysis can also be carried out by implementing sequencing, preferably automatic. The method of the invention is not only very fast, but is also very sensitive. It is therefore particularly suitable for any clinical application which would require the reliable and rapid determination of the genotype of a patient. The method of the invention can for example be used to identify the D alleles of the ACE gene, in relation to a risk of restenosis after the installation of a stent. It can also be useful for determining mutations in tumor cells, by analyzes carried out on samples of tumor cells, during a surgical intervention. The determination of these mutations can then orient the surgeon's gesture, depending on the origin of the cancer thus identified.
La méthode de l'invention revêt en outre un aspect pratique et commercial intéressant : peu coûteuse, elle peut être mise en œuvre sur des prélèvements cellulaires de sujets dont il n'est pas nécessaire de stocker l'ADN. Parmi les exemples non limitatifs d'applications, on peut citer notamment le génotypage d'allèles du gène APOE, à des fins de pronostic du risque de développer la maladie d'Alzheimer (FR 2 716 894).The method of the invention also has an interesting practical and commercial aspect: inexpensive, it can be implemented on cell samples from subjects whose DNA does not need to be stored. Among the nonlimiting examples of applications, there may be mentioned in particular the genotyping of alleles of the APOE gene, for the purpose of prognosis of the risk of developing Alzheimer's disease (FR 2 716 894).
L'exemple suivant illustre l'invention sans en limiter la portée.The following example illustrates the invention without limiting its scope.
EXEMPLE : Génotypage ACEEXAMPLE: ACE Genotyping
Un génotypage pour le gène ACE codant pour l'enzyme de conversion de l'angiotensine a été réalisé sur 345 patients ayant subi des implantations de stent coronarien, dans les 12 à 24 heures précédant l'analyse. 15 μl. de sang total prélevés sur anti-coagulant EDTA ont été mélangés, à 60 μl de Lauryl sarcosyl à 0,5% dans du tampon de lyse de globules rouges (Tris HCI 7,7mM, MgCI2, 6H2O 5m , NaC1 10 mM, pH 7,6).Genotyping for the ACE gene coding for the angiotensin converting enzyme was carried out on 345 patients who underwent coronary stent implantations, in the 12 to 24 hours preceding the analysis. 15 μl. whole blood taken from EDTA anticoagulant were mixed with 60 μl of 0.5% Lauryl sarcosyl in red blood cell lysis buffer (7.7 mM Tris HCI, MgCI 2 , 6H 2 O 5m, 10 mM NaC1 , pH 7.6).
Les échantillons ont ensuite été incubés 40 minutes à 90°C, puis centrifugés 10 minutes à 5600g. Une fraction de 5 μl de surnageant a été utilisée pour l'amplification et déterminer le génotype ACE. Cette analyse a été réalisée selon le protocole décrit dans l'article d'Amant et al, 1997.The samples were then incubated for 40 minutes at 90 ° C, then centrifuged for 10 minutes at 5600g. A fraction of 5 μl of supernatant was used for the amplification and to determine the ACE genotype. This analysis was carried out according to the protocol described in the article by Amant et al, 1997.
Brièvement, le fragment ACE contenant le polymorphisme recherché (ID) a été amplifié au moyen d'un thermocycleur d'ADN de Perkin- Elmer, à l'aide de l'ADN polymérase de Thermus' aquaticus (Amersham). Le polymorphisme ACE a été détecté comme décrit précédemment dans l'article de Tiret et al, 1992, à l'exception de l'addition de DMSO pour augmenter l'amplification de l'allèle ACE I (Fogarty et al, 1994 ; Shanmugam et al, 1993). Les produits de réaction ont été analysés par électrophorèse sur un gel d'agarose pour l'identification allélique. Les patients ont été également génotypes avec un système à trois amorces (Hamon et al, 1995), pour confirmation. Les produits d'amplification ont été déposés sur des gels d'électrophorèse (agarose NuSieve à 4% FMC Bioproducts).Briefly, the ACE fragment containing the desired polymorphism (ID) was amplified using a DNA thermocycler from Perkin-Elmer, using DNA polymerase from Thermus ' aquaticus (Amersham). The ACE polymorphism was detected as described previously in the article by Tiret et al, 1992, except for the addition of DMSO to increase the amplification of the ACE I allele (Fogarty et al, 1994; Shanmugam et al , 1993). The reaction products were analyzed by electrophoresis on an agarose gel for allelic identification. The patients were also genotyped with a three-primer system (Hamon et al, 1995), for confirmation. The amplification products were deposited on electrophoresis gels (NuSieve agarose at 4% FMC Bioproducts).
Pour plus de détails concernant la technique de détection des polymorphismes de l'ACE, l'homme du métier pourra se reporter utilement à la description de la demande WO 90/0345, et plus généralement aux principes décrits dans les brevets US 4,683,202 et US 4,683, 195.For more details concerning the technique for detecting ACE polymorphisms, those skilled in the art may usefully refer to the description of application WO 90/0345, and more generally to the principles described in US Patents 4,683,202 and US 4,683 , 195.
Pour tous les patients, on a vérifié les résultats de la détermination rapide du génotype en utilisation les techniques classiques d'extraction de l'ADN mettant en œuvre notamment une extraction au phénol et une précipitation à l'isopropanol (Amant et al, 1997, et Marcadet et al, 1987).For all the patients, the results of the rapid determination of the genotype were verified using conventional DNA extraction techniques, notably using phenol extraction and isopropanol precipitation (Amant et al, 1997, and Marcadet et al, 1987).
La méthode de génotypage rapide a permis d'obtenir des résultats en 4 heures environ seulement.The rapid genotyping method made it possible to obtain results in only about 4 hours.
16,5% des patients avaient le génotype II, 50% le génotype ID et 33,5% le génotype DD.16.5% of patients had genotype II, 50% had the ID genotype and 33.5% had the DD genotype.
En comparaison avec la méthode conventionnelle de génotypage, le test de détection rapide des porteurs DD présentait une sensibilité de 100%, une spécificité de 98,7%, une valeur prédictive positive de 97,4% et une valeur prédictive négative de 100%. Les patients présentant un génotype DD pour \'ACE ont un risque plus élevé de resténose après l'implantation de l'endoprothèse. Rapidement génotypes, ces patients pourront bénéficier d'une thérapie préventive dans les meilleurs délais. REFERENCES BIBLIOGRAPHIQUESCompared with the conventional genotyping method, the rapid DD carrier detection test had a sensitivity of 100%, a specificity of 98.7%, a positive predictive value of 97.4% and a negative predictive value of 100%. Patients with a DD genotype for CEA have a higher risk of restenosis after implantation of the stent. Quickly genotypes, these patients will be able to benefit from preventive therapy as soon as possible. BIBLIOGRAPHICAL REFERENCES
Amant et al, 1997, Circulation, 96(3), pp 56-60 Amouyel, 2000, Biofutur, n° 206, pp 86-88Amant et al, 1997, Circulation, 96 (3), pp 56-60 Amouyel, 2000, Biofutur, n ° 206, pp 86-88
Fogarty et al, 1994, Lancet, n° 343, p.851Fogarty et al, 1994, Lancet, n ° 343, p.851
Hamon et al, 1995, Circulation, n° 92, pp 296-299Hamon et al, 1995, Circulation, n ° 92, pp 296-299
Lathorp et al, 2000, Biofutur, n° 206, pp 82-85Lathorp et al, 2000, Biofutur, n ° 206, pp 82-85
Marcadet et al, 1987, Histocompatibility Testing, New York, NY Springer-Verlag, pp 553-560Marcadet et al, 1987, Histocompatibility Testing, New York, NY Springer-Verlag, pp 553-560
Sauer et al, 2000, Nucleic Acids Research, 28(5):E13 et 28(3) E100Sauer et al, 2000, Nucleic Acids Research, 28 (5): E13 and 28 (3) E100
Shanmugam et al, 1993, Methods Appl., vol. 3, pp 120-121Shanmugam et al, 1993, Methods Appl., Vol. 3, pp 120-121
Tiret et al, 1992, Am. J. Hum. Genet.vol. 51 , pp 197-205 Tiret et al, 1992, Am. J. Hum. Genet.vol. 51, pp 197-205

Claims

REVENDICATIONS
1. Méthode de génotypage d'un acide nucléique sans étape d'extraction chimique ni d'ajout de protéases, comprenant la mise en présence d'une préparation cellulaire avec un agent détergent, qui permet la lyse des cellules ; le chauffage de la préparation obtenue, puis sa centrifugation ; - le surnageant, récupéré, qui comprend des acides nucléiques, étant enfin soumis à une analyse génotypique.1. A method of genotyping a nucleic acid without a chemical extraction step or the addition of proteases, comprising placing a cell preparation in the presence of a detergent agent, which allows the lysis of cells; heating the preparation obtained, then centrifuging it; - the supernatant, recovered, which comprises nucleic acids, being finally subjected to a genotypic analysis.
2. Méthode selon la revendication 1 , dans laquelle le chauffage de la préparation est réalisé à une température de 70 à 100°C, de préférence à 90°C environ.2. Method according to claim 1, in which the heating of the preparation is carried out at a temperature of 70 to 100 ° C, preferably at approximately 90 ° C.
3. Méthode selon la revendication 1 ou 2, dans laquelle le chauffage de la préparation est réalisé pendant une durée de 30 minutes à 1 heure, de préférence pendant 40 minutes environ.3. Method according to claim 1 or 2, wherein the heating of the preparation is carried out for a period of 30 minutes to 1 hour, preferably for about 40 minutes.
4. Méthode selon l'une des revendications précédentes, dans laquelle la centrifugation est réalisée entre approximativement 4000 et 7000g, de préférence 5600g environ.4. Method according to one of the preceding claims, in which the centrifugation is carried out between approximately 4000 and 7000g, preferably approximately 5600g.
5. Méthode selon l'une des revendications précédentes, dans laquelle la préparation cellulaire consiste en un échantillon de sang total. 5. Method according to one of the preceding claims, in which the cell preparation consists of a whole blood sample.
6. Méthode selon l'une des revendications précédentes, dans laquelle ladite analyse génotypique consiste en la détermination des allèles D du gène ACE codant pour l'enzyme de conversion de l'angiotensine.6. Method according to one of the preceding claims, in which said genotypic analysis consists in determining the D alleles of the ACE gene coding for the angiotensin converting enzyme.
7. Méthode selon l'une des revendications précédentes, dans laquelle ladite analyse génotypique comprend l'amplification d'une région d'ADN où il existe un polymorphisme d'intérêt, la séparation des produits d'amplification dans une matrice, et la détection dudit polymorphisme. 7. Method according to one of the preceding claims, in which said genotypic analysis comprises the amplification of a DNA region where there is a polymorphism of interest, the separation of the amplification products in a matrix, and the detection of said polymorphism.
PCT/FR2002/001016 2001-03-23 2002-03-22 Gene typing method without dna extraction WO2002077273A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
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US5185244A (en) * 1989-12-08 1993-02-09 Emory University Genetic test for hereditary neuromuscular disease
EP0620282A1 (en) * 1989-04-17 1994-10-19 Clinical Diagnostic Systems, Inc. Method of extracting, amplifying and detecting a nucleic acid from whole blood
US5565323A (en) * 1994-03-30 1996-10-15 Mitokor Cytochrome oxidase mutations aiding diagnosis of sporadic alzheimer's disease

Patent Citations (4)

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EP0620282A1 (en) * 1989-04-17 1994-10-19 Clinical Diagnostic Systems, Inc. Method of extracting, amplifying and detecting a nucleic acid from whole blood
EP0428197A2 (en) * 1989-10-18 1991-05-22 Johnson & Johnson Clinical Diagnostics, Inc. Methods of extracting nucleic acids and PCR amplification without using a proteolytic enzyme
US5185244A (en) * 1989-12-08 1993-02-09 Emory University Genetic test for hereditary neuromuscular disease
US5565323A (en) * 1994-03-30 1996-10-15 Mitokor Cytochrome oxidase mutations aiding diagnosis of sporadic alzheimer's disease

Non-Patent Citations (2)

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Title
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MERCIER B ET AL: "DIRECT PCR FROM WHOLE BLOOD, WITHOUT DNA EXTRACTION", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 18, no. 19, 11 October 1990 (1990-10-11), pages 5908, XP000148400, ISSN: 0305-1048 *

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