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WO1996003526A1 - Nucleic acid amplification method using a modified nucleoside, and detection of the amplification product using antibodies - Google Patents

Nucleic acid amplification method using a modified nucleoside, and detection of the amplification product using antibodies Download PDF

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Publication number
WO1996003526A1
WO1996003526A1 PCT/FR1995/000985 FR9500985W WO9603526A1 WO 1996003526 A1 WO1996003526 A1 WO 1996003526A1 FR 9500985 W FR9500985 W FR 9500985W WO 9603526 A1 WO9603526 A1 WO 9603526A1
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WO
WIPO (PCT)
Prior art keywords
nucleoside
modified
amplification
methyl
sequence
Prior art date
Application number
PCT/FR1995/000985
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French (fr)
Inventor
Alain Niveleau
Original Assignee
Parteurop Developpement
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Filing date
Publication date
Application filed by Parteurop Developpement filed Critical Parteurop Developpement
Priority to EP95926413A priority Critical patent/EP0770144A1/en
Priority to JP8505525A priority patent/JPH10505230A/en
Priority to AU30814/95A priority patent/AU3081495A/en
Publication of WO1996003526A1 publication Critical patent/WO1996003526A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • a method of amplifying nucleic acid using a modified nucleoside, and detecting the amplification product using antibodies is provided.
  • the subject of the invention is a method of amplifying a nucleic acid sequence using a nucleoside triphosphate with a modified purine or pyrimide base which is incorporated into the strands of nucleic acid neosynthesized during the amplification operation. .
  • a modified purine or pyrimide base which is incorporated into the strands of nucleic acid neosynthesized during the amplification operation.
  • the amplification product can be used in the preparation of multimers containing several copies of the amplified sequence with modified base, and further containing at least one sequence specific for a sequence of interest.
  • Such a multimer can be used for the detection and / or quantification of any polynucleotide sequence, with amplification of the signal.
  • the enzymatic amplification method which was first described is the polymerase chain amplification, better known under the designation PCR (Polymerase Chain Reaction).
  • PCR Polymerase Chain Reaction
  • two oligonucleotide primers are used, each of which is complementary to a segment of one of the strands of a DNA containing the sequence to be amplified, so that said segments frame said sequence.
  • the primers hybridize with their complementary sequence and, in the presence of a thermostable DNA polymerase and of nucleoside triphosphates in excess, a strand of DNA, complementary to the template strand, is synthesized by elongation from the 3 'end of the hybridized primer.
  • the mixture is then denatured again by heat, and on cooling the primers hybridize on the DNA strands originally present or newly synthesized, a new elongation of the primers takes place, and so on.
  • a method analogous to PCR consists in using as starting material to amplify an RNA, the method then comprising a prior step of reverse transcription of the RNA into complementary DNA.
  • This method is useful in particular when the target is the genome of a retrovirus or when a messenger RNA is used as target to avoid amplifying the introns.
  • Various other amplification methods have been described, and in particular those which consist in using a primer containing an RNA polymerase promoter sequence, so as to obtain by elongation a double strand of DNA whose transcription in the presence of RNA polymerase will lead to l '' obtaining several tens of copies, in the form of transcribed RNA which can enter the amplification cycle.
  • the method includes reverse transcription of the RNA to form a DNA-RNA heteroduplex, then denaturation of the heteroduplex formed, hybridization of a primer to the DNA strand and elongation of the primer, followed by transcription of the double strand obtained, and so on.
  • the strands of RNA formed previously can each give rise to a double strand of DNA which can in turn be transcribed.
  • An isothermal method consists of a target RNA sequence and a primer of
  • RNA-DNA heteroduplex is obtained.
  • a ribonuclease By the action of a ribonuclease, the RNA is hydrolyzed.
  • the single stranded DNA is transformed into a double strand by elongation of an appropriate primer in the presence of a DNA polymerase.
  • the double stranded DNA obtained can then be transcribed and several copies of the target RNA are thus obtained, which can in turn enter the amplification cycle.
  • Another isothermal method consists in using the phenomenon of strand displacement using two primers capable of hybridizing with the same strand of target nucleic acid: the product of extension of a first primer located on the 5 'side relative to a second primer, displaces the extension product of the second primer which is thus released in the form of a single strand, while the extension product of the first primer forms a duplex with the starting target strand.
  • the single strand thus formed can in turn enter an amplification cycle using a similar system of two primers.
  • the first primer may contain on the 5 ′ side, relative to the target recognition site, a recognition site for an RNA polymerase or for a restriction enzyme, which makes it possible to use different routes for the d cycle. 'amplification; see EP-054 3612.
  • Amplification products containing the modified nucleotide are detected or quantified by an avidin-enzyme conjugate in the first case and by an anti-digoxigenin monoclonal antibody in the second case.
  • NTP nucleoside triphosphate designating here both a deoxyribonucleoside triphosphate and a ribonucleoside triphosphate
  • purine or pyrimide base is modified by a simple substitution such as a substitution with a methyl, hydroxymethyl or acetyl group.
  • the modified NTPs can be used by the polymerases to synthesize in vitro new copies of a nucleotide matrix, without any efficiency.
  • the subject of the invention is therefore a method of amplification, by the enzymatic route, of a nucleotide sequence, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, the nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having DNA polymerase and / or RNA polymerase and / or reverse transcriptase activity, under conditions making it possible to carry out, in a manner known per se , an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in an unmodified form, and that, in said modified form, the pyrimidine or purine base is substituted by at least one methyl group,
  • the subject of the invention is also a method for amplifying a nucleotide sequence, and for detecting and / or quantifying the amplification product, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having an activity of DNA polymerase and / or RNA polymerase and / or reverse transcriptase, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in a non-form modified, and that, in said modified form, the pyrimidine or purine base is substituted by
  • the amplification can be carried out by any known method based on the extension of primers to synthesize a double-stranded DNA, a complementary DNA, or to synthesize a transcribed RNA.
  • Amplification methods can be used, for example, which have been described above, and whose implementation details, which are well known, will not be repeated here.
  • modified nucleosides there may be mentioned in particular 5-methyl cytidine, N4-acetyl cytidine, 3-methyl cytidine, N6-methyl adenosine, N6, N6-dimethyl adenosine, 1-methyl guanosine, N2- methyl guanosine, N2, N2-dimethyl guanosine, 7-methyl guanosine, 3-methyl uridine, 5-hydroxymethyl uridine, and 1-methyl inosine.
  • the triphosphates of these modified nucleosides are known or can be prepared according to known methods.
  • Antibodies capable of specifically recognizing these modified nucleosides can be prepared according to known methods. They are preferably monoclonal antibodies.
  • nucleoside triphosphate can be used in the same way as the corresponding nucleoside modified in the elongation of a nucleotide primer.
  • sequence to be amplified contains only a small proportion of the nucleoside which it is desired to replace with the incorporation of the modified nucleoside phosphate, it is in principle possible to use the nucleoside triphosphate considered in modified form only.
  • the nucleoside under consideration is present at several neighboring positions in the sequence to be amplified, the incorporation of the modified nucleoside triphosphate alone may prove difficult. This is why it is generally preferable to use, for the nucleoside considered, a mixture of the modified form and the unmodified form.
  • the proportion of modified nucleoside triphosphate relative to the total proportion of the modified nucleoside and of the corresponding unmodified nucleoside can generally vary from 10 to 100 mol% and in particular from 20 to 75 mol%.
  • the amplification product will contain a variable proportion of the corresponding nucleoside in modified form and in unmodified form, this proportion depending on the nature of the composition of the composition.
  • sequence to be amplified and proportions of triphosphates (modified and unmodified) present during the amplification It has been found that, for a given sequence to be amplified, and for a given proportion of modified and unmodified triphosphates, the respective proportions of modified and unmodified nucleosides in the amplification product are substantially constant. As a result, the detection of the amplification product using antibodies specific for the modified nucleoside also allows quantification.
  • Antibodies prepared by immunization using modified nucleosides, conjugated to a protein have been found to be able to recognize the modified nucleoside incorporated into a strand of DNA or RNA.
  • the antibodies can be used in the form of antibodies labeled with a tracer agent, for example with an enzyme such as peroxidase or alkaline phosphatase, which allows development with the formation of a colored product, according to known methods.
  • a tracer agent for example with an enzyme such as peroxidase or alkaline phosphatase, which allows development with the formation of a colored product, according to known methods.
  • the revelation of the binding of the specific antibody to the amplification product can also be made with an antibody labeled anti-immunoglobulin, in known manner.
  • an antibody labeled anti-immunoglobulin in known manner.
  • the monoclonal antibody used is a mouse IgG
  • a labeled anti-mouse IgG antibody will be used for the revelation.
  • amplification primers linked, in a known manner, to a ligand for example biotin
  • a ligand for example biotin
  • the method of the invention can therefore be used in particular in the field of medical diagnosis or in the food industry, in the search for infections or bacterial or viral contamination.
  • Another advantage of the amplification process is that it leads to products which can be used for the manufacture of multimers which can be used as signal amplification agents in hybridization methods using nucleic probes.
  • the principle of signal amplification is known.
  • the number of copies of the target polynucleotide is low, it is possible, instead of multiplying the number of copies of the target by amplification, to use a detection probe comprising a large quantity of tracer agent, hence amplification of the signal.
  • the specific detection probe for a target to be detected is grafted to a polymer on which are fixed several patterns containing the tracer agent.
  • the signal-amplifying reagent can be produced, for example, in the form of a polymer carrying in branching a plurality of nucleotide sequences in which the modified nucleoside is incorporated.
  • Polymers bearing branching in a fork, comb, etc. can be prepared in a known manner. ; see for example the patent applications mentioned above.
  • the signal amplification reagent thus contains numerous molecules of the modified nucleoside, detection using antibodies specifically recognizing the modified nucleoside will lead to the obtaining of a signal of high intensity.
  • the invention therefore also relates to the use of an amplification product which can be obtained according to the amplification method described above.
  • This use consists in the preparation, in a manner known per se, of a multimer comprising a plurality of units constituted by said amplification product and at least a polynucleotide motif capable of hybridizing with a polynucleotide of interest.
  • a subject of the invention is also the use of an antibody in the detection and / or quantification, according to a method known per se, of an amplification product obtained according to the method described above, or in the detection and / or the quantification, according to a method known per se, of a multimer obtainable as described above, said multimer being hybrid with said polynucleotide of interest.
  • This use is characterized in that the antibody used is an antibody capable of recognizing the modified nucleoside without recognizing the corresponding unmodified nucleoside.
  • Two primers were synthesized located at the ends of a segment of 186 base pairs chosen from the BamHIW region of the EBV genome which is a highly conserved region among the different strains of EBV. Each primer was biotinylated at the 5 ′ end during the synthesis according to the method described by P.R. Langer et al., P.N.A.S. USA Vol. 78 No. 11, 6633-6637 (1981).
  • primers 1 5 'TTT GTC CCC ACG CGC GCA TA 3'
  • Primer 2 5 'AGG TGG CGT AGC AAC GCG AA 3'
  • the position of these primers relative to the BamHIW region of the genome d EBV is described in the article by R. Griffais et al., Nucleic Acids Research, Vol. 19, no.14,
  • Burkitt lymphoma cells of the Namalwa line are used, which have intact EBV DNA in a chromosome; see Matsuo et al., Science 226, 1322-1325 (1984) and Gargano et al., Genes Chrom. Cancer, 4, 205-210 (1992). In fact, it has been shown that two EBV genomes are integrated at a known site on chromosome 1; see Lawrence et al, Cell 52, 51-61 (1988). The cells are lysed by sodium hydroxide in a known manner.
  • Serial dilutions of cell lysates are prepared and used as matrices for amplification according to the PCR method.
  • the cycles are programmed as follows: - 1 cycle consisting of 10 minutes at 99 ° C, 2 minutes at 62 ° C and 2 minutes at
  • the DNA is visualized by fluorescence of ethidium bromide under UV radiation.
  • the DNA is then transferred to nylon membranes (Hybond N +, Amersham International).
  • hybridization was carried out with a detection probe marked with digoxigenin and capable of hybridizing with a sequence of a DNA strand located inside the sequence framed by the primers 1 and 2.
  • This detection probe was obtained by PCR, in the presence of nucleoside triphosphates and of a dUTP labeled with digoxigenin (digoxigenin 1 1 dUTP - Boehringer
  • the membranes are washed three times with PBST (phosphate buffer containing 0.2% Tween 20) and then allowed to incubate for one hour at room temperature with an anti-5-methyl cytidine monoclonal antibody.
  • the monoclonal antibody is that described by C. Reynaud et al., Cancer Letters, Elsevier Scientific Publishers Ireland Ltd, 255-262 (1991).
  • the antibody solution is an undiluted supernatant of hybridoma culture containing between 10 and 15 ⁇ g / ml of antibody.
  • the membranes are then incubated for one hour at room temperature with anti-IgG goat antibodies
  • mice conjugated to peroxidase and purified by affinity chrom atography (Biorad Laboratories). Wash again three times with PBST and then rinse with phosphate buffer (PBS).
  • PBS phosphate buffer
  • the substrate is then added (60 ml of a solution of 4-chloro-1-naphthol at 0.3% in methanol, in 100 ml of phosphate buffer containing 0.02% of hydrogen peroxide) and allowed to incubate at room temperature for 30 minutes.
  • Microtiter plates made of carboxylated polystyrene are used; reference:
  • the plates are washed with phosphate buffer pH 7.2 containing 0.05% Tween 20, then washed 3 times with phosphate buffer.
  • Amplification products denatured by heat are added at the rate of 50 ⁇ l per well.
  • the wells are washed 3 times with PBST.
  • 50 ⁇ l of the anti-5-methyl cytidine monoclonal antibody solution (supernatant of hybridoma culture) are then added. Incubated for 30 minutes at room temperature and then washed 3 times with PBST containing 1% bovine serum albumin
  • the 10-fold serial dilutions of EBV DNA that were used as templates in PCR amplification contained 2-20,000 copies of the EBV genome.
  • PCR amplification results carried out as in c) above, were compared with a mixture of dCTP and 5-MedCTP and biotinylated primers, and the PCR amplification results carried out in a conventional manner with non-NTP. modified and non-biotinylated primers.
  • the amplification products were analyzed by agarose gel electrophoresis and visualized by fluorescence of ethidium bromide under ultraviolet irradiation.
  • the electrophoresis analysis shows that in both cases the amplified DNAs have the expected sizes, according to the migration distances.
  • the sensitivity of the reaction determined by the intensity of the fluorescence on the gel, reaches 200 target molecules.
  • the amplification products obtained by conventional PCR were analyzed with the specific probe conjugated to digoxigenin (obtained in d) above), by Southern hybridization and revelation with an anti-digoxigenin antibody (Boehringer Mannheim). The sensitivity of the reaction reaches 20 molecules of target DNA.
  • the PCR amplification product according to the invention was analyzed in one section after electrophoresis and Southern transfer (see e) above) and in another section on a microtiter plate coated with avidin (see f) above. above).
  • the amplification product is denatured by sodium hydroxide and the detection is carried out using the anti-5 MeC monoclonal antibody, the binding of which is revealed as indicated previously by a anti-mouse IgG antibody, labeled with peroxidase.
  • the sensitivity threshold of the method is then two target DNA molecules.
  • the intensity of the signal increases with the amount of amplification product, which allows quantification.

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Abstract

A nucleotide sequence amplification method wherein a polynucleotide containing the sequence is contacted with oligonucleotide primers, nucleoside triphosphates and/or deoxynucleoside triphosphates containing the various purine and pyrimidine bases needed to synthesise the amplification product, and an enzyme system having DNA polymerase and/or RNA polymerase and/or reverse transcriptase activity, under conditions enabling the sequence of interest to be amplified in a per se known manner. One of the nucleoside triphosphates and/or deoxynucleoside triphosphates is present either in a modified form or in both a modified form and an unmodified form, and, in said modified form, the purine or pyrimidine base is substituted by at least one methyl, hydroxymethyl or acetyl grouping.

Description

Procédé d'amplification d'acide nucléique à l'aide d'un nucleoside modifié, et détection du produit d'amplification à l'aide d'anticorps. A method of amplifying nucleic acid using a modified nucleoside, and detecting the amplification product using antibodies.
L'invention a pour objet un procédé d'amplification d'une séquence d'acide nucléique utilisant un nucleoside triphosphate avec une base purique ou pyrimidique modifiée qui se trouve incorporée dans les brins d'acide nucléique néosynthétisés lors de l'opération d'amplification. A l'aide d'anticorps reconnaissant spécifiquement la base modifiée, il est possible de détecter et/ou de quantifier le produit d'amplification.The subject of the invention is a method of amplifying a nucleic acid sequence using a nucleoside triphosphate with a modified purine or pyrimide base which is incorporated into the strands of nucleic acid neosynthesized during the amplification operation. . Using antibodies specifically recognizing the modified base, it is possible to detect and / or quantify the amplification product.
En outre, le produit d'amplification peut être utilisé dans la préparation de multimères contenant plusieurs copies de la séquence amplifiée avec base modifiée, et contenant en outre au moins une séquence spécifique d'une séquence d'intérêt. Un tel multimère peut être utilisé pour la détection et/ou la quantification d'une séquence polynucléotidique quelconque, avec amplification du signal.In addition, the amplification product can be used in the preparation of multimers containing several copies of the amplified sequence with modified base, and further containing at least one sequence specific for a sequence of interest. Such a multimer can be used for the detection and / or quantification of any polynucleotide sequence, with amplification of the signal.
On sait que les méthodes d'amplification in vitro par voie enzymatique ont pour but notamment d'obtenir un grand nombre de copies d'une séquence d'un polynucleotide présent dans un échantillon en faible quantité, sans qu'il soit nécessaire d'isoler et de cloner ledit polynucleotide.It is known that the in vitro amplification methods by enzymatic route aim in particular to obtain a large number of copies of a sequence of a polynucleotide present in a sample in small quantity, without it being necessary to isolate and cloning said polynucleotide.
La méthode d'amplification par voie enzymatique qui a été décrite en premier est l'amplification en chaîne par polymerase, plus connue sous la désignation PCR (Polymerase Chain Reaction). Dans cette méthode, on utilise deux amorces oligonucléotidiques dont chacune est complémentaire d'un segment d'un des brins d'un DNA contenant la séquence à amplifier, de telle sorte que lesdits segments encadrent ladite séquence. Après chauffage pour dénaturer le DNA les amorces s'hybrident avec leur séquence complémentaire et, en présence d'une DNA polymerase thermostable et de nucléosides triphosphates en excès, un brin de DNA, complémentaire du brin matrice est synthétisé par élongation à partir de l'extrémité 3' de l'amorce hybridée. Le mélange est alors dénaturé à nouveau par la chaleur, et au refroidissement les amorces s'hybrident sur les brins de DNA présents à l'origine ou nouvellement synthétisés, une nouvelle élongation des amorces a lieu, et ainsi de suite. Ces cycles successifs d'appariement des amorces, d'élongation et de dénaturation conduisent à un doublement du nombre de copies de la séquence d'intérêt à chaque cycle.The enzymatic amplification method which was first described is the polymerase chain amplification, better known under the designation PCR (Polymerase Chain Reaction). In this method, two oligonucleotide primers are used, each of which is complementary to a segment of one of the strands of a DNA containing the sequence to be amplified, so that said segments frame said sequence. After heating to denature the DNA, the primers hybridize with their complementary sequence and, in the presence of a thermostable DNA polymerase and of nucleoside triphosphates in excess, a strand of DNA, complementary to the template strand, is synthesized by elongation from the 3 'end of the hybridized primer. The mixture is then denatured again by heat, and on cooling the primers hybridize on the DNA strands originally present or newly synthesized, a new elongation of the primers takes place, and so on. These successive cycles of pairing of primers, elongation and denaturation lead to a doubling of the number of copies of the sequence of interest in each cycle.
Une méthode analogue à la PCR, dite RT-PCR consiste à utiliser comme produit de départ à amplifier un RNA, la méthode comportant alors une étape préalable de transcription inverse du RNA en DNA complémentaire. Cette méthode est utile notamment lorsque la cible est le génome d'un rétrovirus ou lorsqu'on utilise comme cible un RNA messager pour éviter d'amplifier les introns. Diverses autres méthodes d'amplification ont été décrites, et notamment celles qui consistent à utiliser une amorce contenant une séquence de promoteur de RNA polymerase, de façon à obtenir par élongation un double brin de DNA dont la transcription en présence de RNA polymerase conduira à l'obtention de plusieurs dizaines de copies, sous forme de RNA transcrit qui peut entrer dans le cycle d'amplification. La méthode comprend une transcription inverse du RNA pour former un hétéroduplex DNA-RNA, puis la dénaturation de l' hétéroduplex formé, l'hybridation d'une amorce sur le brin de DNA et l'élongation de l'amorce, suivie de la transcription du double brin obtenu, et ainsi de suite. Les brins de RNA formés précédemment peuvent chacun donner naissance à un double brin de DNA qui pourra à son tour être transcrit.A method analogous to PCR, called RT-PCR consists in using as starting material to amplify an RNA, the method then comprising a prior step of reverse transcription of the RNA into complementary DNA. This method is useful in particular when the target is the genome of a retrovirus or when a messenger RNA is used as target to avoid amplifying the introns. Various other amplification methods have been described, and in particular those which consist in using a primer containing an RNA polymerase promoter sequence, so as to obtain by elongation a double strand of DNA whose transcription in the presence of RNA polymerase will lead to l '' obtaining several tens of copies, in the form of transcribed RNA which can enter the amplification cycle. The method includes reverse transcription of the RNA to form a DNA-RNA heteroduplex, then denaturation of the heteroduplex formed, hybridization of a primer to the DNA strand and elongation of the primer, followed by transcription of the double strand obtained, and so on. The strands of RNA formed previously can each give rise to a double strand of DNA which can in turn be transcribed.
L'un des inconvénients des méthodes précédentes est la nécessité de dénaturer les duplex. Cette opération qui se fait généralement à température supérieure à 90°C, nécessite l'emploi d'enzymes thermostables. Pour éviter les étapes de dénaturation, on a imaginé des méthodes d'amplification pouvant être mises en oeuvre de façon isotherme. Une méthode isotherme consiste à partir d'une séquence cible de RNA et d'une amorce deOne of the disadvantages of the previous methods is the need to denature the duplexes. This operation, which is generally done at a temperature above 90 ° C, requires the use of thermostable enzymes. To avoid the denaturation steps, amplification methods have been devised which can be applied isothermally. An isothermal method consists of a target RNA sequence and a primer of
DNA. A l'aide d'une transcriptase inverse, on obtient un hétéroduplex RNA-DNA. Par l'action d'une ribonucléase, on hydrolyse le RNA. Le DNA simple brin est transformé en double brin par élongation d'une amorce appropriée en présence d'une DNA polymerase. Le DNA double brin obtenu peut alors être transcrit et on obtient ainsi plusieurs copies du RNA cible qui peuvent à leur tour entrer dans le cycle d'amplification. Une autre méthode isotherme consiste à utiliser le phénomène de déplacement de brin à l'aide de deux amorces capables de s'hybrider avec un même brin d'acide nucléique cible : le produit d'élongation d'une première amorce situé du côté 5' par rapport à une seconde amorce, déplace le produit d'élongation de la seconde amorce qui est ainsi libéré sous forme de monobrin, tandis que le produit d'élongation de la première amorce forme un duplex avec le brin cible de départ. Le monobrin ainsi formé peut à son tour entrer dans un cycle d'amplification à l'aide d'un système analogue de deux amorces. En outre, la première amorce peut contenir du côté 5', par rapport au site de reconnaissance de la cible, un site de reconnaissance pour une RNA polymerase ou pour une enzyme de restriction, ce qui permet d'utiliser différentes voies pour le cycle d'amplification ; voir EP-054 3612.DNA. Using reverse transcriptase, an RNA-DNA heteroduplex is obtained. By the action of a ribonuclease, the RNA is hydrolyzed. The single stranded DNA is transformed into a double strand by elongation of an appropriate primer in the presence of a DNA polymerase. The double stranded DNA obtained can then be transcribed and several copies of the target RNA are thus obtained, which can in turn enter the amplification cycle. Another isothermal method consists in using the phenomenon of strand displacement using two primers capable of hybridizing with the same strand of target nucleic acid: the product of extension of a first primer located on the 5 'side relative to a second primer, displaces the extension product of the second primer which is thus released in the form of a single strand, while the extension product of the first primer forms a duplex with the starting target strand. The single strand thus formed can in turn enter an amplification cycle using a similar system of two primers. In addition, the first primer may contain on the 5 ′ side, relative to the target recognition site, a recognition site for an RNA polymerase or for a restriction enzyme, which makes it possible to use different routes for the d cycle. 'amplification; see EP-054 3612.
Tous les spécialistes du domaine estiment que les techniques d'amplification sont appelées à jouer un rôle considérable, notamment dans le diagnostic des infections ou contaminations bactériennes ou virales.All specialists in the field believe that amplification techniques are called upon to play a considerable role, in particular in the diagnosis of bacterial or viral infections or contaminations.
Il existe donc un besoin de techniques simples et rapides permettant une évaluation quantitative des produits d'amplification, pour remplacer les techniques d'électrophorèse sur gel et de transfert sur membrane (blotting). Les techniques les plus sensibles actuellement consistent à utiliser un nucleoside triphosphate modifié, à savoir un désoxyuridine triphosphate (dUTP) biotinylé ou un dUTP lié à la digoxigénine. Les produits d'amplification contenant le nucléotide modifié sont détectés ou quantifiés par un conjugué avidine-enzyme dans le premier cas et par un anticorps monoclonal anti-digoxigénine dans le second cas. On a maintenant découvert qu'il est possible de mettre en oeuvre les méthodes d'amplification en utilisant un nucleoside triphosphate, ou NTP (NTP désignant ici aussi bien un désoxyribonucléoside triphosphate qu'un ribonucléoside triphosphate) dont la base purique ou pyrimidique est modifiée par une substitution simple telle qu'une substitution par un groupement méthyle, hydroxyméthyle ou acétyle. On a découvert que les NTP modifiés sont utilisables par les polymérases pour synthétiser in vitro de nouvelles copies d'une matrice nucléotidique, sans peπe d'efficacité. Avec des anticorps qui reconnaissent spécifiquement un nucleoside à base modifiée et qui ne reconnaissent pas un nucleoside contenant ladite base sous forme non modifiée, on peut ainsi détecter une séquence d'intérêt amplifiée dans laquelle le nucleoside modifié a été incorporé. En outre, de façon surprenante, la technique de détection avec des anticorps dirigés contre ces nucléosides modifiés particuliers est, comme on le verra dans la partie expérimentale ci-après, nettement plus sensible que les techniques de détection classique à l'aide d'un conjugué avidine-enzyme (dans le cas d'utilisation d'amorces biotinylées) ou à l'aide du système digoxigénine/anticorps anti-digoxigénine. L'invention a donc pour objet un procédé d'amplification, par voie enzymatique, d'une séquence nucléotidique, dans lequel on met en contact un polynucleotide contenant ladite séquence, des amorces oligonucléotidiques, les nucléosides triphosphates et/ou désoxynucléosides triphosphates contenant les diverses bases puriques et pyrimidiques nécessaires à la synthèse du produit d'amplification, et un système enzymatique ayant une activité de DNA polymerase et/ou de RNA polymerase et/ou de transcriptase inverse, dans des conditions permettant d'effectuer, de façon connue en soi, une amplification de ladite séquence d'intérêt, caractérisé par le fait que l'un desdits nucléosides triphosphates et/ou désoxynucléosides triphosphates est présent soit sous une forme modifiée, soit à la fois sous une forme modifiée et sous une forme non modifiée, et que, dans ladite forme modifiée, la base pyrimidique ou purique est substituée par au moins un groupement méthyle, hydroxyméthyle ou acétyle.There is therefore a need for simple and rapid techniques allowing a quantitative evaluation of the amplification products, to replace the techniques of gel electrophoresis and of membrane transfer (blotting). The most sensitive techniques currently are to use a modified nucleoside triphosphate, namely a biotinylated deoxyuridine triphosphate (dUTP) or a dUTP linked to digoxigenin. Amplification products containing the modified nucleotide are detected or quantified by an avidin-enzyme conjugate in the first case and by an anti-digoxigenin monoclonal antibody in the second case. We have now discovered that it is possible to carry out the amplification methods using a nucleoside triphosphate, or NTP (NTP designating here both a deoxyribonucleoside triphosphate and a ribonucleoside triphosphate) whose purine or pyrimide base is modified by a simple substitution such as a substitution with a methyl, hydroxymethyl or acetyl group. It has been discovered that the modified NTPs can be used by the polymerases to synthesize in vitro new copies of a nucleotide matrix, without any efficiency. With antibodies which specifically recognize a modified base nucleoside and which do not recognize a nucleoside containing said base in unmodified form, it is thus possible to detect an amplified sequence of interest in which the modified nucleoside has been incorporated. In addition, surprisingly, the detection technique with antibodies directed against these particular modified nucleosides is, as will be seen in the experimental part below, much more sensitive than conventional detection techniques using a avidin-enzyme conjugate (when using biotinylated primers) or using the digoxigenin / anti-digoxigenin antibody system. The subject of the invention is therefore a method of amplification, by the enzymatic route, of a nucleotide sequence, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, the nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having DNA polymerase and / or RNA polymerase and / or reverse transcriptase activity, under conditions making it possible to carry out, in a manner known per se , an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in an unmodified form, and that, in said modified form, the pyrimidine or purine base is substituted by at least one methyl group, hydr oxymethyl or acetyl.
L'invention a également pour objet un procédé d'amplification d'une séquence nucléotidique, et de détection et/ou de quantification du produit d'amplification, dans lequel, on met en contact un polynucleotide contenant ladite séquence, des amorces oligonucléotidiques, les nucléosides triphosphates et/ou désoxynucléosides triphosphates contenant les diverses bases puriques et pyrimidiques nécessaires à la synthèse du produit d'amplification, et un système enzymatique ayant une activité de DNA polymerase et/ou de RNA polymerase et/ou de transcriptase inverse, dans des conditions permettant d'effectuer, de façon connue en soi, une amplification de ladite séquence d'intérêt, caractérisé par le fait que l'un desdits nucléosides triphosphates et/ou désoxynucléosides triphosphates est présent soit sous une forme modifiée, soit à la fois sous une forme modifiée et sous une forme non modifiée, et que, dans ladite forme modifiée, la base pyrimidique ou purique est substituée par au moins un groupement méthyle, hydroxyméthyle ou acétyle, et que l'on détecte et/ou quantifie le produit d'amplification, selon des méthodes connues en soi, à l'aide d'un anticorps reconnaissant spécifiquement ledit nucleoside modifié et ne reconnaissant pas ledit nucleoside non modifié.The subject of the invention is also a method for amplifying a nucleotide sequence, and for detecting and / or quantifying the amplification product, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having an activity of DNA polymerase and / or RNA polymerase and / or reverse transcriptase, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in a non-form modified, and that, in said modified form, the pyrimidine or purine base is substituted by at least one methyl, hydroxymethyl or acetyl group, and that the amplification product is detected and / or quantified, according to methods known per se , using an antibody specifically recognizing said modified nucleoside and not recognizing said unmodified nucleoside.
L'amplification peut être effectuée par toute méthode connue basée sur l'élongation d'amorces pour synthétiser un DNA double brin, un DNA complémentaire, ou pour synthétiser un RNA transcrit. On peut utiliser par exemple des méthodes d'amplification qui ont été décrites ci-dessus, et dont les détails de mise en oeuvre, qui sont bien connus, ne seront pas rappelés ici.The amplification can be carried out by any known method based on the extension of primers to synthesize a double-stranded DNA, a complementary DNA, or to synthesize a transcribed RNA. Amplification methods can be used, for example, which have been described above, and whose implementation details, which are well known, will not be repeated here.
Parmi les nucléosides modifiés, on peut citer notamment la 5-méthyl cytidine, la N4-acétyl cytidine, la 3-méthyl cytidine, la N6-méthyl adénosine, la N6,N6-diméthyl adénosine, la 1-méthyl guanosine, la N2-méthyl guanosine, la N2,N2-diméthyl guanosine, la 7-méthyl guanosine, la 3-méthyl uridine, la 5-hydroxyméthyl uridine, et la 1-méthyl inosine.Among the modified nucleosides, there may be mentioned in particular 5-methyl cytidine, N4-acetyl cytidine, 3-methyl cytidine, N6-methyl adenosine, N6, N6-dimethyl adenosine, 1-methyl guanosine, N2- methyl guanosine, N2, N2-dimethyl guanosine, 7-methyl guanosine, 3-methyl uridine, 5-hydroxymethyl uridine, and 1-methyl inosine.
Les triphosphates de ces nucléosides modifiés sont connus ou peuvent être préparés selon les méthodes connues.The triphosphates of these modified nucleosides are known or can be prepared according to known methods.
Les anticorps capables de reconnaître spécifiquement ces nucléosides modifiés peuvent être préparés selon les méthodes connues. Il s'agit de préférence d'anticorps monoclonaux.Antibodies capable of specifically recognizing these modified nucleosides can be prepared according to known methods. They are preferably monoclonal antibodies.
Certains de ces anticorps ont déjà été décrits ; voir C. Reynaud et al., Cancer Letters, 255-262 (1991). Lors de la préparation des anticorps monoclonaux, on a constaté qu'il est possible, de façon générale, d'obtenir des anticorps reconnaissant spécifiquement le nucleoside modifié. C'est ainsi que, par exemple, dans le cas des anticorps anti-5-méthyl cytidine, on a observé qu'environ 3 % des clones d'hybridomes testés sécrétaient des anticorps spécifiques reconnaissant la 5-méthyl cytidine et ne reconnaissant pas la cytidine. De même, dans le cas des anticorps anti-7-méthyl guanosine, 4 % des clones d'hybridome testés sécrétaient des anticorps reconnaissant le nucleoside modifié, mais pas la guanosine. II résulte des études effectuées que le nucleoside triphosphate peut être utilisé au même titre que le nucleoside correspondant modifié dans l'élongation d'une amorce nucléotidique. Lorsque la séquence à amplifier ne contient qu'une faible proportion du nucleoside que l'on souhaite remplacer par l'incorporation du phosphate nucléosidique modifié, il est possible en principe d'utiliser le nucleoside triphosphate considéré sous forme modifiée uniquement. Toutefois, lorsque le nucleoside considéré est présent en plusieurs positions voisines dans la séquence à amplifier, l'incorporation du nucleoside triphosphate modifié seul peut s'avérer difficile. C'est pourquoi il est préférable en général d'utiliser, pour le nucleoside considéré, un mélange de la forme modifiée et de la forme non modifiée.Some of these antibodies have already been described; see C. Reynaud et al., Cancer Letters, 255-262 (1991). During the preparation of the monoclonal antibodies, it has been found that it is generally possible to obtain antibodies which specifically recognize the modified nucleoside. Thus, for example, in the case of anti-5-methyl cytidine antibodies, it was observed that approximately 3% of the hybridoma clones tested secreted specific antibodies recognizing 5-methyl cytidine and not recognizing the cytidine. Similarly, in the case of anti-7-methyl guanosine antibodies, 4% of the hybridoma clones tested secreted antibodies recognizing the modified nucleoside, but not guanosine. It follows from the studies carried out that the nucleoside triphosphate can be used in the same way as the corresponding nucleoside modified in the elongation of a nucleotide primer. When the sequence to be amplified contains only a small proportion of the nucleoside which it is desired to replace with the incorporation of the modified nucleoside phosphate, it is in principle possible to use the nucleoside triphosphate considered in modified form only. However, when the nucleoside under consideration is present at several neighboring positions in the sequence to be amplified, the incorporation of the modified nucleoside triphosphate alone may prove difficult. This is why it is generally preferable to use, for the nucleoside considered, a mixture of the modified form and the unmodified form.
La proportion de nucleoside triphosphate modifié par rapport à la proportion totale du nucleoside modifié et du nucleoside correspondant non modifié, peut varier généralement de 10 à 100 % en moles et en particulier de 20 à 75 % en moles.The proportion of modified nucleoside triphosphate relative to the total proportion of the modified nucleoside and of the corresponding unmodified nucleoside can generally vary from 10 to 100 mol% and in particular from 20 to 75 mol%.
Bien entendu, lorsqu'on utilise un mélange de nucleoside modifié et de nucleoside non modifié, le produit d'amplification contiendra une proportion variable du nucleoside correspondant sous forme modifiée et sous forme non modifiée, cette proportion dépendant de la nature de la composition de la séquence à amplifier et des proportions de triphosphates (modifié et non modifié) présents lors de l'amplification. On a constaté que, pour une séquence à amplifier donnée, et pour une proportion donnée de triphosphates modifié et non modifié, les proportions respectives de nucléosides modifiés et non modifiés dans le produit d'amplification sont sensiblement constantes. Il en résulte que la détection du produit d'amplification à l'aide d'anticorps spécifiques du nucleoside modifié permet aussi une quantification.Of course, when a mixture of modified nucleoside and unmodified nucleoside is used, the amplification product will contain a variable proportion of the corresponding nucleoside in modified form and in unmodified form, this proportion depending on the nature of the composition of the composition. sequence to be amplified and proportions of triphosphates (modified and unmodified) present during the amplification. It has been found that, for a given sequence to be amplified, and for a given proportion of modified and unmodified triphosphates, the respective proportions of modified and unmodified nucleosides in the amplification product are substantially constant. As a result, the detection of the amplification product using antibodies specific for the modified nucleoside also allows quantification.
Pour détecter les produits d'amplification, on utilise les anticorps spécifiques mentionnés ci-dessus. On a constaté que les anticorps préparés par immunisation à l'aide des nucléosides modifiés, conjugués à une protéine, sont capable de reconnaître le nucleoside modifié incorporé dans un brin de DNA ou de RNA.To detect the amplification products, the specific antibodies mentioned above are used. Antibodies prepared by immunization using modified nucleosides, conjugated to a protein, have been found to be able to recognize the modified nucleoside incorporated into a strand of DNA or RNA.
Les anticorps peuvent être utilisés sous la forme d'anticorps marqués avec un agent traceur, par exemple avec une enzyme telle que peroxydase ou phosphatase alcaline, ce qui permet une révélation avec formation d'un produit coloré, selon les méthodes connues.The antibodies can be used in the form of antibodies labeled with a tracer agent, for example with an enzyme such as peroxidase or alkaline phosphatase, which allows development with the formation of a colored product, according to known methods.
La révélation de la fixation de l'anticorps spécifique sur le produit d'amplification peut également être faite avec un anticorps marqué anti-immunoglobuline, de façon connue. Par exemple si l'anticorps monoclonal utilisé est une IgG de souris, on utilisera pour la révélation un anticorps marqué anti-IgG de souris.The revelation of the binding of the specific antibody to the amplification product can also be made with an antibody labeled anti-immunoglobulin, in known manner. For example if the monoclonal antibody used is a mouse IgG, a labeled anti-mouse IgG antibody will be used for the revelation.
La révélation et éventuellement la quantification à l'aide d'anticorps sont des opérations bien connues dont les détails de mise en oeuvre ne seront pas rappelés ici. Par ailleurs, en utilisant des amorces d'amplification liées, de façon connue, à un ligand (par exemple la biotine), il est possible d'effectuer la détection et/ou la quantification sur plaque de microtitration sur laquelle a été fixé un anti-ligand correspondant (par exemple l'avidine). Le procédé de l'invention peut donc être utilisé notamment dans le domaine du diagnostic médical ou dans le domaine agro-alimentaire, dans la recherche d'infections ou de contaminations bactériennes ou virales.Disclosure and possibly quantification using antibodies are well known operations, the details of which will not be repeated here. Furthermore, by using amplification primers linked, in a known manner, to a ligand (for example biotin), it is possible to carry out the detection and / or the quantification on a microtiter plate on which an anti - corresponding ligand (for example avidin). The method of the invention can therefore be used in particular in the field of medical diagnosis or in the food industry, in the search for infections or bacterial or viral contamination.
Un autre intérêt du procédé d'amplification est qu'il conduit à des produits pouvant servir à la fabrication de multimères utilisables comme agents d'amplification de signal dans les méthodes d'hybridation à l'aide de sondes nucléiques.Another advantage of the amplification process is that it leads to products which can be used for the manufacture of multimers which can be used as signal amplification agents in hybridization methods using nucleic probes.
Le principe de l'amplification de signal est connu. Lorsque le nombre de copies du polynucleotide cible est faible, on peut, au lieu de multiplier par amplification le nombre de copies de la cible, utiliser une sonde de détection comprenant une quantité importante d'agent traceur, d'où une amplification de signal. La sonde de détection spécifique d'une cible à détecter, est greffée à un polymère sur lequel sont fixés plusieurs motifs contenant l'agent traceur.The principle of signal amplification is known. When the number of copies of the target polynucleotide is low, it is possible, instead of multiplying the number of copies of the target by amplification, to use a detection probe comprising a large quantity of tracer agent, hence amplification of the signal. The specific detection probe for a target to be detected is grafted to a polymer on which are fixed several patterns containing the tracer agent.
Des exemples de réalisation de multimères utilisables comme réactifs d'amplification de signal sont décrits notamment dans les demandes de brevet EP-0317077, EP-0450594, etc. Dans le cas où l'on souhaite utiliser le produit d'amplification obtenu selon le procédé d'amplification de la présente demande comme réactif d'amplification de signal, il convient de réaliser un multimère comprenant une pluralité de motifs constitués par le produit d'amplification et au moins un motif polynucléotidique capable de s'hybrider avec un polynucleotide d'intérêt. Dans ce cas, la séquence du produit d'amplification peut être quelconque : il suffit qu'elle ne soit pas capable de s'hybrider avec le polynucleotide cible.Examples of embodiments of multimers which can be used as signal amplification reagents are described in particular in patent applications EP-0317077, EP-0450594, etc. In the case where it is desired to use the amplification product obtained according to the amplification method of the present application as signal amplification reagent, it is advisable to produce a multimer comprising a plurality of patterns constituted by the product of amplification and at least one polynucleotide motif capable of hybridizing with a polynucleotide of interest. In this case, the sequence of the amplification product can be arbitrary: it is sufficient that it is not capable of hybridizing with the target polynucleotide.
On peut réaliser le réactif amplificateur de signal par exemple sous la forme d'un polymère portant en ramification une pluralité de séquences nucléotidiques dans lesquelles est incorporé le nucleoside modifié. On peut préparer de façon connue des polymères portant des ramifications en fourchette, en peigne, etc. ; voir par exemple les demandes de brevet mentionnées ci-dessus.The signal-amplifying reagent can be produced, for example, in the form of a polymer carrying in branching a plurality of nucleotide sequences in which the modified nucleoside is incorporated. Polymers bearing branching in a fork, comb, etc. can be prepared in a known manner. ; see for example the patent applications mentioned above.
Le réactif d'amplification de signal contenant ainsi de nombreuses molécules du nucleoside modifié, la détection à l'aide d'anticorps reconnaissant spécifiquement le nucleoside modifié, conduira à l'obtention d'un signal d'intensité élevée.Since the signal amplification reagent thus contains numerous molecules of the modified nucleoside, detection using antibodies specifically recognizing the modified nucleoside will lead to the obtaining of a signal of high intensity.
L'invention a donc également pour objet l'utilisation d'un produit d'amplification pouvant être obtenu selon le procédé d'amplification décrit ci-dessus.The invention therefore also relates to the use of an amplification product which can be obtained according to the amplification method described above.
Cette utilisation consiste en la préparation, de façon connue en soi, d'un multimère comprenant une pluralité de motifs constitué par ledit produit d'amplification et au moins un motif polynucléotidique capable de s'hybrider avec un polynucleotide d'intérêt.This use consists in the preparation, in a manner known per se, of a multimer comprising a plurality of units constituted by said amplification product and at least a polynucleotide motif capable of hybridizing with a polynucleotide of interest.
L'invention a également pour objet l'utilisation d'un anticorps dans la détection et/ou la quantification, selon une méthode connue en soi, d'un produit d'amplification obtenu selon le procédé décrit précédemment, ou dans la détection et/ou la quantification, selon une méthode connue en soi, d'un multimère pouvant être obtenu comme décrit ci-dessus, ledit multimère étant hybride avec ledit polynucleotide d'intérêt. Cette utilisation est caractérisée par le fait que l'anticorps utilisé est un anticorps capable de reconnaître le nucleoside modifié sans reconnaître le nucleoside correspondant non modifié. L'exemple suivant illustre l'invention.A subject of the invention is also the use of an antibody in the detection and / or quantification, according to a method known per se, of an amplification product obtained according to the method described above, or in the detection and / or the quantification, according to a method known per se, of a multimer obtainable as described above, said multimer being hybrid with said polynucleotide of interest. This use is characterized in that the antibody used is an antibody capable of recognizing the modified nucleoside without recognizing the corresponding unmodified nucleoside. The following example illustrates the invention.
EXEMPLE 1 : Amplification du eénome du virus d'Epstein-Barr (EBV)EXAMPLE 1 Amplification of the Epstein-Barr Virus (EBV) Eenoma
a) Amorcesa) Primers
On a synthétisé deux amorces situées aux extrémités d'un segment de 186 paires de bases choisies dans la région BamHIW du génome d'EBV qui est une région hautement conservée parmi les différentes souches d'EBV. Chaque amorce a été biotinylée à l'extrémité 5' lors de la synthèse selon la méthode décrite par P.R. Langer et al., P.N.A.S. USA Vol. 78 n° 11, 6633-6637 (1981).Two primers were synthesized located at the ends of a segment of 186 base pairs chosen from the BamHIW region of the EBV genome which is a highly conserved region among the different strains of EBV. Each primer was biotinylated at the 5 ′ end during the synthesis according to the method described by P.R. Langer et al., P.N.A.S. USA Vol. 78 No. 11, 6633-6637 (1981).
Les séquences des amorces sont les suivantes : Amorce 1 : 5' TTT GTC CCC ACG CGC GCA TA 3' Amorce 2 : 5' AGG TGG CGT AGC AAC GCG AA 3' La position de ces amorces par rapport à la région BamHIW du génome d'EBV est décrite dans l'article de R. Griffais et al., Nucleic Acids Research, Vol. 19, n° 14,The sequences of the primers are as follows: Primer 1: 5 'TTT GTC CCC ACG CGC GCA TA 3' Primer 2: 5 'AGG TGG CGT AGC AAC GCG AA 3' The position of these primers relative to the BamHIW region of the genome d EBV is described in the article by R. Griffais et al., Nucleic Acids Research, Vol. 19, no.14,
3887-3891 (1991).3887-3891 (1991).
b) Préparation de la matrice de DNAb) Preparation of the DNA matrix
On utilise des cellules de lymphome de Burkitt de la lignée Namalwa, qui possèdent le DNA d'EBV intact dans un chromosome ; voir Matsuo et al., Science 226, 1322-1325 (1984) et Gargano et al., Gènes Chrom. Cancer, 4, 205-210 (1992). En fait, il a été montré que deux génomes d'EBV sont intégrés sur un site connu du chromosome 1 ; voir Lawrence et al, Cell 52, 51-61 (1988). Les cellules sont lysées par la soude de façon connue.Burkitt lymphoma cells of the Namalwa line are used, which have intact EBV DNA in a chromosome; see Matsuo et al., Science 226, 1322-1325 (1984) and Gargano et al., Genes Chrom. Cancer, 4, 205-210 (1992). In fact, it has been shown that two EBV genomes are integrated at a known site on chromosome 1; see Lawrence et al, Cell 52, 51-61 (1988). The cells are lysed by sodium hydroxide in a known manner.
Des dilutions en série des lysats cellulaires sont préparées et utilisées comme matrices pour l'amplification selon la méthode PCR.Serial dilutions of cell lysates are prepared and used as matrices for amplification according to the PCR method.
c) Amplificationc) Amplification
Des fractions aliquotes de lysats cellulaires (10 microlitres) sont ajoutées à 100 μl d'une solution tampon contenant 50 mM KC1, 10 mM HC1 (pH 8,4) 1,5 mM MgCl2, 100 μg/ml de gélatine, 200 μM de chacun des nucléosides triphosphates dATP, dTTP et dGTP, 40 μM de dCTP, 40 μM de 5-méthyl dCTP (ci-après : 5-MedCTP) (Boehringer Mannheim) 0,5 μM de chaque amorce et 1 unité de Taq polymerase. On utilise un appareil thermocycleur Perkin-Elmer 180.Aliquots of cell lysates (10 microliters) are added to 100 μl of a buffer solution containing 50 mM KC1, 10 mM HC1 (pH 8.4) 1.5 mM MgCl 2 , 100 μg / ml of gelatin, 200 μM of each of the nucleoside triphosphates dATP, dTTP and dGTP, 40 μM of dCTP, 40 μM of 5-methyl dCTP (hereinafter: 5-MedCTP) (Boehringer Mannheim) 0.5 μM of each primer and 1 unit of Taq polymerase. A Perkin-Elmer 180 thermal cycler is used.
Les cycles sont programmés de la façon suivante : - 1 cycle consistant en 10 minutes à 99°C, 2 minutes à 62°C et 2 minutes àThe cycles are programmed as follows: - 1 cycle consisting of 10 minutes at 99 ° C, 2 minutes at 62 ° C and 2 minutes at
72°C,72 ° C,
- puis 35 cycles consistant en 30 secondes à 95°C, 1 minute à 62°C et 2 minutes à 72°C A la fin de l'opération les échantillons sont maintenus pendant 5 minute à 72°C.- then 35 cycles consisting of 30 seconds at 95 ° C, 1 minute at 62 ° C and 2 minutes at 72 ° C. At the end of the operation, the samples are kept for 5 minutes at 72 ° C.
d) Caractérisationd) Characterization
On soumet des paπies aliquotes (10 μl) de chaque produit d'amplification à une électrophorèse sur gel d'agarose (3 % d'agarose NueSieve et 1 % d'agarose SeaKem).Aliquot parts (10 μl) of each amplification product are subjected to electrophoresis on agarose gel (3% NueSieve agarose and 1% SeaKem agarose).
Après migration, on visualise le DNA par fluorescence du bromure d'ethidium sous iπadiation UV.After migration, the DNA is visualized by fluorescence of ethidium bromide under UV radiation.
Le DNA est ensuite transféré sur des membranes de nylon (Hybond N+, Amersham International).The DNA is then transferred to nylon membranes (Hybond N +, Amersham International).
Pour vérifier la spécificité des produits d'amplification, on a effectué une hybridation avec une sonde de détection marquée à la digoxigénine et capable de s'hybrider avec une séquence d'un brin de DNA située à l'intérieur de la séquence encadrée par les amorces 1 et 2. On a obtenu cette sonde de détection par PCR, en présence de nucléosides triphosphates et d'un dUTP marqué à la digoxigénine (digoxigenin 1 1 dUTP - BoehringerTo verify the specificity of the amplification products, hybridization was carried out with a detection probe marked with digoxigenin and capable of hybridizing with a sequence of a DNA strand located inside the sequence framed by the primers 1 and 2. This detection probe was obtained by PCR, in the presence of nucleoside triphosphates and of a dUTP labeled with digoxigenin (digoxigenin 1 1 dUTP - Boehringer
Mannheim), avec les amorces suivantes :Mannheim), with the following primers:
- amorce 3 : 5' CCA GGA AGC GGG TCT ATG 3'- primer 3: 5 'CCA GGA AGC GGG TCT ATG 3'
- amorce 4 : 5* CCT TTG GTG AAG TCA CAA ACA 3'. On obtient ainsi un amplimèrc de 106 nucléotides dont la séquence est incluse dans la séquence de 186 nucléotides amplifiée précédemment.- primer 4: 5 * CCT TTG GTG AAG TCA CAA ACA 3 '. An amplimèrc of 106 nucleotides is thus obtained, the sequence of which is included in the sequence of 186 nucleotides amplified previously.
On a vérifié que la sonde ainsi obtenue s'hybride spécifiquement avec les produits d'amplification PCR obtenus en c) ci-dessus. La révélation de la sonde hybridée marquée à la digoxigénine est effectuée à l'aide d'anticorps anti-digoxigénine conjugués à la phosphatase alcaline, en présence de NBT/X-phosphate qui fournit un précipité bleu (kit de détection d'acides nucléiques, Boehringer Mannheim).It has been verified that the probe thus obtained specifically hybridizes with the PCR amplification products obtained in c) above. The revelation of the digoxigenin-labeled hybridized probe is carried out using anti-digoxigenin antibodies conjugated with alkaline phosphatase, in the presence of NBT / X-phosphate which provides a blue precipitate (nucleic acid detection kit, Boehringer Mannheim).
e) Immunodétection de la 5-méthyl cytidinee) Immunodetection of 5-methyl cytidine
L'incorporation de 5-méthyl cytidine dans le DNA amplifié a été démontrée de la façon suivante. Après électrophorèse, puis transfeπs sur membranes de nylon comme mentionné précédemment, les membranes sont immergées pendant 30 minutes dans une solution à 0,5 % de réactif bloquant (Boehringer Mannheim) dilué dans un tampon TRIS 0,05 M pH 7,4.The incorporation of 5-methyl cytidine into the amplified DNA was demonstrated as follows. After electrophoresis, then transfer to nylon membranes as mentioned above, the membranes are immersed for 30 minutes in a 0.5% solution of blocking reagent (Boehringer Mannheim) diluted in 0.05 M TRIS buffer pH 7.4.
Les membranes sont lavées trois fois avec du PBST (tampon phosphate contenant 0,2 % de Tween 20) puis on laisse incuber pendant une heure à température ambiante avec un anticorps monoclonal anti-5-méthyl cytidine. L'anticorps monoclonal est celui décrit par C. Reynaud et al., Cancer Letters, Elsevier Scientific Publishers Ireland Ltd, 255-262 (1991). La solution d'anticorps est un surnageant non dilué de culture d'hybridome contenant entre 10 et 15 μg/ml d'anticorps. Les membranes sont ensuite incubées pendant une heure à température ambiante avec des anticorps de chèvre anti-IgGThe membranes are washed three times with PBST (phosphate buffer containing 0.2% Tween 20) and then allowed to incubate for one hour at room temperature with an anti-5-methyl cytidine monoclonal antibody. The monoclonal antibody is that described by C. Reynaud et al., Cancer Letters, Elsevier Scientific Publishers Ireland Ltd, 255-262 (1991). The antibody solution is an undiluted supernatant of hybridoma culture containing between 10 and 15 μg / ml of antibody. The membranes are then incubated for one hour at room temperature with anti-IgG goat antibodies
(H+L) de souris conjugués à la peroxydase et purifiés par chrom atographie d'affinité (Biorad Laboratories). On lave à nouveau trois fois avec du PBST puis on rince avec du tampon phosphate (PBS).(H + L) of mice conjugated to peroxidase and purified by affinity chrom atography (Biorad Laboratories). Wash again three times with PBST and then rinse with phosphate buffer (PBS).
On ajoute alors le substrat (60 ml d'une solution de 4-chloro-l-naphtol à 0,3 % dans le méthanol, dans 100 ml de tampon phosphate contenant 0,02 % de peroxyde d'hydrogène) et on laisse incuber à température ambiante pendant 30 minutes.The substrate is then added (60 ml of a solution of 4-chloro-1-naphthol at 0.3% in methanol, in 100 ml of phosphate buffer containing 0.02% of hydrogen peroxide) and allowed to incubate at room temperature for 30 minutes.
f) Préparation de microplaquesf) Preparation of microplates
On utilise des plaques de microtitration en polystyrène carboxylé ; référence :Microtiter plates made of carboxylated polystyrene are used; reference:
Niveleau et al. J. Immunol. Methods, 159, 177-189 (1993).Niveleau et al. J. Immunol. Methods, 159, 177-189 (1993).
On ajoute dans chaque puits 100 μl d'une solution à 10 μg/ml de chlorhydrate de N-(3-diméthylaminopropyl)-N'-éthylcarbodiimide (Merck) dilué dans du tampon phosphate pH 7,2. Après 10 minutes à température ambiante, on ajoute dans chaque puits 100 μl d'une solution à 10 μg/ml d'avidine (Sigma) dans un tampon phosphate de pH 7,2.100 μl of a 10 μg / ml solution of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (Merck) diluted in phosphate buffer pH 7.2 is added to each well. After 10 minutes at room temperature, 100 μl of a solution containing 10 μg / ml of avidin (Sigma) in a phosphate buffer of pH 7.2 are added to each well.
Au bout d'une heure, les plaques sont lavées avec du tampon phosphate pH 7,2 contenant 0,05 % de Tween 20, puis lavées 3 fois avec du tampon phosphate. Les produits d'amplification dénaturés par la chaleur sont ajoutés à raison de 50 μl par puits. Après 15 minutes à température ambiante, les puits sont lavés 3 fois par du PBST. On ajoute alors 50 μl de la solution d'anticorps monoclonal anti-5-méthyl cytidine (surnageant de culture d'hybridome). On laisse incuber pendant 30 minutes à température ambiante puis on lave 3 fois avec du PBST contenant 1 % de sérum albumine bovineAfter one hour, the plates are washed with phosphate buffer pH 7.2 containing 0.05% Tween 20, then washed 3 times with phosphate buffer. Amplification products denatured by heat are added at the rate of 50 μl per well. After 15 minutes at room temperature, the wells are washed 3 times with PBST. 50 μl of the anti-5-methyl cytidine monoclonal antibody solution (supernatant of hybridoma culture) are then added. Incubated for 30 minutes at room temperature and then washed 3 times with PBST containing 1% bovine serum albumin
(PBST-BSA).(PBST-BSA).
On ajoute ensuite 50 μl d'anticorps de chèvre anti-IgG (H+L) de souris conjugué à la peroxydase (Biorad Laboratories). Après 30 minutes à température ambiante, on rince 3 fois avec du PBST-BSA puis on ajoute le substrat de la peroxydase qui est la 3,3',5,5'-tétraméthyl- benzidine, en solution dans le diméthylformamide, mélangé à 10 % (en volume) de peroxyde d'hydrogène. 100 μl de ce mélange sont ajoutés dans chaque puits et on laisse incuber à température ambiante pendant 17-20 minutes, jusqu'à ce qu'une coloration bleu intense du produit de réaction apparaisse. La réaction est stoppée par addition d'acide sulfurique IN (100 μl par puits). La densité optique à 655 nm est mesurée par spectrophotométrie.Then added 50 μl of goat anti-mouse IgG (H + L) antibody conjugated with peroxidase (Biorad Laboratories). After 30 minutes at room temperature, rinsing 3 times with PBST-BSA and then adding the peroxidase substrate which is 3,3 ', 5,5'-tetramethylbenzidine, in solution in dimethylformamide, mixed with 10 % (by volume) of hydrogen peroxide. 100 μl of this mixture are added to each well and the mixture is left to incubate at room temperature for 17-20 minutes, until an intense blue coloration of the reaction product appears. The reaction is stopped by adding IN sulfuric acid (100 μl per well). The optical density at 655 nm is measured by spectrophotometry.
RésultatsResults
Les dilutions en série de 10 en 10 du DNA d'EBV qui ont été utilisées comme matrices dans l'amplification par PCR, contenaient de 2 à 20.000 copies du génome d'EBV.The 10-fold serial dilutions of EBV DNA that were used as templates in PCR amplification contained 2-20,000 copies of the EBV genome.
On a comparé les résultats d'amplification par PCR, effectuée comme en c) ci-dessus, avec un mélange de dCTP et de 5-MedCTP et des amorces biotinylées, et les résultats d'amplification par PCR effectuée de façon classique avec NTP non modifiés et amorces non biotinylées.The PCR amplification results, carried out as in c) above, were compared with a mixture of dCTP and 5-MedCTP and biotinylated primers, and the PCR amplification results carried out in a conventional manner with non-NTP. modified and non-biotinylated primers.
Les produits d'amplification ont été analysés par électrophorèse sur gel d'agarose et visualisés par fluorescence du bromure d'éthidium sous irradiation ultraviolette. L'analyse en électrophorèse montre que dans les deux cas les DNA amplifiés ont les tailles attendues, d'après les distances de migration. La sensibilité de la réaction, déterminée par l'intensité de la fluorescence sur le gel, atteint 200 molécules cibles deThe amplification products were analyzed by agarose gel electrophoresis and visualized by fluorescence of ethidium bromide under ultraviolet irradiation. The electrophoresis analysis shows that in both cases the amplified DNAs have the expected sizes, according to the migration distances. The sensitivity of the reaction, determined by the intensity of the fluorescence on the gel, reaches 200 target molecules.
DNA, aussi bien avec la PCR selon l'invention qu'avec la PCR classique.DNA, both with the PCR according to the invention and with conventional PCR.
Par ailleurs, à titre de comparaison, les produits d'amplification obtenus par PCR classique ont été analysés avec la sonde spécifique conjuguée à la digoxigénine (obtenue en d) ci-dessus), par hybridation de Southern et révélation avec un anticorps anti-digoxigénine (Boehringer Mannheim). La sensibilité de la réaction atteint 20 molécules de DNA cible. On a analysé le produit d'amplification PCR selon l'invention d'une pan après électrophorèse et transfert de Southern (voir e) ci-dessus) et d'autre pan sur plaque de microtitration revêtue d'avidine (voir f) ci-dessus).Furthermore, by way of comparison, the amplification products obtained by conventional PCR were analyzed with the specific probe conjugated to digoxigenin (obtained in d) above), by Southern hybridization and revelation with an anti-digoxigenin antibody (Boehringer Mannheim). The sensitivity of the reaction reaches 20 molecules of target DNA. The PCR amplification product according to the invention was analyzed in one section after electrophoresis and Southern transfer (see e) above) and in another section on a microtiter plate coated with avidin (see f) above. above).
Dans le premier cas (électrophorèse et transfeπ de Southern) le produit d'amplification est dénaturé par la soude et on effectue la détection à l'aide de l'anticorps monoclonal anti-5 MeC, dont la fixation est révélée comme indiqué précédemment par un anticorps anti-IgG de souris, marqué à la peroxydase. Le seuil de sensibilité de la méthode est alors de deux molécules de DNA cible.In the first case (electrophoresis and Southern transfer) the amplification product is denatured by sodium hydroxide and the detection is carried out using the anti-5 MeC monoclonal antibody, the binding of which is revealed as indicated previously by a anti-mouse IgG antibody, labeled with peroxidase. The sensitivity threshold of the method is then two target DNA molecules.
Lorsque l'analyse est faite par dépôt du produit d'amplification sur plaque de microtitration revêtue d'avidine, et détection à l'aide de l'anticorps monoclonal anti-5When the analysis is made by depositing the amplification product on a microtitration plate coated with avidin, and detection using the anti-5 monoclonal antibody
MeC comme précédemment, on a trouvé que le seuil de détection est cette fois inférieur à deux molécules de DNA cible.MeC as before, it was found that the detection threshold is this time lower than two target DNA molecules.
L'intensité du signal augmente avec la quantité de produit d'amplification, ce qui permet la quantification. The intensity of the signal increases with the amount of amplification product, which allows quantification.

Claims

REVENDICATIONS
1. Procédé d'amplification d'une séquence nucléotidique, dans lequel on met en contact un polynucleotide contenant ladite séquence, des amorces oligonucléotidiques, les nucléosides triphosphates et/ou désoxynucléosides triphosphates contenant les diverses bases puriques et pyrimidiques nécessaires à la synthèse du produit d'amplification, et un système enzymatique ayant une activité de DNA polymerase et/ou de RNA polymerase et/ou de transcriptase inverse, dans des conditions permettant d'effectuer, de façon connue en soi, une amplification de ladite séquence d'intérêt, caractérisé par le fait que l'un desdits nucléosides triphosphates et/ou désoxynucléosides triphosphates est présent soit sous une forme modifiée, soit à la fois sous une forme modifiée et sous une forme non modifiée, et que, dans ladite forme modifiée, la base pyrimidique ou purique est substituée par au moins un groupement méthyle, hydroxyméthyle ou acétyle.1. Method for amplifying a nucleotide sequence, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, the nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the product d amplification, and an enzymatic system having DNA polymerase and / or RNA polymerase and / or reverse transcriptase activity, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized by the fact that one of said nucleoside triphosphates and / or deoxynucleosides triphosphates is present either in a modified form, or both in a modified form and in an unmodified form, and that, in said modified form, the pyrimidine base or purine is substituted by at least one methyl, hydroxymethyl or acetyl group.
2. Procédé d'amplification d'une séquence nucléotidique, et de détection et/ou de quantification du produit d'amplification, dans lequel on met en contact un polynucleotide contenant ladite séquence, des amorces oligonucléotidiques, les nucléosides triphosphates et/ou désoxynucléosides triphosphates contenant les diverses bases puriques et pyrimidiques nécessaires à la synthèse du produit d'amplification, et un système enzymatique ayant une activité de DNA polymerase et/ou de RNA polymerase et/ou de transcriptase inverse, dans des conditions permettant d'effectuer, de façon connue en soi, une amplification de ladite séquence d'intérêt, caractérisé par le fait que l'un desdits nucléosides triphosphates et/ou désoxynucléosides triphosphates est présent soit sous une forme modifiée, soit à la fois sous une forme modifiée et sous une forme non modifiée, et que, dans ladite forme modifiée, la base pyrimidique ou purique est substituée par au moins un groupement méthyle, hydroxyméthyle ou acétyle, et que l'on détecte et/ou quantifie le produit d'amplification, selon des méthodes connues en soi, à l'aide d'un anticorps reconnaissant spécifiquement ledit nucleoside modifié et ne reconnaissant pas ledit nucleoside non modifié.2. Method for amplifying a nucleotide sequence, and for detecting and / or quantifying the amplification product, in which a polynucleotide containing said sequence is contacted, oligonucleotide primers, nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having an activity of DNA polymerase and / or RNA polymerase and / or reverse transcriptase, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in a non-form modified, and that, in said modified form, the pyrimidine or purine base is substituted by at least one group t methyl, hydroxymethyl or acetyl, and that the amplification product is detected and / or quantified, according to methods known per se, using an antibody specifically recognizing said modified nucleoside and not recognizing said non-nucleoside amended.
3. Procédé selon la revendication 1 ou 2, caractérisé par le fait que ledit nucleoside modifié est choisi parmi la 5-méthyl cytidine, la N4-acétyl cytidine, la 3-méthyl cytidine, la N6-méthyl adénosine, la N6,N6-diméthyl adénosine, la 1-méthyl guanosine, la N2-méthyl guanosine, la N2,N2-diméthyl guanosine, la 7-méthyl guanosine, la 3-méthyl uridine, la 5-hydroxyméthyl uridine, et la 1-méthyl inosine. 3. Method according to claim 1 or 2, characterized in that said modified nucleoside is chosen from 5-methyl cytidine, N4-acetyl cytidine, 3-methyl cytidine, N6-methyl adenosine, N6, N6- dimethyl adenosine, 1-methyl guanosine, N2-methyl guanosine, N2, N2-dimethyl guanosine, 7-methyl guanosine, 3-methyl uridine, 5-hydroxymethyl uridine, and 1-methyl inosine.
4. Procédé selon l'une quelconque des revendications précédentes, caractérisé par le fait que la propoπion de nucleoside triphosphate modifié par rappon à la propoπion totale de nucleoside modifié et de nucleoside correspondant non modifié est de 10 à 100 % en moles. 4. Method according to any one of the preceding claims, characterized in that the proportion of nucleoside triphosphate modified with respect to the total proportion of modified nucleoside and corresponding unmodified nucleoside is from 10 to 100 mol%.
5. Procédé selon la revendication 4, caractérisé par le fait que ladite propoπion est de 20 à 75 % en moles.5. Method according to claim 4, characterized in that said propoπion is from 20 to 75 mol%.
6. Utilisation d'un produit d'amplification pouvant être obtenu selon le procédé de l'une quelconque des revendications 1 et 3 à 5, dans la préparation, de façon connue en soi, d'un multimère comprenant une pluralité de motifs constitués par ledit produit d'amplification et au moins un motif polynucléotidique capable de s'hybrider avec un polynucleotide d'intérêt.6. Use of an amplification product obtainable according to the method of any one of claims 1 and 3 to 5, in the preparation, in a manner known per se, of a multimer comprising a plurality of units consisting of said amplification product and at least one polynucleotide motif capable of hybridizing with a polynucleotide of interest.
7. Utilisation d'un anticorps dans la détection et/ou la quantification, selon une méthode connue en soi, d'un produit d'amplification obtenu selon le procédé de l'une quelconque des revendications 1 à 3, ou dans la détection et/ou la quantification, selon une méthode connue en soi, d'un multimère pouvant être obtenu selon la revendication 6, ledit multimère étant hybride avec ledit polynucleotide d'intérêt, caractérisée par le fait que ledit anticorps est un anticorps capable de reconnaître ledit nucleoside modifié sans reconnaître le nucleoside correspondant non modifié. 7. Use of an antibody in the detection and / or quantification, according to a method known per se, of an amplification product obtained according to the method of any one of claims 1 to 3, or in the detection and / or the quantification, according to a method known per se, of a multimer obtainable according to claim 6, said multimer being hybrid with said polynucleotide of interest, characterized in that said antibody is an antibody capable of recognizing said nucleoside modified without recognizing the corresponding unmodified nucleoside.
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