Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• PROCESS FOR MARKING A PRODUCT USING A PLURALITY OF POLYNUCLEOTIDES METHOD FOR IDENTIFYING THE MARKING AND PRODUCT
• the present invention relates to a method of marking a product, a method of identifying the marking and a product labeled by the method of the invention.
• the labeling used in the present invention is based on single-stranded nucleic acids.
• the present invention makes it possible to distinguish an authentic product from a counterfeit.
• it makes it possible to mark the authentic product so that it can be traced and identified.
• counterfeiting or illegal reproduction of objects entails serious financial consequences for companies, but also in terms of jobs, health security or even social life. This is why manufacturers are working to combat this scourge by developing new marking and authentication techniques for their products in order to track and destroy counterfeit products.
• This type of marking must have particular properties: the marking must be done in a transparent manner vis-à-vis the end user of the product, it must not alter the physical properties. product and should not present a hazard to the end user of the product. It must also be, as far as possible, undetrable and / or tamper-proof to avoid being falsified at the same time as the product.
• markers There is no reliable technique today, stable over time, in particular for the very many chemical substances known to be aggressive for markers, such as perfumes, cosmetic creams or on materials such as leather, fabric, etc. To date, there are no tagging techniques that actually have great difficulty decrypting for counterfeiters.
• At least one target polynucleotide consisting of a single-stranded polynucleotide of defined length and sequence
• decoy polynucleotides which are of identical or different determined lengths and of identical or different determined sequences, said decoy polynucleotides being of identical length (s) or different (s) and of different sequences of the sequence of said at least one target polynucleotide wherein each of the target polynucleotides and lures does not hybridize with any of the other polynucleotides of said plurality of polynucleotides.
• said plurality of polynucleotides further comprises at least one recognition polynucleotide consisting of a single-stranded polynucleotide of defined length and sequence of nature identification and the sequence of the at least one target polynucleotide, wherein each recognition polynucleotide does not hybridize with any of the other polynucleotides of said plurality of polynucleotides.
• the marking of the present invention therefore consists of said plurality of polynucleotides as defined in the present description. These polynucleotides are single-stranded polynucleotides. In the present description, this polynucleotide is also designated by the term "marker”.
• the present invention also relates to a labeled product that can be obtained by the method of the invention and to a method for detecting the marking of this product.
• the present invention relates in particular to the determination of the marker used to implement the process of the invention, the manufacture of these markers, the marking of the products, as well as the techniques for detecting markers in the marked products.
• the present invention makes it possible to distinguish a counterfeit from an authentic product or to identify diversions of distribution circuits or unauthorized parallel circuits.
• the polynucleotides of said plurality of polynucleotides may be of several types: they may be single-stranded ribonucleotide or ribonucleic acid (RNA) polymers or deoxyribonucleotides or single-stranded deoxyribonucleic acid (DNA) or a mixture thereof.
• plural of polynucleotides is meant herein the set formed polynucleotide (s) target (s), lures, and, if appropriate, recognition.
• s target polynucleotide (s) target (s), lures, and, if appropriate, recognition are used.
• several target polynucleotides, several decoy polynucleotides and, where appropriate, several recognition polynucleotides are used.
• 2 to 100 polynucleotides for example from 2 to 50, can be used.
• target polynucleotide is meant herein a polynucleotide whose sequence has been determined and constructed to constitute a reference sequence for labeling the product according to the present invention, then to be specifically sought in this product to authenticate it.
• the marker comprises several identical or different target polynucleotides, preferably different.
• the target polynucleotides are referenced in a confidential target / product database that establishes the link between the target polynucleotide (s) and the labeled product.
• confidential database is meant a database to which only the manufacturer of the marking according to the present invention for a given product and / or only the manufacturer / creator of said product has (have) access (the one and / or or the other is hereinafter referred to as "the one who puts implement the present invention ").
• This is a correspondence base in which, for each product or each product family is assigned a particular signature determined according to the present invention. In the case where only target polynucleotides and decoys are used for the signature of the products, this database or correspondence may be called “confidential target / product base” or "target / product base”.
• the target / product base is first searched for the target sequence (s) assigned to said product, and then it is sought if the or the target sequence (s) is (are) actually present in said product, for example using one of the methods described below. If the target sequence (s) is (are) actually present, the product is declared authentic. On the other hand, if the target sequence (s) is (are) not present, the product is declared to be counterfeit. Of course, this only works if all authentic products are marked according to the present invention.
• the correspondence database may be constructed by the signature manufacturer of the present invention and / or by the manufacturer / creator from a list of signatures according to the present invention, for example by matching each signature to a particular product. In this case, the authentication is direct.
• decoy polynucleotide is meant herein a polynucleotide whose sequence has been chosen and constructed to be different from the target sequence (s).
• the decoy sequences are intended to scramble the marking according to the present invention, but not to be searched in the product to authenticate it.
• the decoy sequences are there to complicate the work of a counterfeiter to attempt to reproduce the signature of the present invention. Indeed, only one who implements the present invention knows the target sequence (s).
• sequences (s) target (s) for reproduction are made all the more difficult or impossible that, according to the invention, the Decoy sequences are present, which target (s) and decoys are short, single stranded sequences and do not hybridize with each other.
• the greater the number of decoys the more difficult the reproduction of the signature as a whole and, statistically, the less chance there is of randomly determining the sequence (s) of the target polynucleotide (s).
• sequences of these decoy polynucleotides are also known to those who practice the present invention, but these polynucleotides are not in a database to be assigned to a product.
• recognition polynucleotide is used herein to mean a polynucleotide of a sequence and of a determined length which allows the identification of the target polynucleotide (s). These may have a nature or be present in concentrations facilitating quick search and identification, which can serve as a first test of authentication: their absence is a first sign of counterfeiting. This is one or more polynucleotide (s) whose sequence (s) has (have) been chosen and constructed to constitute a code used in a confidential database of identification of the target polynucleotide (s) or "target identification base” to provide information on the number, nature, sequence and length of the target polynucleotide (s).
• a set of recognition polynucleotides In this case, in the target identification base, for each recognition polynucleotide or recognition polynucleotide set, is assigned a target polynucleotide or a set of target polynucleotides according to the present invention.
• the sequences of the recognition polynucleotides are known only to those who implement the present invention.
• the presence of polynucleotide (s) of recognition in the signature of the The present invention is optional, it corresponds to a particular embodiment of the method of the invention.
• a confidential target identification base is created by the one who implements the present invention. This base makes it possible to identify the target polynucleotide (s) present in a product to be authenticated from the recognition polynucleotide (s). In this case, the authentication of a product is indirect. Indeed, if it is desired to authenticate a product intended to include a signature according to the invention, the recognition polynucleotide (s) is identified according to one of the methods described below of the present invention, it is then sought in the database.
• target identification (s) target (s) assigned (s) to said product and then it is sought if the target sequence (s) is (are) actually present (s) in said produced, for example using one of the methods described below. If the target sequence (s) is (are) actually present, the product is declared authentic. On the other hand, if the target sequence (s) is (are) not present, the product is declared to be counterfeit. Note in this embodiment of the present invention that there are two steps of identifying polynucleotides before being able to authenticate a product, which complicates the task of any counterfeiters. Of course, this only works if the authentic products are marked according to the present invention.
• the sequence of the recognition polynucleotides may contain a sub-sequence bearing the code which enables the user of the invention to find which are the target polynucleotides, and which are the polynucleotides luring from a base of target recognition.
• these recognition polynucleotides are extracted from the product to be authenticated, or directly identified in or on the product.
• the identification of the target polynucleotides can be done by reading the sequence of a recognition polynucleotide, or by identifying a set of recognition polynucleotides present among a plurality of putative coding polynucleotides.
• the user of the present invention will refer in a correspondence table (database of recognition of target polynucleotides), and read therein the nature of the target polynucleotides theoretically present in the product to be authenticated.
• This table can for example, and without this example being limiting, be stored in a secure database and computerized (to ensure confidentiality), which database was created when marking the product, and in which couples ("read recognition polynucleotide code" - "target polynucleotides to be searched for").
• the user of the invention can deduce the exact nature of the target markers that should be present in the product to be authenticated.
• the target polynucleotides are then extracted and identified. If the detected target polynucleotides exactly match the theoretical code read in the table, then the product is authenticated. If other target polynucleotides are present, the user of the invention may suspect a mixture of labeled products.
• the target polynucleotides detected are totally different from those expected, it may be a counterfeit. If there is no recognition polynucleotide used in a signature according to the invention, only a target / product database is useful.
• the polynucleotides of the plurality of polynucleotides can be designed for example by methods known to those skilled in the art, for example using a software implementing an algorithm as presented below, such that, for a given polynucleotide, selected from the plurality of polynucleotides constituting the labeling, no other polynucleotide of this plurality, nor any complementary polynucleotide inverted thereof.
• polynucleotides consists of a nucleotide sequence complementary to this given polynucleotide.
• no double-stranded complex for example a nucleic acid double helix, such as for example a DNA double helix, nor any hybridization between the polynucleotides of the signature of the present invention can be formed even at the temperature and in the molecular environment of product conditioning, and at the temperature and molecular environment of polynucleotide discovery.
• formation of a double-stranded complex is meant a pairing of complementary, energetically stable nucleotides, including under the conditions mentioned above.
• inverse complementary polynucleotide of a given polynucleotide is meant a new polynucleotide, existing or theoretical, where each nucleotide of the given polynucleotide is replaced by a complementary nucleotide that can be paired with the first, such as, for example, adenine replacing a thymine. , or a thymine replacing an adenine, or a cytosine replacing a guanine, or a guanine replacing a cytosine in the case of a deoxyribonucleic acid polynucleotide.
• hybridization is meant the association by non-covalent bonds of two complementary single-stranded polynucleotides. This hybridization can be perfect, ie the sequences are totally complementary, or imperfect, that is to say that the sequences are not totally complementary but sufficiently complementary to hybridize with each other and form a double structure. -strand.
• non-hybridization means the non-association by non-covalent bonds of two single-stranded polynucleotides because they are not complementary and / or because the complementarity is insufficient for the formation of a double strand.
• the plurality of polynucleotides of the tag of the present invention to be incorporated in a product or a substance of interest are not all assigned in a database to directly or indirectly authenticate a product. It is thus possible to use only a small number of target polynucleotides, these target polynucleotides being those which are sought during an authentication of the product, and to incorporate them into the product at the same time as a large number of decoys.
• the target polynucleotide (s) can be "embedded" in a mass of decoy polynucleotides that are extraordinarily scrambling the tracks in case of attempted malicious decoding of the target polynucleotides in order to reproduce the signature.
• recognition polynucleotides in which recognition polynucleotides are used, only reading and decryption of the code carried by the recognition polynucleotides can incriminate, among a mixture of target polynucleotides and polynucleotides lures. , which actually correspond to the target sequences, and by elimination which are only decoys intended to deceive the counterfeiter.
• the labeling method of the present invention can be carried out using markers which are deoxy and / or ribonucleic acids.
• the said polynucleotides of the plurality of polynucleotides may thus be single-stranded deoxyribonucleic acid or single-stranded ribonucleic acid sequences or a mixture of deoxyribonucleic acid and ribonucleic acid sequences.
• a marker according to the present invention can therefore be composed of common bases, called “natural bases”, for example those present in DNA: adenine, guanine, thymine, cytosine, or in RNA: adenine, guanine, uracil, cytosine (see, for example, Molecular Cloning, Maniatis, Spring-Harbor CoId, 2nd Edition, pp C3-C14 [1]).
• a marker according to the The present invention may also include less frequent natural or synthetic compounds, referred to as "modified bases”, such as, for example, dihydrouridine (DHU), inosine, or pseudouracil resulting from modifications, for example deamination, carried out on the previously presented bases.
• DHU dihydrouridine
• inosine inosine
• pseudouracil resulting from modifications, for example deamination
• the nitrogenous bases can be made from natural isotopes and / or from stable isotopes of different atomic mass and / or be modified to establish a number of hydrogen bonds different from normal during the hybridization processes.
• the deoxyribonucleic acid sequences may comprise, in their sequence, the same proportion of the four bases A, C, G and T, natural or modified.
• the ribonucleic acid sequences may contain, in their sequence, the same proportion of the four bases A, C, G and U, natural or modified.
• the set of polynucleotides comprising the marking have the same number of nucleotides, and the same molecular weight.
• This particular embodiment of the invention advantageously makes it even more difficult or impossible to separate and identify the polymers by a possible counterfeiter. For example, separation and identification as a function of size and / or molecular weight by techniques such as electrophoresis, for example on agarose or polyacrylamide gel and / or mass spectrometry is impossible to realize on a signature according to these last embodiments, in particular the last.
• a single-stranded (or oligomeric) polynucleotide of 20 nucleotides, each of the nucleotides being selected from 4 possible bases makes it possible to make 420 different sequences, ie about 1, 1 ⁇ 10 12 combinations, that is, to say a thousand billion combinations.
• the probability of extracting a target marker, according to the invention, at random for example a plurality of polynucleotides of size 20 in a marking according to the present invention, and that this marker is the one that has been assigned to the product in a base of target / product data is therefore almost zero.
• the labeling of the present invention is composed of several target molecules of determined sequences and length, of several decoys, and, if appropriate, of several recognition polynucleotides, which ensures both a very high safety and an inviolability. outstanding marking.
• the polynucleotides used may therefore comprise, for example, an oriented combination of 4 nitrogenous bases, the nature of which is to be defined by the one who implements the present invention.
• This combination which is at the origin of the specificity of each polynucleotide of the marker of the present invention, and which can bear the information relating to the labeled product, can be calculated in a computerized manner, according to the needs (complexity of the code, type information that the markers bear) and the physico-chemical properties of these markers (hybridization properties, molecular weight, fragment size, composition of nitrogenous bases).
• one or more target polynucleotide (s) may be used.
• the invention therefore allows a considerable number of signature or marking variants.
• the target polynucleotides of the first set there may be mentioned the following different forms: one or more target single-stranded polynucleotide (s) of large size (s), that is to say comprising for example 500 to 5000 nucleotides or bases; one or more target single-stranded polynucleotide (s) of small size (s), ie for example comprising from 5 to 200 nucleotides or bases, for example from 15 to 200, for example from 20 to 200, for example from 5 to 50; one or more target single-stranded polynucleotide (s) of average size (s), ie comprising, for example, from 201 to 499 nucleotides or bases; one or more target single-stranded polynucleot
• At least two target polynucleotides can be used, one being a circular polynucleotide and the other a linear polynucleotide.
• a plurality of circular or linear target polynucleotides or a mixture thereof may be used, depending on the chosen complexity of the labeling by the one implementing the present invention.
• the single-stranded polynucleotides when some or all of the single-stranded polynucleotides are linear, they may comprise a variable end of one polynucleotide to another and a constant end of one polynucleotide to another .
• constant end is meant a part of the polynucleotide sequence including one of the two ends of said sequence and having a determined and constant sequence, that is to say identical for part of the target sequences or for all the target sequences of the the present invention.
• variable end is meant a part of the polynucleotide sequence including the other two ends of said sequence and having a variable sequence variable from one target sequence to another in the marker of the present invention.
• a solid support for decryption can be used, on which support polynucleotides complementary to the variable ends of the target polynucleotides are fixed, as for a DNA chip.
• the constant ends can be used to highlight the hybridization of target polynucleotides on the solid support, for example by means of biotin / streptavidin. This detection mode is described below.
• the number and nature of the target polynucleotides, combined with their size, makes it possible to define the complexity of the marking, and in a combinatorial manner, the number of possible combinations.
• the number of possible combinations increases exponentially with the size of these polynucleotides.
• the labeling information may consist of: target polynucleotide sequences, each assigned in a target / product-to-product database, and / or one or more combination (s) of target polynucleotide sequences, said (said) combination (s) being assigned in a target / product database to a product.
• target polynucleotides of determined sequences it is possible to use several target polynucleotides of determined sequences.
• decoy polynucleotide sequences to form the label according to the present invention.
• the decoy polynucleotides do not hybridize with the target polynucleotides and their role is to make it even more difficult for a counterfeiter to decrypt the marking of the present invention in order to copy it.
• These decoy polynucleotides may be in linear or circular form or a mixture of circular polynucleotides and circular polynucleotides as indicated above for the target polynucleotides.
• the number of lure polynucleotides added to the marker depends on the desired interference. Preferably this number is greater than the number of target polynucleotides. Examples are given above.
• the decoy polynucleotides are of the same or different length (s) from one another, preferably of identical length to the one, to the one or to the target sequence (s) present in the the present invention, for example, as indicated above for the target polynucleotides, from 15 to 5000 bases, for example from 15 to 200, for example from 20 to 200, for example from 201 to 499, for example from 500 to 5000. bases, or a mixture of these lengths.
• recognition polynucleotides allowing, by means of a database, to discriminate the target polynucleotides of the decoy polynucleotides as indicated above.
• the number of recognition polynucleotides depends in particular on the complexity of the desired marking.
• the recognition polynucleotides may be circular or linear. They may be of the same or different length (s) and of identical or different length (s) from those of the target polynucleotides and decoys, for example, as indicated above.
• target polynucleotides of 5 to 5000 bases for example from 15 to 200, for example from 20 to 200, for example from 201 to 499, for example from 500 to 5000 bases, or a mixture of these lengths.
• said polynucleotides of the plurality of polynucleotides may be circular, linear, or a mixture of circular and linear polynucleotides, for example with a free 3'OH end and a free 5 'phosphate end.
• the length of the polynucleotides of the labeling of the present invention is from 5 to 5000 nucleotides, for example from 5 to 100 nucleotides, for example from 5 to 50 nucleotides, for example from 20 to 50 nucleotides.
• said polynucleotides of the plurality of polynucleotides are single-stranded polynucleotides.
• one of the peculiarities of the present invention is that the use of single strands makes it more difficult to decrypt the marking of the present invention.
• the marking method of the present invention thus makes it possible to manufacture a very large number of markers.
• Each marker comprises a code consisting of the target polynucleotide (s) and, if appropriate, recognition polynucleotides.
• sequences of the polynucleotides of the marker of the present invention can be created empirically or, preferably, especially for a matter of speed, by appropriate software that can be generated for this purpose. In the latter case, it is a computer design or "Design In Silico" marker of the present invention.
• step (1) If the set of scores does not exceed a threshold given by the user, add p in E. Return to step (1) as long as E is not of a desired size.
• This threshold is a minimum alignment score above which two sequences are considered sufficiently identical to hybridize with each other.
• these target polynucleotides and decoys can be manufactured by any existing method known to those skilled in the art.
• One or more protocols may be used depending on the nature of the polynucleotides manufactured and according to the chosen marking: synthesis of ribonucleic and / or deoxyribonucleic acids, single-stranded, circular and / or linear, of a size may vary for example from 5 bases to 5000 bases. Examples of protocols that can be used to implement the present invention are those making it possible to synthesize single-strand circular polynucleotides [3] or the protocols for in silico synthesis of polynucleotides [4].
• sequences of the recognition polynucleotides are desired for labeling, their respective sequences can be determined empirically, or using an algorithm as described above, so that the recognition polynucleotides do not hybridize with each other or with each other. with the target polynucleotides, or with the decoy polynucleotides.
• the labeling of a solution or a compound using markers of ribonucleic acid or deoxyribonucleic acid according to the process of the invention can be done in different ways depending on the desired labeling complexity.
• Each target polynucleotide carries specific information, inherent to its sequence.
• Each target polynucleotide or target polynucleotide combination can be used uniquely.
• the first possible level of coding may be that of using one or more target polynucleotides.
• Several batches of labeled products can then be traced by one or more polynucleotide (s) of defined size (s), and of sequence (s) determined (s) but different from each other.
• the second possible level of coding is the use of several target polynucleotides, chosen from an initial pool of target polynucleotides. The code then no longer comes from each target polynucleotide sequence but from the combination of the target polynucleotides found in a product.
• a labeled product can be labeled with n target polynucleotides selected from a possibility of N different polynucleotides, n being in the range [0; NOT].
• the tag of the present invention may be supplemented by a third level of coding, consisting of using recognition polynucleotides, then indicating, by means of a target recognition base, which markers among the polynucleotides.
• the targets which may be used by the manufacturer and which may be different in nature and concentration, are present if the product is authentic and marked with the process of the present invention.
• each step of the product marking process can be subject to specific traceability. This traceability can be ensured by introducing specific data at each stage into one or more confidential databases.
• Each marker batch container followed by means of its identifier, can be attached in a database for example to the reference of a customer's order and to the delivery references of this container to said customer. A confirmation of receipt from the customer can also be entered into this database.
• nucleic acids do not alter the physicochemical properties of marked products. In addition, the nucleic acids have no effect on their container, i.e., the labeled product. Finally, the nucleic acids have proved very stable in the numerous tests carried out by the inventors. This property of stability is demonstrated in the examples presented below.
• the single-stranded DNA and RNA markers included in the present invention can be included in a very wide range of products and substances that are likely to be victims of illegal reproduction (counterfeiting), abusive, trafficked on a parallel market and / or where it is necessary to follow the track (tracing of products).
• the process of the invention is applicable to the marking of any industrial or consumer product, liquid, semi-liquid or solid.
• the invention can be applied to a whole range of products in the luxury and cosmetics industry: perfumes, perfume waters, toilet waters, essential oils, creams, masks, ointments, etc.
• the invention can also be used to trace various substances such as inks, resins, varnishes, paints, dyes, additives, flavors, glues, powders.
• the present invention finds application to luxury products that may be targets of counterfeiting or cheating (mixtures), such as liqueurs, spirits, wines, or any product that is important to ensure authenticity for security reasons for example.
• Markers can also be used in the pharmaceutical industry to mark and trace drugs and other drugs.
• the invention can also be used in tracing biological samples in a hospital context. This may involve, for example, implementing a traceability protocol for blood samples in a biochemistry laboratory, tumor samples in an anatomopathology laboratory, or in order to constitute a a bank of directly authenticated human material that can be stored and traced for many years in a biological resource center.
• the products can be marked either as such or as a component of a product.
• a paper document can be marked through the ink used which has been previously marked.
• the step of adding the marker of the present invention may be carried out by any suitable means for adding to the product to be labeled the polynucleotides constituting the marker of the present invention.
• the products or substances to be marked can be marked in the mass, by incorporating the markers whose final concentration is studied and planned or on the surface of the product.
• the markers are polymers of ribo or deoxyribonucleic acids having the physicochemical properties deriving from their nature: they are hydrophilic and negatively charged molecules.
• they may be pre-diluted or added directly into the product. They can also be encapsulated. They can still be deposited or incorporated on the surface of the product.
• This addition of the marker of the present invention to the product can be carried out by adding said plurality of polynucleotides in said product during its manufacture and / or in or on (ie on the surface) the finished product.
• the present invention thus also relates to a labeled product that can be obtained by the marking method according to the invention.
• the addition of the marker when the addition of the marker is carried out on the surface of the product to be marked, it may be carried out for example by soaking the finished product in a solution comprising the said marker or by spraying or spraying such a solution on the final product.
• the solution is a protic solvent such as ethanol, methanol or diethylene glycol, or a polar aprotic solvent such as acetone or tetrahydrofuran.
• This mode of addition is suitable for example for solid products after manufacture such as fabrics, leather, wood, paper, cardboard, tobacco, cigarettes, cigars, etc.
• the addition can also be carried out during the manufacture of the product, by mixing said marker with the compounds or components constituting the product.
• This introduction of the plurality of polynucleotides can be carried out on or in a component of said product.
• This type of addition is suitable for any product or substance whose manufacture passes through a liquid or semi-liquid phase in which the markers may be incorporated. This may be the case for example for the marking in the mass of a cosmetic product or a drug.
• a step of encapsulation of said plurality of polynucleotides in lipid vectors can be carried out prior to the addition step.
• This encapsulation step makes it possible to maintain the polynucleotides in a favorable medium, or to facilitate their future extraction.
• a product chosen from the group comprising a vector of cationic lipid nature such as dioleoxyloxypropyltrimethylammonium bromide (DOTMA) and dioleoylphosphatidylethanolamine (DOPE), or polynucleotide complexes may be used as an encapsulation product. with molecules like polylysine, protamine, or polyethyleneimine (PEI) called polyplexes.
• Bioch the technique described in Bioch can be used for this purpose.
• a step of encapsulating and / or protecting said plurality of polynucleotides in lipid vectors or the like may be carried out prior to the addition step.
• This step also makes it possible to ensure stability and / or facilitate its recovery.
• the term "protection" herein refers to the protection of the polynucleotides of the present invention, in particular any physicochemical attack originating from the medium in which the signature of the present invention (perfume, food) is found, for example in charged polymers or carbon nanotubes.
• the marker of the present invention is preferably added to the product for its labeling at very small concentrations ranging from micro-molar to femto-molar.
• the respective concentrations of each target polynucleotide of the first set may be different and compose a coding subset.
• the concentration of the plurality of polynucleotides after its addition in said product can be, but is not limited to, 10 -6 moles to 10 -18 moles / dm- 3 .
• this corresponds to the amount of markers mixed with the product per unit volume.
• this corresponds to the amount of markers mixed in the product per unit volume or deposited on the surface of the product. reduced to the product surface, this amount can also be defined per unit area, or 10 "6 moles to 10" 18 moles / dm 2.
• concentrations can be obtained by dilution of a more concentrated solution. the solution may be as defined above.
• the present invention also relates to a method for detecting the labeling of a product that can be obtained by the labeling method of the invention, said method comprising a step of analyzing the plurality of polynucleotides making it possible to specifically detect the at least one target polynucleotide.
• the invention further provides several methods for detecting markers of the present invention. The detection can be carried out laboratory or laboratory, for example by means of a portable system, for example by means of a DNA chip specially designed for the detection of target polynucleotides of the marker of the present invention.
• the analysis step can be carried out for example by immunodetection.
• the analysis step may comprise, for example, a step of sequencing the coding polynucleotides.
• the analysis step may comprise, for example, a retro-transcription of the ribonucleic acid into deoxyribonucleic acid, in particular when the target polynucleotides are ribonucleic acid.
• the analysis step can comprise a colorimetric or luminescent detection, or fluorimetric, coupled to a specific hybridization. In other words, the analysis step may use a specific means for detecting the target polynucleotides.
• the analysis technique used can implement the physico-chemical properties, the code and the specificity of the marker.
• This may be, for example, a sandwich assay technique, a detection technique using microarrays, or any other technique known to those skilled in the art to reveal the presence of polynucleotides and / or identify them.
• target polynucleotides and decoys can be polynucleotides of small size, that is to say 8 to 30 nucleotides / nucleosides and single strands.
• these polynucleotides by their nature can not be used as a template strand to be amplified and detected by exponential amplification techniques such as chain polymerization reactions (" PCR ", Polymerase Chain Reaction). Thus, their detection by PCR amplification is impossible.
• the detection of the polynucleotides can be carried out by hybridization methods and direct revelation without amplification is achievable only by the user who knows the signature used.
• the counterfeiter who would try to extract, amplify and reproduce the plurality of polynucleotides present in the labeled product will be held in check.
• the revelation methods comprising only hybridization and revelation stages of this hybridization can be implemented much more rapidly (a chain polymerization reaction takes several hours, whereas simple hybridization polynucleotides is almost instantaneously) insofar as the detection is specific to the polynucleotides to be revealed.
• the analysis technique used can be based on the physicochemical properties, the code and the specificity of the marker. It can include amplification of the target polynucleotides, exponentially or linearly, any other technique can be used to reveal the presence of the markers.
• the analysis step may comprise a step of linear extension of the target polynucleotides.
• the method of the invention may further comprise the following steps, prior to the analysis step: (a) taking a sample of the product; and
• the extraction step (b) can be carried out by any technique known to those skilled in the art to extract the polynucleotides from a sample.
• the polynucleotides can be extracted according to a protocol depending on the nature of the labeled product. Any type of ribonucleic or deoxyribonucleic acid extraction technique existing or future, can be used to extract the polynucleotides from the mass of the labeled product. It may be for example a phenol chloroform extraction. Extraction techniques that can be used in the present invention are for example described in Molecular Cloning.Maniatis, CoId Spring-Harbor, 2nd edition, pp E3 to E4 [8].
• the methods of analysis of the labeled products can therefore consist in extracting the polynucleotides of these products, in detecting the code carried by the recognition polynucleotides, in referring to the database of recognition of the target polynucleotides, in thus decrypting the code carried.
• the recognition polynucleotides then exploiting this information in order to find the target polynucleotides among the plurality of target polynucleotides, including the decoy polynucleotides, and then to detect the presence of the target polynucleotides, which is characteristic of the labeled product (in case of detection of the marking) or to conclude that a counterfeit product (in the absence of the target polynucleotides, or of non-conforming marking to the target / product database).
• the specific detection of the analysis step can comprise for example the following successive steps:
• step (ii) removing non-hybridized polynucleotides by step (i); and (iii) detecting the presence on the support of the target polynucleotides.
• the detection carried out in step (iii) may be carried out by a suitable specific means, for example it may be for example it may be a detection using a fluorescent molecule, or a detection using a luminescent molecule, or detection using an enzyme whose reaction product may be colored, or detection using an enzyme whose catalyzed reaction is exothermic, or detection using an enzyme whose catalyzed reaction releases light, or detection using a specific protein target polynucleotides, such as an antibody, an enzyme.
• the specific detection may furthermore comprise, between steps (i) and (ii), a step of capturing the plurality of polynucleotides on a support, by at least one specific capture system of the polynucleotide (s).
• the method of this particular embodiment may further comprise, before step (i), a step (x) for identifying these recognition polynucleotides and a step (y) of choice of a solid support, according to the identified recognition polynucleotides, solid support chosen because it comprises the complementary probe sequences of the target sequences identified by the recognition polynucleotides.
• This detection method may advantageously be used with a marker according to the invention which comprises target polynucleotides having a constant end and a variable end. These target polynucleotides are defined above.
• the polynucleotides complementary to the target polynucleotides can be fixed on the solid support by any means known to those skilled in the art.
• These techniques of detection on support and the types of supports that can be used in the present invention are, for example, those described in Molecular Cloning, Maniatis, CoId Spring Harbor, 2nd Edition, pp. 9.47 to 9.57 [9].
• the fixing of the probe sequences on the support can be carried out by means of a biotin / streptavidin binding, the probe being coupled to a biotin molecule and the support having streptavidin molecules.
• the attachment of the probe sequences can also be accomplished by covalent bonding to a charged nylon membrane, said membrane forming the solid support.
• a charged nylon membrane said membrane forming the solid support.
• It may be, for example, a detection by means of polynucleotides labeled with a labeling agent and complementary at the other end of the target polynucleotides, the labeling agent being able to be chosen from the group comprising a fluorochrome, a d colloidal gold and an enzyme.
• the detection method of the invention may furthermore comprise a step of comparing the results of the step of analysis of the target polynucleotides with the content of a database which makes it possible to identify the target polynucleotides, and of analysis target polynucleotides which makes it possible to identify and authenticate said product, and which can also make it possible to determine the origin of said product.
• the database and the decryption of the code carried by the Coding polynucleotides can identify a counterfeit of an original product.
• the user manufacturer of a marker according to the present invention can associate with the identifier of the marker batch container references relating for example to a production lot labeled with the help from this lot of markers. This association can be done for example in a database identical or separate from previous databases.
• the information inserted in this database and relating to the production batch is preferably sufficient, depending on the information system implemented at this user, to allow to trace this lot unequivocally.
• the revelation of the identified polynucleotide sequences can be compared with the data entered into the databases during the production and delivery of markers. Counterfeiting is for example characterized if no marker is identified. Counterfeiting can also be demonstrated if at least one marker in the marker set that has been partly revealed and whose precise composition has been obtained by querying the database is missing. Counterfeiting can still be characterized if all the markers are present the batch of markers, but that the goods tested do not come from the manufacturer who took delivery of the batch of markers which has just been revealed.
• the products are authentic and the batches of markers revealed actually correspond to batches of markers delivered to the manufacturer of the tested product.
• the method of the invention makes it possible to obtain, by query in the manufacturing database, markers of the identifier of the batch of markers.
• this identifier advantageously allows the manufacturer to compare the theoretical allocation of the marked production batch to one of his customers with the actual allocation found during the taking of a product sample suspected of parallelism. If the theoretical assignment of the products does not correspond to the actual allocation, there may be diversion of the distribution channel.
• FIG. 2 represents an addressing of the probes on the chip.
• FIG. 3 represents emission and absorption spectra of Cy5.
• FIG. 4 appended represents a method for detecting the markers according to the present invention on a support:
• the markers M1 and M2 are present in a mixture. They make "bridge” between the probe attached to the chip, and the Universal probes: the signal (Cy5 fluorescence) is then detected.
• the signal Cy5 fluorescence
• FIG. 5 reveals a revelation of the markers with the NanoChip station (registered trademark) (Nanogen Inc.).
• FIG. 8 represents a detection scheme of a marking according to the invention and authentication of a labeled product according to the invention.
• the target polynucleotides and decoys used are single-stranded sequences of deoxyribonucleic acid with a size of 28 nucleotides.
• the encoding polynucleotide is a circularized DNA sequence, 4.3 kilobase (kb) in size.
• the marked product is a perfumed solution: J'Adore perfume (registered trademark, perfumes Christian Dior).
• Three markings are made, illustrating three possible scenarios during the authentication process: an example where the product is authenticated, as well as two examples where the product is not authenticated.
• Ten single-stranded deoxyribonucleic acid polynucleotides are generated as follows: The ten 5 'nucleotides are all identical, defined by the user. This is the sequence GCAACTCCAG. The eighteen nucleotides at 3 'are then generated using the algorithm presented in the "Presentation of the invention" section, using the following parameters: a length of words equal to 18 nucleotides, five nucleotides G, five nucleotides C, four nucleotides A and four nucleotides T.
• Each new polynucleotide is then generated in a random manner, so that it contains this determined number of each of the bases.
• An alignment score between this new polynucleotide and the set of polynucleotides already validated, as well as the polynucleotides resulting from the concatenation of each of the two by two polynucleotides of this set, this step not having any place if it is is the first polynucleotide. is calculated according to the Smith and Waterman algorithm, with the following parameters:
• the minimum score selected is 3, which means that if the new polynucleotide aligns with a polynucleotide of the set, or a concatenation of two polynucleotides of the set with a score greater than 3, it is discarded. Otherwise, it is validated and then added to all the polynucleotides. This step is repeated until ten polynucleotides are obtained.
• Primer- 1 5 1 -GCAACTCCAGGCACTCCATGAGTCATGG-3 I (SEQ ID NO :: 1)
• Primer-2 5'-GCAACTCCAGGTGGCGACTCATACGTCA-3 1 (SEQ ID NO: 2)
• Primer-3 5 I -GCAACTCCAGCTCAGGGGGACTCTATCA-3 1
• Primer-4 5 I -GCAACTCCAGGCTCTAGGGCAAGTCTCA-3 I
• Primer-5 5'-GCAACTCCAGGCAGACTCTGGATCTCAG-3 1
• Primer-6 5 1 -GCAACTCCAGGCAGCATGAGGTCTCATC-3 I
• Primer-7 5 I -GCAACTCCAGGCAGCAGGAGTCTCATTC-3 1
• Primer-8 5 1 -GCAACTCCAGGGTGGCTCAGCAATACTC-3 1
• Primer-9 5 I -GCAACTCCAGCTCAGGGCAGTGATCTCA-3 I
• the first five polynucleotides (Primer-1 to Primer-5) are considered as target polynucleotides.
• the last five (Primer-6 to Primer-5) are considered as target polynucleotides.
• Primer-10 are considered lure polynucleotides. These markers are then injected at a final concentration of 10-12 moles per dm 3 into the perfume, along with the coding marker.
• the ten polynucleotides are mixed in three pre-labeling solutions, diluted in deionized water, without nuclease, to a final concentration of 10 -9 moles of markers per liter, per label (ie 5.10 9 mol / l of total markers). .
• each label is pre-diluted twice to 1/100 in water without nuclease (1 .mu.l using a P10 pipette, in 99 .mu.l using a P100 pipette) or an intermediate dilution at 1/10000.
• each intermediate solution is added to a 1.5 ml tube (Eppendorf, registered trademark), and the mixture is then added to 100 ⁇ L by addition of 50 ⁇ L of water (using a pipette P100).
• Table 1.2 shows the three pre-labeling solutions (A, B, C) each containing a combination of five target polynucleotides selected from ten possible, target or decoys.
• the encoding polynucleotide is a circular single-stranded nucleic acid sequence of 4.3 kilobases (kb) in length. It is, for the example, synthesized from a plasmid pBR322. This polynucleotide contains within it a specific sequence following nucleotides (A, T, G, C), known to the user of the invention. It constitutes a unique code, which subsequently allows the user to know which combination of target markers is in theory present in the marked product.
• the coding sequence is a portion of 20 nucleotides located exactly 50 bases upstream (5 'side) of a known and universal sequence which remains constant regardless of the coding sequence, and whose sequence is: 5' -CTGTAAGCGGATGCC-3 '(SEQ ID NO: 11).
• the user has an associative table that allows him to link the coding polynucleotide coding sequence, and the target polynucleotide combination expected in the labeled product.
• the plasmid is first digested with the restriction enzyme Nb.BpuiOI, which cuts a single strand of the DNA molecule, recognizing the restriction site shown in Figure 1: NbBpuiOI cleavage site.
• the polynucleotide is then digested a second time with exonuclease III, releasing the nucleotides from the free 3 'hydroxyl end of the cut strand by Nb.BpuiOI.
• the strand which has not been digested with Nb.BpuiOI is then spared, because circular.
• Plasmid pBR322 Invitrogen (registered trademark)
• Buffer R 10 X (buffer Nb.BpuiOI) reference # BR5, (Fermentas) - Exonuclease III (1200 U) reference # EN0191, (Fermentas)
• the markers taken up in 20 ⁇ L are assayed using a Nanodrop spectrophotometer (registered trademark).
• the concentration is then reduced to 10-9 mol / L by adding a suitable volume of demineralized water without nuclease using a P10.
• the polynucleotides are injected into the mass of the products, at a final concentration of 10 -12 M for the target polynucleotides and decoys, and 10 -12 M for the coding polynucleotides.
• the first solution is labeled with the pre-labeling solution A, containing the target polynucleotides 1 and 2 as well as the decoy polynucleotides 8, 9, 10 and the encoding polynucleotide.
• This solution corresponds to normal labeling, where the encoding polynucleotide is present, and where the combination of the target polynucleotides present actually correspond to the information carried by the encoding polynucleotide.
• the second solution is labeled with the pre-labeling solution B, containing the target polynucleotides 3, 4 and 5, as well as the decoy polynucleotides 6 and 7 and the encoding polynucleotide.
• This solution is an example of incoherent labeling: although the coding polynucleotide is present, the combination of target polynucleotides does not correspond to the information it contains.
• the third solution is labeled with the pre-labeling solution C, containing the target polynucleotides 1, 2, 3, 4 and 5, no decoy polynucleotide, and no coding polynucleotide.
• This solution is also an example of incoherent labeling: on the one hand it does not comprise coding polynucleotides, and on the other hand, although it has the target polynucleotides 1 and 2 of the normal labeling, it contains other unexpected polynucleotides.
• the solutions are stored at room temperature or at 4 ° C.
• the markers are extracted from their alcoholic medium (perfume) and then recovered in an aqueous medium in order to then be able to use molecular identification techniques.
• the extraction should preferably not only have a good yield (recover a maximum of markers, ideally: 100% yield), but it should also preferably rid the markers of any "polluting" substances that can interfere with the detection techniques .
• the technique used is that of phenol chloroform extraction.
• the markers are extracted from 500 ⁇ L of labeled perfume.
• Vi volume of water is 250 ⁇ l using a P1000 (Gilson Pipetman, registered trademark) and then vortex for 10 seconds. Centrifuge for 5 minutes at 10,000 rcf (in Centrifuge 5415 R centrifuge, Eppendorf, registered trademark).
• the information carried by the polynucleotides coding, relative to the target polynucleotides, is read in the nucleotide sequence of these first (by a sequencing technique). It then allows the user of the invention to know the exact nature of the target polynucleotides carrying the product authentication information by referring to the associative table presented in 1.b.1.
• Matrix extracted polynucleotide solution.
• sequence reaction is carried out from the solution of markers extracted in a 200 ⁇ L tube (Eppendorf, registered trademark), according to the protocol set forth in Table 1.5.
• the reactions are then sequenced on an ABI3100 capillary sequencer 16 (Applied Biosystem). Analysis of the results then enables the user of the invention to read the nucleotide sequences of interest, and to correlate the read sequences with the target polynucleotides theoretically present in the labeled product.
• a support (or DNA chip) is used to detect the presence of target polynucleotides.
• This support has a battery of several probes, fixed in a covalent manner, and complementary complementary inverse 3 'variable regions of the markers.
• the appended FIG. 2 represents the addressing of the probes on the chip. When the extracted solution, containing the putative markers, is brought into contact with this support, the present markers hybridize with their inverse complementary on the chip. After washing, the support is then brought into contact with a solution containing probes (polynucleotides) coupled to a fluorophore (Cy5), thus the sequence is the exact complementary inverse of the 5 'universal region of the markers.
• the appended FIG. 3 represents the emission (667 nm) and excitation (650 nm) spectra of the Cy5.
• FIG. 4 represents the principle of detection of the markers.
• Anti-1 probe SEQ ID NO: 14
• Anti-3 probe SEQ ID NO: 16
• Anti-4 probe SEQ ID NO: 17
• Anti-5 probe (SEQ ID NO: 18) 5 '(B) -TGGATCCCGCACACGACTGACTGAGATCCAGAGTCTGC 3'
• Anti-6 probe SEQ ID NO: 19
• Anti-8 probe SEQ ID NO: 21
• AntMO probe SEQ ID NO: 23
• NanoChip (registered trademark) Electronic Microarray, 100-Site NanoChip (Trademark) Cartridge)
• Nanochip processing station registered trademark
• Molecular biology Workstation Nanochip (registered trademark) Reader, Nanochip (registered trademark) Loader
• High salinity buffer 500 mM sodium phosphate, 500 mM sodium chloride, Nanogen TM
• Low salinity buffer 50 mM sodium phosphate, Nanogen®
• the probes complementary to the specific regions of the markers are purified on the Multiscreen filtration system (from Millipore (registered trademark)) and then taken up in 60 ⁇ l of 50 mM L-histidine buffer. They are then transferred to the cartridge. Each of them is then addressed to a specific site of the cartridge for a period of 120 seconds (protocol managed by the workstation). The 5 'biotin binds to the streptavidin of the support.
• the user After reading the information carried by the encoding polynucleotide present in the first and the second product, the user refers to the associative table indicating that the code of the encoding polynucleotide corresponds to the presence of the target polynucleotides 1 and 2 (no matter the lure polynucleotides present). These polynucleotides are well detected in the first labeled solution, and only these two polynucleotides: the first solution can be authenticated. As for the second solution, the unexpected presence of the target polynucleotides 3, 4 and 5 does not make it possible to authenticate the product.
• This example illustrates a labeling using several single-stranded deoxyribonucleic acid polynucleotides, injected into a skin cream, and detected according to the technique presented in Example 1.
• the markers are first prepared nuclease-free distilled water and then incorporated the cream, to a final concentration of 10-12 moles per tablet of dm 3 cream for target markers and decoys, and at a concentration of 10 " 14 moles per dm 3 of cream for the coding markers.
• a preliminary mixture of target, coding and decoy polynucleotides is injected using a P10 pipette (Gilson Pipetman, registered trademark) into 1 cm 3 samples of Thermal Fix (registered trademark) creams, leading to a final concentration of 10-12 moles of marker per dm 3 for target markers and decoys, and a final concentration of 10-14 moles per dm3 for the encoding polynucleotides, the samples are then stored for identification.
• P10 pipette Gilson Pipetman, registered trademark
• Thermal Fix registered trademark
• the markers are detected in the same way as in Example 1, except for the extraction step of the polynucleotides.
• the markers are extracted from the cream by breaking the emulsion and recovering the aqueous phase.
• a high temperature above 80 ° C. is sufficient to reduce the emulsion constituting the cream and thus separate the aqueous and lipid phases.
• the markers, very polar, are found in the aqueous phase from which they are extracted.
• the labeled cream is heated for 15 minutes at a temperature of 95 ° C. and then centrifuged for 5 minutes at 10,000 rcf.
• the aqueous phase is recovered, and is used for the detection of markers.
• the polynucleotides can then be detected according to the method described in Example 1.
• This example illustrates a technique for labeling a spirit with a mixture of target, coding and decoy polynucleotides.
• the first type of marker (target polynucleotides) consists of a pool of 20 single-stranded deoxyribonucleic acids of a size of 20 bases.
• the second marker is a single-stranded circular nucleic acid, 1000 bases in size, whose sequence contains the target marker search instructions.
• this sequence makes it possible to know which target markers, among the 20, are significant for the coding of the spirits, the other markers being lures added in a semi-random manner.
• markers of single-stranded deoxyribonucleic acid are generated by means of the algorithm presented in the description of the invention. These markers differ from each other, do not self-hybridize, and do not are not likely to hybridize each other. They constitute the target polynucleotides.
• the polynucleotide encoding is a 1000 base deoxyribonucleic acid, circular.
• the sequence of this nucleic acid contains, at a given location, a cassette containing the combination of target polynucleotides to be searched in the product.
• FIG. 6 shows the principle of coupled labeling: the coding marker, in circular form, contains a general information site, as well as a site allowing to know which of the target markers, also present in the mixture, carry the information. code (the others are only decoys).
• the sequence of the coding marker therefore contains a coding box whose position is hidden. Reading this sequence allows the user to know which markers are to be searched in the target marker pool via cross-referencing in a lookup table.
• the combination of the target markers makes it possible to identify the product unitarily: only one possible combination for a possible product.
• a second sequence codes for general product information, such as the batch, year of production, etc.
• the markers are then injected into the spirits to be marked.
• the detection then takes place in two phases. The first is to detect the specific sequences of the coding marker.
• the first information that comes out of it is: • Product authentication. In case of coarse counterfeiting, no coding marker is present • Product information: Batch number, date of manufacture, ...
• Target markers consist of 20 different polynucleotides, obtained by chemical synthesis (Eurofins MWG GmbH). This example is realized with the marking of 5 different products. In each of these products, the markers can be used:
• Primer-1 5 1 -AGTCGAGAGCCGATTCCGCT-3 1 (SEQ ID NO: 25)
• Primer-2 ⁇ '-GTCCGAGCAAAGGCTTCCGT-S 1 (SEQ ID NO: 26)
• Primer-3 5 I -AGACCCGTGGGCTCCATTAG 3-1 (SEQ ID NO: 27)
• primer-4 5 I -CCACCCAGAGGGCTTAGGTT ⁇ 3 1 (SEQ ID NO: 28)
• primer-5 5 I -ATCCCACGAGGGTGATCTCG 3-1 (SEQ ID NO: 29)
• Primer-6 5 I -GGAATCCGACCGTGCATGTC 3-1 (SEQ ID NO: 30)
• Primer-7 5 I -CAGAGACGTGACCCGCTGTT 3-1 (SEQ ID NO: 31)
• Primer-8 5 -GACCCAGGGGTACATTCTCG I-3 I ( SEQ ID NO: 32)
• Primer-9 5 I -AAACGAGCCCGTTCCGTGTG-3 1 (SEQ ID NO: 33)
• the products 1 to 4 are samples of Mo ⁇ t et Chandon Champagne, cuvée Im Southernale 2005 (registered trademark), the product number 5 is a sample of Mo ⁇ t et Chandon Champagne, vintage Imperiale 2002 (registered trademark).
• Table 3.1 illustrates the marking of the 5 products.
• the dash indicates that the marker is not injected.
• the letter M indicates that the marker is injected, and that it serves in the signature of the marking.
• the letter L means that the marker is injected, but acts as a decoy.
• products 1 and 2 even if they contain different lures, contain the same markers. They present the same signature.
• products 3 and 4 have the same decoys, but the markers are different. They have a different signature.
• the product 5 has the same signature as the product 1. Only the coding marker makes the difference between the two products.
• the markers are injected at the rate of 100 ⁇ L (using a Pipette P100) so as to obtain a final concentration of
• the final volume is, for example, 10 ml_.
• the product thus contains 10 -8 moles of labels per liter, ie a total of 10 -10 moles of labels in 10 ml.
• the coding markers are single-stranded deoxyribonucleic acids with a total length of 1000 bases. Their sequence is as follows (SEQ ID NO: 45):
• Mark 1 codes for the combination of target markers to look for in the product.
• the mark 2 codes for a generic information such as the vintage from which the samples originate. Below (Table 3.2), the legend of type 1 marks.
• Table 3.2 Coding of the target markers according to the variable sites of the coding markers
• Table 3.3 Coding of the product type according to the secondary information of coding markers
• a specific coding marker which indicates which are the target authentication markers coding for the information, and which indicates, in our example, the vintage from which the sample originates, at a final concentration. 10 ⁇ 11 mol / L according to the technique described in Example 1. They contain the following variable sequences:
• the detection is done in two steps.
• the first is to detect coding markers. It allows a first authentication of products, thanks to the second mark. Reading the first mark then makes it possible to know the combination of target markers to be searched.
• the markers are extracted from their environment (here, a Champagne wine), then recovered in an aqueous medium in order to then be able to use molecular identification techniques.
• the extraction should preferably not only have a good performance
• the technique used is that of phenol chloroform extraction.
• the markers are extracted from 500 ⁇ l_ of product (Champagne).
• - In a 2 mL tube (Eppendorf, registered trademark) containing 500 ⁇ l_ labeled product, add ⁇ A phenol volumes (250 ⁇ l_), ⁇ A volume of chloroform (250 ⁇ l_) with the aid of a P1000 pipette, then vortex for 10 seconds. Centrifuge for 5 minutes at 10000 rcf.
• the coding marker is detected by a chain polymerization technique. Two primers are needed to do this.
• a first type of primer is complementary to a region located 5 'of the variable sequence 1. This primer is called universal because it does not depend on the variability of the target markers (it recognizes a site common to all these markers).
• the second primer is in turn complementary to the variable sites 2.
• variable sequence types 2 here, two pairs).
• FIG. 7 represents the detection of the primary labeling of the coding markers by PCR. This figure shows the two types of primers, the matrix as well as the complementary strand that is generated during the first PCR cycle.
• a chain polymerization reaction is carried out on the markers extracted from the products.
• the specific amplification of the marker coding for a primer pair reveals the presence of a type 2 sequence.
• the absence of amplification is indicative of an unlabeled product, which is therefore probably counterfeited.
• An amplification of the marker with the wrong pair of primer, or with several pairs of primers reveals a cheating on the product (cheating on the vintage, mixtures, ...)
• variable sequences V2 the antisense primers are their reverse counterparts
• Eurofins MWG GmbH 100 pmol / ⁇ L
• the chain polymerization reactions are then launched on a GeneAmp PCR System 9700 device (Applied Biosystem) by respecting the following cycles:
• the polymerase chain reaction products are then deposited on a 0.5% agarose gel prepared in 0.5X TBE and migrated in 0.5 X TBE buffer at 10 V.cm- 1 . migration is carried out, the gel is put in a bath containing BET, rinsed and then revealed with UV.For each product, the absence of band reveals that the marker was not detected, or that the sequence 2 does not correspond to the type of primer used A band (358 bp in size) corresponds to an amplification, thus to the detection of a specific sequence of the coding marker.
• the amplicons are conserved for possible detection of target markers.
• Target markers are searched in the product for further detection of the mark. Only the reading of the coding marker makes it possible to detect the target markers. This detection is done on the amplicons obtained previously by polymerization chain reaction.
• the first step is to read the information contained on the coding markers, and thus on the amplicons.
• This information (sequences 1) makes it possible to know, thanks to a correlation table, which are the target markers present in the product whose presence carries the information of the marking. Knowing this, a detection of the total target markers is performed, using a DNA chip. After revealing this detection, the user is able to know which target markers are present or not in the product, and can compare these results to theoretically expected results obtained by reading the information on the coding markers.
• the sequence reaction is carried out on each of the amplicons, in a 200 ⁇ l tube (Eppendorf, registered trademark), according to the protocol set out in Table 3.6.
• variable sequences 1 or Marques 1).
• the target markers are then detected according to the technique described in Example 1. This detection reveals, of course, the ten or so markers present in each product, but it is by comparing with the coding markers that it is possible to remove the relevant information. .
• Example 4 Labeling According to the Process of the Invention of a Medicament
• This example shows how to label drugs in the form of tablets such as those used for many drug formulations, according to the labeling method of the present invention.
• the protocol of Example 1 is used.
• the markers are first put in an ethanolic solution (80%) and then incorporated during the tableting process, to a final concentration of 10-12 moles per 400 mg tablet for the target markers and lures, and at a concentration of 10-14 moles per tablet for the coding markers.
• the tablets do not contain any active ingredient.
• the markers are prepared in the same manner as in Example 1.
• Preliminary mixing is however carried out in an 80% ethanolic solution for the incorporation of the polynucleotides into the tablets.
• the tablets are prepared from 300 g of grains.
• the different powders composing the grains are first weighed and then mixed ("Lodigge” type swirling and blasting mixer), according to the following formula:
• the wetting solution is then prepared from 100 g of sugar syrup and 750 ⁇ l of the marker solution. Gradually add this wetting solution to the powder mixture until a moist mass is obtained which looks like a coarse semolina, then granulate the preceding mixture to obtain a grain having the appearance of a moist vermiculate ( using an oscillating Granulator).
• the grain is then dried at a temperature of 60 ° C. until a hygrometry of 4 to 6% is obtained and then sieved on a sieve column to remove the fine particles. After lubrication with 1% magnesium stearate, the grains are pressed and compressed in press.
• the markers are extracted by grinding the tablets and then recovering the polynucleotides in the aqueous phase.
• the tablets are crushed using a mortar and pestle, so as to obtain a very fine powder. This powder is then mixed with
• Example 1 1 mL of distilled water, in a 1.5 mL tube (Eppendorf, registered trademark). After heating for 15 minutes at 70 ° C., the tube is centrifuged for 5 minutes at 5000 rcf in order to remove the solid particles. The aqueous phase is then taken up in a new tube for the polynucleotide authentication step. The polynucleotides can then be detected according to the method described in Example 1.
• This example shows how to mark and extract markers in a food product, such as pizza dough.
• the markers are injected into the fresh dough and being prepared. They can be detected later in the finished product, ready for consumption, cooked or not.
• the markers are pre-diluted in deionized water. Mixtures of target, coding and decoy polynucleotides are used, as shown in Example 1. They are then incorporated into a pizza dough recipe, which is then cooked.
• a preliminary solution of target markers, coders and decoys is added, prepared as described in Example 1, is added using a P200 pipette (Gilson Pipetman, registered trademark) to the pizza dough during its manufacture, in sufficient quantity to obtain a final marker concentration of 1 E- 10 mol / kg (for each non-decoying marker, ie at 2 E- 9 mol / kg of polynucleotides).
• the following table shows the different amounts of ingredients to obtain 828 grams of raw pizza dough.
• the dough is then baked for 15 minutes at an average temperature of 240 ° C.
• the markers are extracted by dissolving a cooked portion of dough in demineralized water: 1 gram of cooked dough is reduced to powder and then taken up in 10 ml of water. The whole is then mixed vigorously and then heated for 15 minutes at 94 ° C. After centrifugation for 5 minutes at 10,000 rcf, the aqueous phase recovered and stored at 4 ° C. for the detection of the markers. These markers were detected according to the technique presented in Example 1.
• Example 6 Marking according to the process of the present invention of tobacco
• This example shows how to mark tobacco. It is labeled with polynucleotides as presented in Example 1 by direct adsorption on tobacco.
• the marking is on one of the cigarettes of cigarettes called “blondes”. On this sample is adsorbed a mixture of target polynucleotides, coding polynucleotides and decoy polynucleotides as described in Example 1.
• the tobacco is labeled with the solution of polynucleotides prepared according to the method described in Example 1.
• One gram of tobacco extracted from cigarettes is labeled so as to obtain, in the end, 10 -12 moles of label per gram of tobacco: a sufficient quantity of pre-marking solution is injected using a P200 pipette ( Gilson Pipetman, registered trademark) directly on the tobacco, then the whole is mixed vigorously, at a temperature of 37 ° C for 15 minutes.
• a P200 pipette Gilson Pipetman, registered trademark
• the tobacco thus labeled is then stored at a temperature of 160 ° C. and 70% hygrometry.
• the markers are extracted by soaking marked tobacco
• This example shows how to mark a hydrocarbon (crude oil) and what technique can be used to extract the markers and reveal the marking.
• a crude oil sample is labeled with polynucleotides previously prepared in a polar organic solvent.
• the labeling is composed of target polynucleotides, coding polynucleotides and decoy polynucleotides as described in Example 1.
• a solution of target, coding and decoy single-stranded polynucleotides are prepared as described in Example 1.
• a second preliminary mixture is then carried out in DMSO before inserting the markers into the oil.
• the second preliminary solution of markers is then mixed with the oil, using a precision pipette (Gilson Pipetman, registered trademark) in sufficient quantity to obtain a final marker concentration of 10 -10 mol / l.
• the labeled oil is then stored at room temperature for the purpose of identifying the markers.
• the markers are extracted from their apolar medium and then recovered in an aqueous medium in order to then be able to use an identification technique.
• the technique used is that of phenol chloroform extraction.
• the markers are extracted from 500 ⁇ l of crude oil.
• Example 8 Labeling according to the method of the invention of a fresh food product
• a fresh food product is labeled with a polynucleotide mixture.
• the product is a dairy product: a yoghurt.
• the markers are pre-diluted in deionized water.
• the labeling is composed of target polynucleotides, coding polynucleotides and decoy polynucleotides as described in Example 1.
• a preliminary solution of target, coding and decoy polynucleotides is added to the yogurt, in the mass, in an amount sufficient to obtain a final marker concentration of 1 E- 10 mol / kg (for each non-decoying marker, ie at 2 E- 9 moles / kilogram of polynucleotides) using a P10 pipette (Gilson Pipetman, registered trademark), 2 ⁇ L of preliminary solution are then injected into the mass of the product. Everything is well homogenized using a sterile spatula. The marked yogurt is then stored at 4 ° C pending identification of the markers.
• the markers are extracted by dissolving a portion of yoghurt in demineralised water: 1 gram of yoghurt is taken up in 10 ml of water. The whole is then mixed vigorously and then allowed to warm 15 minutes at 94 C C. After centrifugation for 5 minutes at 10,000 rcf, the aqueous phase recovered and stored at 4 ° C for the revelation of the markers. These markers can be detected according to the technique presented in Example 1.
• a non-alcoholic soft drink is labeled with polynucleotides as described in Example 1: Orangina (registered trademark), Schweppes International Limited.
• Target, coding and decoy markers are pre-diluted in demineralized water. They are then mixed directly into the drink.
• Preliminary mixing is performed in deionized water before inserting the markers into the beverage.
• the preliminary solution of markers is mixed with the beverage, in an amount sufficient to obtain a final marker concentration of 1 E- 10 mol / L (for each non-decoying marker, ie at 2 E- 9 mol / l of polynucleotides).
• the labels are extracted using the phenol-chloroform technique presented in Example 1.
• This example shows how to mark and extract markers in a paper form.
• the markers are directly adsorbed on the paper. They are subsequently detected by dissolving the marked paper.
• Example 1 10. a.1 Principles
• the target, coding and decoy polynucleotides as described in Example 1 are pre-diluted in demineralized water. A drop is then adsorbed on the surface of a sheet of paper.
• Preliminary mixing as set forth in Example 1 is carried out in deionized water prior to deposition on the paper sheet.
• the preliminary solution of markers is then deposited on the paper, so that 1 E-12 moles of labels are deposited. 1 ⁇ l of preliminary solution is deposited using a precision pipette
• the paper is then dried in the open air and then stored for the detection of markers.
• the markers are extracted by dissolving a section of paper in demineralized water: 1 cm 2 of paper containing the marker disk is cut into very small pieces, and then immersed in 10 ml of water. Everything is then heated to 15 minutes at 94 ° C.
• the paper pulp is mixed very vigorously, then homogenized by suction-discharge using a pipette.
• the aqueous phase recovered and stored at 4 ° C. for revealing the markers. These markers can be detected according to the technique presented in Example 1.
• Stability and aging tests were carried out jointly with a cosmetics manufacturer. Olfactory tests have made it possible to verify that the addition of target polynucleotides, coding agents and decoys by carrying out a marking according to the present invention does not in any way alter the physicochemical and olfactory properties of the perfumes. These tests were carried out in parallel on several perfumes, and according to several conditions: a month at 5 ° C which served as a reference, a month at 50 c C, which simulated an accelerated aging of the perfume, as well as a month exposed to the light of day.
• FIG. 8 represents a detection scheme of a marking according to the invention and of authentication of a labeled product according to the invention.
• the coding markers are identified.
• the first level of authentication of the product lies in their presence. If the product does not contain a coding marker, the product is counterfeit.
• the target markers and decoys are not differentiated (it is impossible to say that a polynucleotide is a target polynucleotide or a decoy polynucleotide).
• the nature of the coding polynucleotide detected during the first step (here the polynucleotide A) is then sent to the supplier of the marking.
• the tagging provider retrieves from a database the tag decryption key, which will define, in the series of polynucleotides lures and targets, which are decoys and which are target.
• the user is interested in target polynucleotides only. It can then read the code that makes up the presence or absence of target polynucleotides (in this example, the code is -, -, + and -). - Reading the code then allows the user to check, using a secure database, if the identification code corresponds to the product of interest. Otherwise, it may be illegal reproduction, mixing or misappropriation of the product.

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