GB2387437A - A method of authenticating an article or its origin - Google Patents

Abstract

Methods for labelling an article or substance, comprising securing oligomers thereto, at least two being binding oligomers, A and B, carrying binding sites for a third, key substance, C, a coordination complex between said binding oligomers and said third substance being detectable to confirm the presence of said binding oligomers on said article or substance, a plurality of non-binding oligomers being present in excess to render copying of the binding oligomers unattractive provide a greater level of security than that currently offered by tagging techniques. Preferably, the oligomers are polynucleotides ie DNA oligonucleotides, and A and B may be used as primers in a PCR. Preferably, the article to be authenticated is a precious stone e.g. a diamond.

Description

A METHOD FOR AUTHENTICATING AN ARTICLE OR ITS ORIGIN

The present invention relates to a method of labelling an article the identity of which it may be subsequently desired to authenticate, comprising labelling the article or substance with unique oligomers.

Such methods of tagging or labelling articles are now commonplace, and allow for high-accuracy recognition in order to prevent or minimise counterfeiting, so called "grey marketing" or other illicit trading.

The problems associated with counterfeit goods are well known and have resulted in substantial losses in revenue throughout the world. This applies to all products, but these losses can be particularly damaging in the case of high value, luxury goods.

The use of polymers to tag liquids and solids is well known in the art. For instance the use of DNA to tag crude or refined oil is well known and is described in WO 91/17265. WO 87/06383 describes a method of labelling crude oil using one or more predetermined DNA tags and hybridising these with a specific DNA probe.

These tags may effectively be hidden by use of non-specific tags. The drawback to this technique is highlighted in WO 90/14441 which illustrates that by using Polynerase Chain Reaction (PCR) technology, the sequence of the tag can readily be determined and, therefore, the origin of the tag can be worked out. In most cases, the DNA strands are attached to beads, for instance polystyrene beads.

WO 94/04918 describes the use of DNA tags for labelling liquids, whilst WO 95/02702 teaches that DNA tags can be used in an aerosol to label individuals trying to steal an article, for instance. Furthermore, this disclosure teaches that a

number of identical beads can be attached to an article, and that each bead can have a number of strands of DNA attached thereto. Each bead has a number of strands, each with a specific primer site. The sequences of these primer sites may be those

! commonly used in industry. In this case, another region of DNA on the strand provides the specificity. This is assessed by sequencing the strand after PCR.

Alternatively, the primer sites themselves may confer the specificity. By using a number of non-standard primer sites, PCR can only be achieved by using the correct primer. Therefore, by assaying which primers anneal to the strand and result in PCR initiation, the identity of the tag may be determined, particularly if the primers are chosen from within a number of groups, so that a type of code is established.

The problem with these methods of labelling lies in the fact that sequencing the security sequences and their reproduction is possible, the techniques required being well known in the art. The more valuable the article or substance to be labelled, the greater the incentive to counterfeit or pass off.

Thus, there is a need to provide a still greater level of security than that currently offered by tagging techniques.

Surprisingly, we have now discovered that it is possible to provide such security by requiring paired oligomers which each have a recognition site for a key, the coordination complex of the three being confirmable to identify the substance, wherein the presence of further unrelated oligomers obscures and hinders any efforts to overcome security.

Thus, in a first aspect, the present invention provides a method for labelling an article or substance, comprising securing oligomers thereto, at least two being binding oligomers, A and B. carrying binding sites for a third, key substance, C, a coordination complex between said binding oligomers and said third substance being detectable to confirm the presence of said binding oligomers on said article or substance, a plurality of non-binding oligomers being present in excess to render copying of the binding oligomers unattractive.

In general, the oligomers may be of any desired make up, configuration or conformation, provided that they are able to bind with the third substance, or key.

They may usefully be polynucleotides, especially DNA, but may also be oligosaccharides or oligopeptides, for example.

Regardless of the nature of the oligomers, it will be appreciated that the individual chains need to be disposed sufficiently close to each other that the key be able to bind the pair to form the coordination complex. If the test is to be performed in situ, such as where the origin of a diamond is confirmed at each way station, then this may be achieved by ensuring that the individual oligomers are present in sufficient quantity, or are disposed together on individual beads, for example. Where the substance to be labelled is oil, for example, then the mobility of the tags ensures a close relationship for testing.

Alternatively, or in addition, the tags may be releasably secured to the article, so that they may be removed by a suitable washing step and subject to an appropriate assay. Unless the washing is so selected as to leave some label in place, this may result in the substance being verifiable only once. However, this is often sufficient.

The nature of the coordination complex may be any that is suitable to be detected, either directly or indirectly. For example, the key may be fluorescent or luminescent, and the presence of the binding oligomers may be simply established by exposing the article to the key, washing off excess key, and assaying for fluorescence/luminescence. Many suitable substances are known for this purpose, and these may be bound to antibodies, for peptides for example, lectins, for saccharides for example, or DNA and bound to the binding oligomers under suitable conditions.

Direct assay of this nature typically requires large amounts of binding oligomers, which can be expensive and/or inconvenient, so is not generally preferred.

More usually, there is little of the binding oligomers present, so other techniques, such as ELISA or PCR, are preferred.

PCR is a well established and sensitive technique for determining the presence of vanishingly small amounts of DNA. A primer sequence starts the polymerase chain in one direction and, when the extending chain reaches the primer sequence for the

opposite direction, the reverse chain is generated. This multiplies up very rapidly and provides readily detectable amounts of characteristic DNA. In the present invention, this may be achieved by providing two oligomers with standard primers. The two primers sequences may be the same or different. What is important is that there be provided unique recognition sequences thereon to recognise a third sequence. The third sequence may consist merely of the antisense versions of the two recognition sequences linked together or, more usually, will have a linker sequence therebetween.

This may be sufficiently long to allow for physical separation of the two binding oligomers. Before PCR can be effected, it is necessary to ligate the coordination complex, so that the two binding oligomers are physically joined by a sequence of DNA which is the antisense counterpart of that part of the key situated between the two antisense versions of the binding sites, or recognition sequences. Once this bridge has been established, then PCR can be initiated from the two primer sites. Lack of PCR indicates that at least one of the unique recognition sequences is not present.

It will be appreciated that the binding conditions of the present invention should generally be selected such that the key will readily bind to the binding oligomers, but will not bind to other oligomers not carrying the specific recognition sequence. In order to ensure ease of use, it may be desirable to ensure that any non-

binding oligomers do not have sequences remotely resembling those of the binding oligomers, so that the binding conditions can be very diverse and still give the correct result. Alternatively, the conditions may need to be highly stringent, if it is desired to use a large number of very similar sequences in order to make it even more difficult to counterfeit the article and the security provided by the present invention, In order to increase security, it is desired to increase the number of non-

binding oligomers in excess of the binding oligomers. It is only necessary that two types of binding oligomers be provided in small amounts, but sufficient to be detectable. The remaining oligomers are selected so as to add diversity and to render copying difficult and/or unattractive. Thus, they may be selected to have specific sequences, or may simply be digestion products from a nucleotide source. The only

requirement is that they should not have the same recognition sequence(s) as the binding oligomers. This may be achieved by running the digest over a column under the desired degree of stringency to remove any oligomers having identical andlor similar sequences. The remaining oligomers can then be used to provide an effective smokescreen. The length of the oligomers is not an essential feature of the present invention, and is mainly decided by the convenience of use combined with a sufficient length to make sequencing an unattractive option.

It will be appreciated that, while it is generally convenient to have only the pair of binding oligomers, it may be desirable to have three or more binding oligomers.

There may be two pairs, for example, or there may be two each relying on a third binding oligomers, there being two keys, but only three binding oligomers.

an alternative aspect of the present invention, there is provided a method of labelling an article it is wished to authenticate, wherein a number of unique oligomers have been attached to the article, including two unique target oligomers, A and B. characterized in that - each of A and B contain a specific region suitable for binding a complementary region on a third unique key oligomer, C, so that a complex A-C-B can be formed, the method comprising: - binding oligomer C, to oligomers A and B. to create a complex, ACE, - polymerising the complex ACB to form AC'B, and - detecting the presence of AC'B.

Possession of the key oligomer, C, is necessary to establish whether the target oligomers, A and B are present. This can only be done using C because of the two different complementary regions on C that are highly specific for complementary regions on A and on B. The key oligomer, C, comprises at least two portions or sections. These are the portions capable of binding the two target oligomers A and B that are attached to the

article. Preferably, the key oligomer may additionally comprise an intervening section or portion.

In order to hamper a third party from duplicating the labels, the article may be provided with a large number of other, unique oligomers to obfuscate the target oligomers, A and B. to which the key, oligomer C, is directed. Preferably these obfuscating oligomers should be the same type of polymer as the target oligomers, for instance, both should be DNA. Therefore, it is a preferred embodiment of the present invention that a number of obfuscating oligomers are also used, in addition to the target oligomers, A and B. to tag or label the article.

A further way in which a third party can be prevented from duplicating the labels is to replace the tags on the article according to a secret, predetermined pattern.

For example, the first set of target oligomers and the key, called A', B', C' here for reference, may be used for a certain time period, for instance, three months. After the time period has lapsed, the target oligomers Al and B' may be removed and replaced with a new, unique set of target oligomers, A2 and B2, but preferably a whole new tag according to the present invention, including A2 and B2. Furthermore, a new key, C2, will be required. Therefore, it is a preferred embodiment of the present invention that the target oligomers and the key are replaced. It is also a preferred embodiment of the present invention that the frequency of replacement and the sequence of the new target oligomers are predetermined and that this information is kept secret. In a further embodiment of the present invention, the target oligomers and any obfuscating oligomers are replaced.

The time, effort and cost required to determine the sequence of all of the oligomers on the article will be significant and in most cases will exceed the cost of the genuine article itself. For articles of increased value, the number of obfuscating oligomers on the tag can be increased. Furthennore, a number of different key oligomers and a corresponding number of target oligomers can be used to authenticate one article.

Similarly, for articles of lower value, it may be desired to decrease the number of unique sequences added in addition to oligomers A and B. allowing costs to be reduced. In general, it is preferred to have sufficient obfuscating oligomers that the time, effort and cost required to determine the sequence of all the oligomers on the article will be significant and in most cases may exceed the cost of the genuine article itself. In a preferred embodiment of the present invention, this method may be directed to unique articles such as works of art, diamonds and jewellery. It is particularly preferred to apply the present invention to precious stones, especially rough or polished diamonds. However, the method according to the present invention can also be applied to articles that have a high value, but of which a number of copies are produced. Furthermore, in another preferred embodiment of the present invention, this method may also be applied to mass-produced articles, where it may be preferential to tag only a small number of the articles. In a further preferred embodiment of the present invention, the article may be naturally produced rather than manufactured. Articles to which the method according to the present invention can also be applied include, explosives, aerosols, organic solvents, paper goods, inks, perfumes, paint products, refined and unrefined oil of all grades, pharmaceutical products, wood, foodstuffs, as well as biological material such as protein, oligosaccharides, polynucleotides and liposomes. The articles may be classed as solids, liquids or gases.

The oligomers may be polynucleotides, proteins, or oligosaccharides, for example.

In a preferred embodiment of the present invention, the oligomers of the present invention are 20-50mers, preferably Comers, or thereabouts. It is a further preferred embodiment of the present invention that the oligomers attached to the article and the key oligomer, C, are polynucleotides. Although this may include

polynucleotides such as RNA, pDNA or DNA-RNA hybrids, it is particularly preferred that the oligomers used are DNA. This may be double-stranded, but is preferably single-stranded.

DNA synthesis is well known in the art. In designing the binding oligomers, it should be borne in mind that the oligomers need to be unique and it is essential that the oligomers used to label the article are not complementary, so that they do not hybridise. It is especially important that the two target oligomers, A and B do not hybridise. A simple computer program, for example, can be used to ensure that when the oligomers are designed, they are unique and also that there is no complementarily and, hence, no possibility of hybridization between them.

In one embodiment of the present invention, the tag or label comprises other oligomers. For example, oligomers other than oligonucleotides, such as oligopeptides or oligosaccharides. Therefore, if polypeptides are used, the key, C, may be any suitable polypeptide binding moiety, for instance, an antibody, such as a monoclonal or polyclonal antibody. If oligosaccharides are used, the key, C, may be any oligosaccharide-binding moiety such as a lectin. In both these cases, the key must be highly specific and the sequence of the peptide or saccharide monomers of the oligomers used for tagging or labelling the article must follow the same rules as set out above for polynucleotide oligomers.

It should be noted here that C' is complementary, in the sense that it is able to base pair to the intervening portion of C, only where the oligomers used are polynucleotides. If the oligomers used are polypeptides or oligosaccharides, then base or residue pairing is not experienced and so the polymerization step cannot occur in the same way as for polynucleotides.

Thus, in a further aspect of the present invention, there is provided a method of labelling an article it is wished to authenticate, wherein a number of unique target oligomers have been attached to the article, including two unique oligomers, A and B. hrzr..tf.ri1 in that

- each of A and B contain a specific region suitable for binding a complementary region on a third unique key oligomer, C, so that a complex ACB can be formed, the method comprising: - binding oligomer C, to oligomers A and B. to create a complex, ACB, and - detecting the presence of ACB.

It is also a further aspect of the present invention, that the key oligomer, C, in these cases, for instance, an antibody or a lectin, is able to bind to oligomers A and B in a highly specific manner.

The oligosaccharide-binding moiety and the polypeptide-binding moiety can be attached, directly or indirectly to an isolating means, such as biotin, so that the ACB complex can be readily isolated.

Oligomers according to the present invention may be attached to the article to be labelled by any of a number of means. These are also well known in the art, but may include direct immersion, bonding with adhesives, painting or spraying, for instance using an aerosol, of the oligomers onto the article. Alternatively, the oligomers may be attached indirectly to beads, for instance, such as polystyrene beads.

This may be achieved by using biotin/avidin methods well known in the art. In a preferred embodiment of the present invention the method of attachment is reversible so that the oligomers may be recovered from the article by washing in a suitable buffer solution for instance, to aid in the subsequent steps of formation of the ACB complex, and detection of the presence of AC'B.

The oligomers attached to the article may also be encapsulated in addition to, or in place of the attachment methods described above. Encapsulating materials may include Araldite, other glues, amber and polysaccharides.

However, it is generally preferred that the oligomers are not encapsulated, although they may be attached to the article by indirect means such as a bead. In one embodiment, oligomers are bonded directly to the article.

In another preferred embodiment of the present invention, a system of cross-

verification can be employed for an added degree of security. For example, a system of documentation accompanying the labelled article may be similarly labelled. For instance, the application of DNA to paper may be effected by trivial means such as painting or spraying, so that the DNA becomes absorbed in to the paper's cellulose matrix. The DNA is then readily recoverable by careful washing. By cross-verifying the results of the method according to the present invention from the article and its accompanying paperwork, counterfeiting can be further hampered.

Once the AC'B strand has been formed, in the case where the oligomer is a polynucleotide, it is a preferred embodiment of the present invention that this strand is amplified by the Polymerase Chain Reaction (PCR), a standard technique in molecular biology. Although not essential, it aids the sequencing process by providing a substantial amount of the AC'B strand for analysis. However, any technique that has a similar amplifying effect can be used.

Similarly, many techniques for are available for sequencing of oligomers, such as the Maxxam-Gilbert method and the Sanger method for the sequencing of polynucleotides. However, it is a preferred embodiment of the present invention that an automated system of oligomer sequencing is used, as these are rapid, highly accurate and commonly available. Polynucleotide sequencing methods such as the Edman degradation reaction and sequencers using this technology, are also well known to those skilled in the art. Similarly, oligosaccharide sequencing methods such as sequential degradation and analysis are also well known to those skilled in the art.

Although it is a preferred embodiment of the present invention that the sequence of the oligomers is determined by the sequencing methods described, it is also another preferred embodiment of the present invention that microarrays can be used to identify the ACB or AC'B oligomer by binding or hybridising to it with a very high specificity. Given the high specificity of a microarray to detect the ACB or AC'B oligomers, little or no amplification of this oligomer may be necessary.

Microarrays suitable for binding polynucleotide oligomers can contain a large number of complementary DNA molecules that are capable of binding to the ACB, but preferably the AC'B oligomer, whether it has been amplified or not, and therefore microarrays are particularly suited to detecting the ACB or AC'B polynucleotide oligomers. The microarray may also use a polynucleotide-binding protein or any other such polynucleotide-binding moiety to bind the ACB or AC'B polynucleotide oligomers. In particular, microarrays are suitable for use in a kit, such as a field kit, for

assaying the presence of the AC'B oligomer. Given that a large number of specific probes can be bound to a single microarray, it is possible to authenticate a number of tags or labels simultaneously. This may be done providing that the appropriate complementary sequence of polynucleotide, polypeptide, or oligosaccharide, to the ACB or AC'B oligomer or amplified AC'B oligomer, is used, according to the oligomers used on each article.

Therefore, it is a preferred embodiment of the present invention that a kit comprising a microarray is used in accordance with the method according to the present invention. This may be a simple, robust kit for use "in the field" for

authentication outside of a laboratory. This is particularly desirable as it may not always be possible, due to time or geographical constraints, to authenticate the tag or label in a laboratory, so that it is necessary to bring the authentication process to the article, rather than vice versa.

Non-natural analogues of DNA such a pDNA are known to be more stable than natural DNA, whilst still retaining many of the same properties as natural DNA.

For instance, pDNA can be amplified by the PCR technique discussed above. More stable DNA may be particularly useful in embodiments of the present invention, but in particular, the field kit described above. Therefore, it is a preferred embodiment of

the present invention that the oligomers are pDNA polynucleotides.

The microarray may be used in combination with the amplification procedure or without it. Similarly, it is another a preferred embodiment of the present invention that a microarray is used in addition to, or in place of the sequencing step.

As the number of monomers in the oligomers increases, the number of possible combinations rises rapidly. For instance, if polynucleotides are used, an oligomer comprising 4 randomly chosen nucleotides, in a linear sequence, can have 256 possible combinations. However, a polynucleotide comprising 12 nucleotides can have a total of over twenty thousand possible combinations.

In addition, the formula for the number of pairwise combinations of target oligomers is given as: nXr= tn(n-l)(n-2). (n-r+l)}/r! where X = the total number of combinations possible, n = the total number of oligomers added to the article and r = the number of target oligomers to be used in tagging the article (in this case, 2).

Therefore, if 10 oligomers are added to the article, i.e. n = 10 n X 2 = {n(n-1)}/2, X = 45.

Therefore, if A,B and 8 obfuscating oligomers are added to tag or label the article, there are 45 possible combinations of oligomers that could be A and B. The method according to the present invention is further described below, using DNA as an example of the oligomers to be used.

A and B cannot hybridise together under any conditions. For ease of use, one end of the A and the opposite end of B (in terms of 3' to 5' orientation) will comprise a readily-utilisable primer sequence, for instance, one commonly used in industry.

Although in some circumstances it may be desirable to use unique, nonstandard

primer sequences for added security, this is not essential, as it would be possible for a third party to determine the sequence of A or B anyway.

The opposite end of A and B to the 'primer end' is the 'key-binding end'. As the name suggests, it is at this end that the region complementary to another region on the key oligomer, C, is located. However, it is preferred that there is no recognisable primer sequence or site at this key-binding end of A or B. as this would unnecessarily aid a third party in fortuitously choosing primers for PCR amplification and subsequent sequencing of A or B. The same applies for the obfuscating oligomers; it is preferred that these are designed so that they do not contain easily recognisable primer sites.

Oligomers A, B and C should be designed such that the complementary oligomer-binding regions are at least 12 nucleotide monomers in length. Therefore, C will comprise two such regions, one at either end for binding A and B respectively.

Furthermore, the intervening section of C should also be designed to be unique to C. It is this intervening portion of C that, when the ACB complex is formed and polymerization is initiated, will be used as the template for C'.

Therefore, once polymerization has taken place, under conditions well known in the art, and C' has been ligated to A and B to form the AC'B polynucleotide oligomer, the identity of AC'B can be determined. One method of identifying AC'B is to amplify AC'B via PCR using the primer sequences at the primer of A and B. In this way, a large amount of AC'B can be quickly generated and sequenced according to the methods discussed above.

Alternatively, a microarray can be used to bind to AC'B and thereby isolate it.

It is preferred that the polynucleotide or polynucleotide-binding protein used in the microarray is highly specific for AC'B.

A further embodiment of the present invention relates to a method for detecting oligomers A and B. as previously described, except that in the case of polynucleotide oligomers, detection of a large amount of polynucleotide, following

amplification, of the approximate molecular weight of AC'B is an indication that A and B are present, as C must have hybridised with A and B. It will be appreciated that the present invention further provides articles or substances prepared as described herein, or which carry two oligomers capable of being identified by their interaction with a third, key substance.

The term oligomer as used herein, refers to any polymer suitable for use in the present invention, for instance a polynucleotide, a polypeptide or a oligosaccharide, or a combination thereof.

The term "complementary", as used herein, refers to the ability of polymers to bind non-covalently to each other with a high degree of specificity or with a high affinity. In reference to polynucleotides, this means that that the two polyoucleotides are capable of base pairing or hybridising to each other. the case of polypeptides, this means that the polypeptide-binding moiety binds the polypeptide with a high degree of specificity or with a high affinity. In the case of oligosaccharides, this means that the oligosaccharide-binding moiety binds the oligosaccharide with a high degree of specificity or with a high affinity.

Claims (12)

1. A method for labelling an article or substance, comprising securing oligomers thereto, at least two being binding oligomers, A and B. carrying binding sites for a third, key substance, C, a coordination complex between said binding oligomers and said third substance being detectable to confirm the presence of said binding oligomers on said article or substance, a plurality of non-binding oligomers being present in excess to render copying of the binding oligomers unattractive.

2. A method of labelling an article it is wished to authenticate, wherein a number of unique oligomers have been attached to the article, including two unique target oligomers, A and B. characterized in that - each of A and B contain a specific region suitable for binding a complementary region on a third unique key oligomer, C, so that a complex A-C-B can be formed, the method comprising: - binding oligomer C, to oligomers A and B. to create a complex, ACE, - polymerising the complex ACB to form AC'B, and - detecting the presence of AC'B.

3. A method according to claim 1 or 2, wherein the oligomers are oligonucleotides, and the detection involves amplification of the ligated product of A and B.

4. A method according to any preceding claim, wherein each of A and B contain at least one primer sequence.

5. A method according to any preceding claim, wherein oligomers are DNA and the method further comprises isolating and sequencing the amplified AC'B DNA.

6. A method according to claim 5, wherein the amplification step is PCR.

7. A method according to any preceding claim, wherein the oligomers for attachment to the article are polynucleotides and the key oligomer, C, is any polynucleotide-binding moiety.

8. A method according to claim 7, wherein the polynucleotide binding moiety is a polynucleotide.

9. A method according to claim 8, wherein the polynucleotide is DNA.

10. A method according to any preceding claim, wherein the article is a precious stone.

11. A method according to claim to claim 10, wherein the stone is a diamond.

12. A precious stone as defined in claim 10 or 11.