EP1483411A2

EP1483411A2 - Improvements in and relating to marking

Info

Publication number: EP1483411A2
Application number: EP03712311A
Authority: EP
Inventors: Jonathan Harold Forensic Science Service WETTON; Andrew John Forensic Science Service HOPWOOD
Original assignee: UK Secretary of State for the Home Department
Current assignee: Forensic Science Service Ltd
Priority date: 2002-03-01
Filing date: 2003-03-03
Publication date: 2004-12-08
Also published as: WO2003074733A3; AU2003216989A1; WO2003074733A2

Abstract

A marking system, marker and methods of detecting marking are provided based around a marking system, the marking system comprising a plurality of different DNA fragment types, each of the plurality of different DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence.

Description

IMPROVEMENTS IN AND RELATING TO MARKING

This invention concerns improvements in and relating to marking, particularly but not exclusively, to the marking, labelling or identification of items by the use of DNA.

A great variety of applications and situations make use of some form of marking of an item for security or other reasons. Some markings are intended to be visible, whilst a number of forms call for the marking to be invisible during normal use and only become visible in certain circumstances. Examples include inks which become visible under certain light conditions.

Certain other situations call for items to become marked in the event of certain circumstances arising and may additionally involve the transfer of the marking to individuals who come into contact with the marked item and/or to other locations which contact the item. Examples include the marking of bank notes with highly visible dye in the event of a robbery.

The applicant's earlier filed PCT application, PCT/GBO 1/03929, was aimed at these issues. That technique used one of a series of possible markers to mark an item when needed. The marker included a number of DNA fragments, with each DNA fragment in a marker having a different length. The variations in length may be supplemented by limited variations in the sequence of selected parts of the DNA fragments. Following amplification and size based separation the different DNA fragment which make up the marker can be determined and compared with records of markers deployed. Use of different length DNA fragments is central to the technique.

The present invention has amongst its aims to provide a marking system which is covert but can readily be inspected The present invention has amongst its aims the provision of a marking system which readily transfers and yet can be traced. The present invention has amongst its aims a marking system which can be readily examined using a minimum of investigating agents (such as primers) and/or investigation steps (such as sets of amplifications). The present invention has amongst its aims to provide a marking system which is easy to produce and use. The present invention has amongst its aims to provide a marking system which can readily provide a vast number of individual markers. According to a first aspect of the present invention we provide a marking system, the marking system comprising a plurality of different DNA fragment types, each of the plurality of different DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence.

According to a second aspect of the invention we provide a marker, the marker including a DNA fragment type, the DNA fragment type including a plurality of different

DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence.

The first and second aspects of the invention may include any of the features, options and possibilities set out in this document. The marker system may include at least 1x10° possible different combinations of fragments, more preferably at least lOOxlO⁶ possible combinations of fragments and ideally at least lxlO¹² possible combinations of fragments. Preferably these numbers of possibilities equate to the number of possible fragment types in the marker system.

The marker system may be produced by mixing together separately produced fragments to produce fragment types as required. Preferably equal quantities of each of the fragments are mixed together.

A fragment type, potentially in combination with one or more other components, may form a marker. The components may include one or more liquids, such as buffers.

The fragment types may differ from one another due to the different fragments they include. Preferably each fragment type includes at least 5 different fragments, more preferably at least 8 different fragments and ideally at least 10 different fragments. The fragments may differ due to the differences in an identity variable portion included within them.

Preferably the marker system and/or markers and/or fragment types are formed of double stranded fragments. Preferably one strand has a sequence which pairs fully with the other.

Preferably all the fragments in a fragment type are of the same length in terms of the number of bases. Preferably all the fragment types in the marker system are of the same length in terms of the number of bases in each of the fragments. Preferably the fragments are between 45 and 100 bases long, more preferably between 50 and 80 bases long. Preferably the fragments differ from one another in terms of the identity of a part of the sequence forming the fragments, particularly the identity of one or more identity variable portions of the fragments. Preferably the different identity variable portions are of the same length as one another. Preferably a 5' end portion is provided between the identity variable portion and the 5' end of the fragment. Preferably a 3' end portion is provided between the identity variable portion and the 3' end of the fragment. Preferably the identity variable portion is provided between a 5' end portion and a 3' end portion of the fragment. The reference to the 3' and 5' ends, here and in the document as a whole, is preferably a reference to the orientation of the upper strand (unless stated otherwise) and is represented by the right hand and left hand of the fragments as illustrated.

Preferably each of the fragments are provided with a portion which has a common sequence in all of the fragments. Preferably each of the fragments have two portions which have a common sequences in all of the fragments. The sequences of the two portions are preferably different from one another. One of the common sequence portions may be provided on the 5' end side of the identity variable portion. One of the common sequences may be provided at or near the 5¹ end of the fragment. The common sequence may be or may be included in the 5' end portion. One of the common sequence portions may be provided on the 3' end side of the identity variable portion. One of the common sequences may be provided at or near the 3' end of the fragment. The common sequence may be or may be included in the 3' end portion. Ideally each fragment is formed of a 5' end portion, identity variable portion and 3' end portion, the identity variable portion being different for each fragment, the 5' end portion being the same for each fragment and the 3' end portion being the same for each fragment. Preferably the different identities for the identity variable portions are due to a variation in one or more of the bases forming the sequence of the identity variable portions. Preferably the identity of the identity variable portion may vary due to differences in five or more bases of the identity variable portion sequence, more particularly in ten or more bases. Preferably no other identity variable portions will anneal to and/or hybridise with a sequence which anneals to or hybridises with a given identity variable portion.

Preferably each fragment in a marker of fragment type has a different identity variable portion to each of the others. Preferably each fragment in a marker system has a different identity variable portion to each of the others. The number of different identity variable portions in a fragment type and/or marker may be at least 5, is more preferably at least 8 and ideally is at least 10. The number of different identity variable portions in a marker system may be at least 25, is more preferably at least 50 and is ideally at least 100. Each identity variable portion preferably has a GC base ratio within a predetermined variation range about that ratio. The predetermined variation range may be the ratio +/-10% of the ratio value. Ideally the GC base ratio is the same between the different identity variable portions.

Preferably fragments of the same length are provided in each of the different fragment types. Preferably all the fragments in a fragment type correspond in length with a fragment in the other fragment types. Preferably the same number of fragments are provided in each of the fragment types. Ideally the same number of fragments of the same lengths are provided in each of the fragment types.

According to a third aspect of the invention we provide a method of marking an article, the method including providing a marking system, the marking system including a plurality of different DNA fragment types, each of the plurality of DNA fragment types including a plurality of different DNA fragments, a known one of the different DNA fragment type being applied to the article, the different DNA fragment types differing from one another in terms of at least part of their sequence. According to an fourth aspect of the invention we provide a method of marking an article, the method including providing a known DNA fragment type, the DNA fragment type including a plurality of different DNA fragments, the DNA fragment type being applied to the article, the different DNA fragment types differing from one another in terms of at least part of their sequence. The third and / or fourth aspects of the invention may include any of the features, options or possibilities set out elsewhere, particularly the following. The article may be a person and/or an item and/or a location. The details of the known fragment type applied to the article may be recorded, for instance in a database. Details of the article may also be recorded, such as a description of the article and/or the articles owner.

The fragment type may be applied by contacting the fragment type, for instance in liquid form, with the article. The article may be wetted and/or soaked in the fragment type. The fragment type may be applied by painting or printing of the fragment type on the article. The fragment may be applied from solution to the article. The fragment may be applied to the article as an aerosol.

The fragment type may be applied to a part or the entirety of the article. The fragment type may be applied to the external surface of the article and/or to an internal location of the article.

The fragment type may be applied during the article's production, for instance during the formation of the article and / or during the finishing of the article and/or during the packaging of the article. The fragment type may be applied to the article after production, for instance by the purchaser and/or on behalf of the purchaser.

According to a fifth aspect of the invention we provide a method of providing a potential marking for an article, the article being provided in proximity with a unit, the method comprising providing a known DNA fragment type, the DNA fragment type including a plurality of different DNA fragments, the DNA fragment type being applied to the article as a result of a disturbance to the unit, the different DNA fragment types differing from one another in terms of at least part of their sequence.

The fifth aspect of the invention may include any of the features, options or possibilities set out elsewhere.

The article may be provided in the unit. The unit may be a container, box, case or canister. The unit may be a cash transport box or an automated telling machine. The unit may enclose the article against access and / or from view. The unit may be open able, for instance using a key, security code or other activating device. In this way authorised access to the article(s) may be obtained and / or access to the article(s) may be obtained without disturbing the unit. The article(s) may be bank notes, cheques, vouchers or other paper or paper type goods having financial value. The article may be bank cards, credit cards, security cards or the like. A significant number of articles of the same or similar type may be provided within the unit.

A disturbance to the unit may include entry by unauthorised persons an/or entry at an unauthorised time and/or entry by unauthorised means and/or entry otherwise than by an activating device. Disturbance to the unit may include the breaking of the unit or a part thereof, forced access to the unit, damage to the unit, the removal of the unit from a location or a change in inclination to the unit. Disturbance may comprise the removal of the unit from a particular person or type of person's, such as security staff, possession. The fragment type may be applied to the article by the broaching of a barrier between the fragment type and the article. The barrier may comprise an element separating a portion of the unit containing the articles from the portion of the unit containing the fragment type and / or the breakage of a vessel containing the fragment type and / or the breaking or removal of a portion thereof. The fragment type may be provided within the unit and / or attached thereto. The fragment type may be distributed over the article by an explosive charge. The fragment type may be applied to the article by wetting of the article by the

DNA fragment type. The DNA fragment type may flow and / or be sprayed and / or drop on to the article.

The fragment type may be applied to items present at the time of the disturbance. The item may be money within a container. One or more items also present at the time of the disturbance may also be marked, for instance the person causing the disturbance or persons in proximity to the disturbance. Preferably the fragment type does not transfer to other items after application to the item or items. Preferably contact of a further item with an item which bears the fragment type does not result in the fragment type being present on the further item. According to a sixth aspect of the present invention we provide a method of detecting the marking of an article by a DNA fragment type from amongst a plurality of different DNA fragment types, each of the plurality of DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragment types differing from one another in terms of at least part of their sequence, the method comprising obtaining a sample of the DNA fragment type from the article; contacting the sample with an amplifying mixture, the amplifying mixture comprising a least one forward primer and at least one reverse primer; amplifying the DNA fragment type; contacting the amplified DNA fragment type with a plurality of different probes, each different probe having a sequence which hybridises to a particular amplified DNA fragment and each of the different probes having a distinct detectable element; hybridising those probes and amplified DNA fragments whose sequences hybridise to produce a hybridisation product; analysing the hybridisation product to identify the detectable elements present. The sixth aspect of the invention may include any of the features, options or possibilities set out elsewhere.

The sample may be obtained from the article by touching the article with an item, particularly a damp item, for instance a swab. The sample may be removed from the item by washing. The sample may be removed from the article by washing. The DNA fragments may be recovered by centrifuging or filtration, particularly by centrifugal micro filtration.

The article may be solid or liquid. Examples of solid articles include paper goods, such as bank notes, cheques and other printed matter having or providing financial value. Examples of other articles include plastic goods; personal possessions such as jewellery, antiques and the like; precious goods such as paintings, antiques, furniture, jewellery and works of art; electronic goods, such as computers, computer peripheral devices, printers, microchips, disc drives and the like; goods requiring protection against counterfeiting such as clothing, watches, perfumes and the like.

The article may be a person who has been in direct or indirect contact with an article having the fragment type. The article may be a location which has been in direct or indirect contact with an article having the fragment type.

The sample or one or more parts thereof may be amplified using PCR. Preferably the amplification process is performed using suitable primers for the DNA under consideration. A single forward primer and single reverse primer on their own are preferably used to achieve amplification. Preferably the forward primer has a sequence which anneals to the common sequence between fragments or a part there of. Preferably the forward primer anneals to the fragments near their 5^* end and more preferably at the 5' end. Preferably the forward primer anneals to the fragment between the identity variable portion and the 5' end. Preferably the forward primer anneals to the 5' end portion or a part thereof. Preferably the reverse primer has a sequence which anneals to the common sequence between fragments or a part there of. Preferably the reverse primer anneals to the fragments near their 3' end and more preferably at the 3' end. Preferably the reverse primer anneals to the fragment between the identity variable portion and the 3' end. Preferably the reverse primer anneals to the 3' end portion or a part thereof.

Preferably one of the primers is provided with a distinctive unit. Preferably the reverse primer is so provided. Preferably the distinctive unit is incorporated into the amplified fragment type by the reverse primer. Preferably the distinctive unit is introduced at the 5' end of the primer. Preferably the distinctive unit is a dye, for instance a green dye. Preferably the dye fluoresces in response to the application of radiation, such as laser light. Preferably the fluorescence is detectable by an analysis instrument. Preferably a measurement of a detectable element is only taken into account in determining the identity of the detectable element if a distinctive unit is also detected. If no distinctive unit is detected then the measurement may be attributed to an unhybridised probe.

The probes may be DNA based probes or may consist of DNA. The probes may be formed wholly or in part from DNA analogues. The probes may be of or include LNA or PNA Preferably the probes are single stranded.

Preferably a probe is provided for each identity of fragment which might be present. Thus in the preferred forms at least 25, more preferably at least 50 and ideally at least 100 different probes are provided.

The primers may be of different lengths, but are preferably of the same length. The primers may be between 15 and 25 bases long, preferably between 16 and 24 bases in length and ideally 20+/- 1 base.

Preferably the probe sequence is perfectly complimentary to at least a part of its respective fragment, more preferably to at least a part incorporated at least a part of the identity variable portion and ideally to the identity variable portion thereof. Preferably each different probe is provided with a different detectable element.

Preferably the detectable element on each probe includes one or more particles, such as a micro-sphere. The particle may be of polystyrene. Preferably the particle is covalently linked to the probe.

The detectable element may be rendered detectable by the presence of one or more colours. The colour and/or the amount of colour and/or the relative proportions of two or more of the colours may vary between different detectable elements to give them differences. Red and orange may be the preferred two colours. One colour may be provided within the particle. One colour may be provided on the surface of the particle. Preferably both colours are provided within the particle.

Preferably the probes and the part of the fragments with which they are complimentary have melting temperatures within 2°C of one another, more preferably within 1°C of one another and ideally have the same melting temperature.

The detectable elements may be visible to the naked eye and/or more preferably to an analysis instrument. The colour may be immediately visible or require subsequent processing or action to render it visible. The detectable elements may be of other form, including radio emitters. The identity of the detectable elements and/or the presence of the distinctive unit may be considered using a human eye, instrumentation for detecting colouration or instrumentation for detecting radio emissions or other characteristics of the detectable elements or distinctive element.

The analysis instrument may consider the hybridised probes and fragments one by one. Preferably the hybridised probes and fragments pass through a monitoring location in single file. The hybridised probes and fragments may pass through the monitoring location in a stream of liquid. Radiation, such as light, preferably from a laser, may be introduced to the monitoring location. The monitoring location may include one, two, or more detectors. Preferably the detectors detect light given out by the distinctive unit and/or detectable element. Three different colours are preferably considered. Preferably the colour and/or the relative proportions of at least two colours are considered.

The detectable elements detected may indicate the particular probes involved in the hybridisation and preferably all such probes. The number of probes detected should equal the number of fragments in the fragment type. The detected results may be compared with records or a database for a marking system or systems to determine a match between the particular fragment type of the sample analysed and a known fragment type and/or one or more recorded fragment types. A match or a lack of a match may be used to confirm or deny the source of the article and / or the genuine nature of the article and / or contact of the article with an article marked with the fragment type. The results may, therefore, be used to confirm physical contact between an article, such as a person, vehicle or the like with an article marked with the fragment type, such as bank notes or the like, either directly or indirectly. The results may be used as evidence in the prosecution of a suspect in legal proceedings. The results may be used to confirm that one or more articles were part of a set of articles, for instance notes from a bundle or box of cash. The results may be used in insurance claim proceedings. According to a seventh aspect of the invention we provide a method for establishing the amount or concentration of a DNA fragment type in a sample, the method including: obtaining a sample; adding to the sample a known amount of a reference DNA fragment type; amplifying the DNA fragment type and reference DNA fragment type in the presence of one another; comparing the detected amount of the reference DNA fragment type with the detected amount of the DNA fragment type, the comparison providing an indication of the amount or concentration of the DNA fragment type.

Preferably the DNA fragment type and reference DNA fragment type are such that even amplification of the two occurs. Preferably the DNA fragment type and reference

DNA fragment type are of the same length. Preferably the DNA fragment type and the reference DNA fragment type have the same 3' end portion. Preferably the DNA fragment type and the reference DNA fragment type have the same 5' end portion. Preferably the DNA fragments of the reference DNA fragment type are different from the DNA fragments of the DNA fragment type, ideally in terms of their identity variable portion, preferably all of the DNA fragments are different from each other.

Preferably a known volume of a known concentration of reference DNA fragment type is added to the sample. Preferably the volume of the sample is known, preferably the reference DNA fragment type is throughly mixed with the sample. Preferably the reference DNA fragment type is not one used as a marker.

Preferably the DNA fragment type and reference DNA fragment type are extracted from the sample and then amplified. In certain cases the amplification may occur in the sample.

Preferably the level of reference DNA fragment type detected in the results is compared with the level of marking DNA fragment type detected in the sample to give the amount or concentration of DNA fragment type present. The result may be used to establish whether the concentration of the DNA fragment type in the sample was the same at the time of analysis as at the time of marking.

The DNA fragment type could be formed of a number of DNA fragments of different lengths to one another. The possibilities with regard to the various forms of DNA fragment set out in PCT/GBO 1/03929, the contents of which are incorporated herein by reference.

The seventh aspect may include any of the features, options or possibilities set out elsewhere in this application.

According to an eighth aspect of the invention we provide a method of producing a marking system, the method including: selecting a sequence for a DNA fragment; synthesising a DNA fragment having that sequence; incorporating the DNA fragment in a vector; using the vector to replicate the DNA fragment; obtaining the DNA fragment; producing a plurality of different DNA fragments according to this method.

Preferably the method includes the step of producing a DNA fragment type, the DNA fragment type being produced by mixing together a plurality of different DNA fragments. Preferably at least 5, more preferably at least 8 and ideally at least 10 different

DNA fragments are mixed to form the DNA fragment type.

Preferably the method of producing a marker system includes producing at least 40, more preferably at least 70 and ideally more than 90 different DNA fragments, preferably the number of different DNA fragments is less than 150. The sequence may be just that of the identity variable portion. The sequence of the 3 ' end portion and/or 5' end portion may come from the vector. The sequence is preferably introduced to the vector by ligation. Primers directed to the vector sequence could be used to achieve amplification.

Preferably the sequence of the DNA fragment synthesised is checked against the selected sequence after forming the vector. Preferably the DNA fragment within the vector is only produced in large quantities if the sequence is as selected. The method may be used to produce DNA fragments differing from one another in terms of their seqeunce and particularly a sequence variable portion thereof. The method may be used to produce DNA fragments differing from one another in terms of their length and particularly a length variable portion thereof, for instance as set out in PCT/GB01/03929, the contents of which are incorporated herein by reference, particularly as they relate to the DNA fragment forms.

According to a ninth aspect of the invention we provide a marking system, the marking system comprising a plurality of different DNA fragments, different combinations of DNA fragments being used to form DNA fragment types, different DNA fragment types being used to distinctly mark, the marking system further including a plurality of organisation identifying DNA fragments, the organisation identifying DNA fragment of an organisation being present in all DNA fragment types of that organisation.

According to a tenth aspect of the invention we provide a method of marking, the method using a DNA fragment type to mark, the DNA fragment type including a plurality of different DNA fragments and at least one organisation identifying fragment.

The ninth and tenth aspects of the invention may include any of the features, options or possibilities set out elsewhere in this application and particularly from amongst the following.

Preferably the marking system includes at least 50 DNA fragment types for use by the organisations and at least one organisation identifying DNA fragment for each organisation using the marking system.

Preferably a DNA fragment type is made of 9 DNA fragments and one organisation identifying DNA fragment.

Preferably the organisation identifying DNA fragments differ from one another in the same way the DNA fragments differ from one another, for instance a variation in sequence, preferably in the sequence variable portion, or a variation in length, preferably in the length variable portion. The organisation identifying DNA fragment length, particularly the length variable portion thereof may be 2 bases different in length compared with the nearest length DNA fragments. Preferably the nearest DNA fragments differing in length relative to one another by 4 bases.

Various aspects of the invention will now be described, by way of example only, and with reference to the accompanying drawings in which :- Figure la illustrates schematically an example of one DNA fragment from a marker according to a first embodiment of the present invention;

Figure lb illustrates schematically an example of a second DNA fragment from a marker according to the first embodiment of the present invention;

Figure 2 illustrates schematically the primers added to the marker of the first embodiment to achieve amplification; and

Figure 3 illustrates schematically the probes added to the amplified marker of the first aspect of the invention to achieve labelling. The invention aims to provide a marking system which is versatile and capable of use in a variety of situations, some of which are described in more detail below.

The general concept behind the invention is the provision of a distinct marker in each case where specific identification is required. The marker is selected from the markers which make up a marker system. Each marker is provided by a DNA fragment type which is formed by a combination of different double stranded DNA fragments so giving a very large number of marker/DNA fragment type permutations in the marker system.

In the preferred embodiments of the invention, each DNA fragment type is formed of a number of DNA fragments which differ in terms of their sequence. The DNA fragments have the same overall length and have standard sequence portions at the 3' and 5' ends of each DNA fragment. The two strands pair to one another and so each fragment has one strand with the same 3' end and 5' end sequence as each other and a pairing strand having the same 3' end and 5' end sequence as each other. A specified number of different sequence DNA fragments may be used in each DNA fragment type, with different mid part sequences for each DNA fragment. Thus a given DNA fragment only varies in terms of the sequence of its mid section, but the potential variations in those sequences allow a significant number of different DNA fragments to be provided which in varying combinations with one another give a very substantial number of possible markers/DNA fragment types. This very large number of possible permutations allows a very individual marker/DNA fragment type to be used in each particular case where marking is required.

In this form the invention is significantly different from the invention set out in applicant's PCT application, PCT/01/03929. In that case each DNA fragment type is formed of a number of different sized DNA fragments so as to provide the desired variation. Further variation occurs in terms of one or both of the 3' and 5* end sequences of each DNA fragment also being different. A given number of different sized DNA fragments is used in each DNA fragment type, with certain possible identities for the 3' and or 5' end sequence. Thus a given DNA fragment will have a certain size (selected from the possible sizes used and different from all the other DNA fragment sizes) and a certain 3* and/or 5' end sequence (selected from the possible sequences used). A significant number of different sizes and different 3' and/or 5' sequences soon leads to a very large number of possible permutations for the make-up of an individual DNA fragment type which is used to mark in a particular case.

The form used in the present invention offers significant advantages over this prior form. In its most preferred form the common 3' end sequences of all the DNA fragments and the common 5' end sequences of all the DNA fragments means that only one forward primer and one reverse primer are needed to effect the amplification of the marker. This assists in getting even efficiency of amplification of all the fragments, minimises primer dimer formation and reduces primer production costs. The use of identical lengths for the fragments also eliminates the chance of preferential amplification and minimises the chance of DNA degradation. Even without the presence of these features in their most preferred form significant improvements over the prior form occur. The technique is also not reliant on a size based separation and hence the need for a manually intensive task is removed.

By obtaining the variation through different sequences, the very large number of permutations is achieved whilst still needing only one forward and one reverse primer to effect the analysis process. This means that the cost of providing the primers and the time and cost involved in performing the analysis is kept low. This contrast with a potential system which could be based around a very large number of different and unrelated DNA sequences to make up the marker system.

Some of the forms of DNA fragment, marker/DNA fragment type and marker system possible according to the present invention are now described in more detail. As illustrated in conjunction with Figure la a DNA fragment 200 is made up of two strands 200 and its complement 200^b each having three portions. These are, a 5' end portion 202^a and its compliment 202^b and a 3' end portion 204^a and its compliment 204^b. The left portion 202 and right portion 204 are joined by an identity variable portion 206^a and 206^b. The a and b strands pair to one another.

Another DNA fragment, for which a single strand only is shown is provided in Figure lb. The fragment 208 is suitable for use in the same DNA fragment type as the fragment 200 of Figure la. The DNA fragment 208 is provided with 5' end portion 202 which has the same sequence and length as the portion 202 in fragment 200. The DNA fragment 208 is provided with a 3' end portion 204 which has the same sequence and length as the portion 204 in fragment 200. Only the identity variable portion 210 of fragment 208 is different in sequence when compared with the identity variable portion 206 of fragment 200.

Whilst the DNA fragments 200, 208 and others, differ from one another in terms of the sequence of the identity variable portion 206, 210 and others it is preferred that the length of this identity variable portion is the same for each of the strands of each of the DNA fragments in a DNA fragment type. Indeed it is most preferred that each of the DNA fragments in each of the DNA fragment types in the marking system have identity variable portions of the same length, and are otherwise the same as one another apart from the sequence of the identity variable portion.

In each case, the 5' end portion 202 is a target for a 20 base forward primer sequence, and the 3' end portion 204 is a target for a 20 base reverse primer sequence. The identity variable portion 206, 210, etc can be set at between 20 and 30 bases in length as desired, but with a common length being used for all DNA fragments of a fragment type once the number of bases has been selected. As a further refinement, the ratio of the occurrence of the nucleotide bases G and C within the identity variable portion 206, 210, etc is also the same between the different DNA fragments even though their overall sequence within the identity variable portion 206 changes between fragments.

In a preferred form, the marking system includes 100 DNA fragments each of which has a different identity variable portion 206, 210 etc, but common 5' end and 3' end portions in the manner outlined above. In a preferred form a DNA fragment type is made up of a selection of 10 of the 100 unique DNA fragments. It is a DNA fragment type which is used to mark a location, item or the like. As a consequence this selection of 10 out of 100 possibilities gives 1.73 x 10¹³ possible different DNA fragment types that can be made using the preferred form of the marking system. A marker system offering a very large number of markers is thus provided. Increasing the number of identity variable portions can easily be used to increase the number of possible variations if necessary. This massive number of DNA fragment types which can be used for specific markings is achieved using only a very limited number of different DNA fragments, one hundred. This compares markedly with systems in which the DNA fragment type is formed of only one or two DNA fragments, as those one or two fragments need to be selected from a marker system made up of many thousands or tens of thousands of different DNA fragments so as to get the desired number of possible permutations. By requiring a relatively low number of different DNA fragments, a hundred or so, the present invention also enables the marker system to be produced at lower cost and with better quality assurance than prior art systems. It is far easier to manufacture accurately and check thoroughly the sequences for a hundred DNA fragments which are then used to make the marker system, rather than having to check many tens of thousands of such individual fragments.

A variety of ways of deploying the marker and a variety of possible uses exist. Some of these are discussed in more detail below.

Once a sample of the DNA tag has been recovered from an item, location or person (the possible manner of which is described below) it can be amplified.

The amplification technique in this case, however, only involves using a single forward primer 300 and a single reverse primer 302. Only one of each primer is needed to replicate the marker as all of the different DNA fragments within that marker have common 5' end portions 202 and 3' end portions 204. Thus PCR can be used to readily amplify a sample which consequently ensures that the efficiency of amplification is substantially equal between all amplicons, that primer dimer formation is easier to minimise, whilst also ensuring that production and quality control costs are reduced by minimising the number of ingredients involved. A suitable forward primer 300 and reverse primer 302 are illustrated in Figure 2 and will anneal to the portion 202^b and end portion 204^a. The full match between their sequence and the fragment ends allows the two to anneal well and selectively and give rise to the desired extension and hence replication during PCR. Because all of the DNA fragments which are to be amplified are of equivalent length, preferential amplification is minimised, as is the effect of DNA degradation. The evenness of amplification for all of the DNA fragments which make up a DNA fragment type and a part of the marker system means that it is possible to use the present invention to mark items in a quantitative approach. In such an approach, a known amount of a known DNA fragment type is mixed with a material to mark it. The material may particularly be a liquid, for instance oil, perfume or the like. The DNA fragment types should be thoroughly mixed with the material being marked so as to have an even concentration therein.

Under this approach, when a sample is recovered a reference DNA fragment type is added to the sample and mixed in. Again, the concentration of this reference DNA fragment type is known. Furthermore, the reference DNA fragment type is of the same length as the marking DNA fragment type and has the same 3' end and 5' end sequences to the marking DNA fragment type. The reference DNA fragment type is one which is specifically held back for the quantitative analysis and which is not used as a marker. As a consequence, there is no possibility of the reference DNA fragment type being the same as the DNA fragment type which was used to mark the sample.

After mixing with the sample, the markers are either amplified in the sample, or more normally extracted for amplification. The extraction process will be equally effective with both DNA fragment types and all their constituent DNA fragments due to the equivalent nature of the DNA fragments employed. Similarly, amplification will be equally effective for the item marking DNA fragment type and the reference DNA fragment type added as part of the quantitative measurement. The levels of reference DNA fragment type detected in the results can be compared with the level of marking DNA fragment type detected in the sample. From the known concentration of the reference DNA fragment type added to the sample, the concentration of the marking DNA fragment type in that sample can be determined. As a consequence, it is possible to establish whether the concentration of the DNA fragment type in the sample was the same at the time of analysis as at the time of marking. Reassurance that no adulteration or dilution of the sample has occurred in the meantime and / or evidence of adulteration or dilution can be provided as a result. Checks could also be made to ensure that only the one marker is present, a plurality of markers indicating blending of two or more samples. This quantitative approach is particularly suited to use in conjunction with the sequence varying DNA fragments of the present invention. However, it could also be used with the length variation approach of PCT/GB01/03929 as well.

Returning to the analysis technique, it is desirable that the reverse primer 302 incoφorate a distinctive unit 306, for instance in the form of a green fluorescent dye at its 5' end with that dye consequently being attached to amplification products once the sequence extension has occurred. The function of the green fluorescent dye is explained in more detail below. The technique is advantageous in that only one primer needs labelling in the case of the primers used in the amplification process, with consequential cost reductions. Rather than relying upon electrophoretic separation due to size to analyse the results with consequential inspection of the colours occurring for each of the bands, an alternative technique which reduces manual work and analysis time, improves data collection and a degree of automated processing is possible. This technique involves introducing to the amplified products, represented in Figure 3 by a strand 350 made up of sequence 202^b, 206^b, 204^b and dye 306, a mixture of single stranded DNA probes 400. Probes formed partially from DNA and/or from DNA analogues, such as PNA and LNA are also possible. A probe 400 with a sequence pairing to each of the possible variable identity portions 206, 210, etc is provided. Thus in the preferred form mentioned above 100 different probes 400¹, 400², 400\ 400¹⁰⁰ are provided. Each of the different types of probe 400 has a sequence which is perfectly complimentary to one and only one of the identity variable portions 206, 210, etc.

A probe 400 having the perfect complimentary sequence to one of the identity variable portions 206, 210, etc will hybridise thereto as illustrated in Figure 3. This will occur for 10 different probes 400 from amongst the 100 different probes 400 introduced, in the case of the preferred form. The other probes 400 do not hybridise due to their sequences not matching one of the variable identity portions 206, 210, etc actually present in the marker.

Each of the probes 400 is provided with a distinct label 402 when compared with the other probes 400. Thus a different label is provided for each different identity variable portion in the marker system.

In one embodiment the labelling is provided by the probe being attached covalently to a micro-sphere. Such micro-spheres, which can be obtained from Luminex Corporation, 12212 Technology Blvd, Austin, Texas,78727, USA for instance, provide a distinctive recognition pattern. This can be due to the presence of two different colours which are provided in different proportions relative to one another and also in different absolute amounts. Both colours can conveniently be provided within the micro-spheres. Once hybridised to the denatured amplification product of the PCR, the amplification products are ready for analysis. Analysis itself can be achieved using an instrument capable of identifying the label attached to the amplification product. For instance, in an instrument such as a Luminex 100 from Luminex Corporation, a fine stream of liquid is generated in which the micro-beads are aligned in single file and pass a pair of fluorescence detectors. Laser illumination allows the colours associated with a micro-bead to be detected. Because each of the different probes is provided with a distinct micro- sphere, the colour or colours of that micro-sphere revealed by the instrument reveal the identity of the probe and hence of the variable identity portion as a result.

As a development over that technique, to ensure that unattached micro-spheres are not being considered in the analysis, a result is only taken into account when the colour pattern detected for the micro-spheres is also accompanied by the detection of the green fluorescent label introduced into the amplification products by the reverse primer during amplification.

In the preferred form, with ten different DNA fragments in the DNA fragment type, ten different micro-sphere colours with green fluorescence associated should be detected with these giving the distinct designation associated with that particular DNA fragment type.

A typical result might thus be the presence of the label associated with probes 400 , 400²⁶, 400³¹, 400³², 400⁴⁷, 400⁶⁹, 400⁷³, 400⁹⁰, 400⁹⁴, and 400⁹⁸ being detected. In the refined form, where the lengths of the identity variable portions are all the same and the GC base content of the identity variable portions is identical then all the probes and all the fragments can be provided with identical melting temperatures. This ensures that hybridisation conditions between the two are optimised as a result.

As the markers and probes are designed to have the same melting temperatures, Tm, and must not adversely react with each other, there is also the possibility of using the zip code sequences published by Jingwen Chen et al (2000) "A Micro-Sphere Based Assay for Multiplex Single Nucleotide Polymorphism Analysis Using Single Based Extension", www.genomeresearch. Zip codes are pre- validated 24mer sequences of the same Tm which do not interact, hence development of new systems can progress rapidly using such technology in combination with the distinct features of the present invention. Similar technology has been developed by Luminex and could be incorporated into the present invention in a similar way.

It is, of course, possible to synthesise as much as required of each of the DNA fragments which are used to form the marker system and from which combinations are selected to form DNA fragment types. Synthesising significant quantities of such fragments is relatively expensive, and given the limited number of DNA fragments which need to be produced, other production techniques can be employed. These techniques are not viable possibilities for situations which require thousands of different DNA fragments, as they only become viable when significant quantities of a particular DNA fragment are required. In the case of a hundred or so such DNA fragments, such production techniques are commercially attractive. In basic terms, a limited amount of the desired sequence DNA fragment is synthesised. The DNA fragment is then spliced together with a vector. A very limited amount of the vector is produced and the DNA fragment contained within is carefully checked to ensure that its sequence is as intended. The result can then be combined with an appropriate expression system to ensure large scale replication of the DNA fragment of interest. The accuracy of replication is high and hence good quality control of the DNA fragment obtained as corresponding to the DNA fragment desired is achieved. This approach can be pursued whether it is a fixed length, sequence varying tag of the present type which is of interest, or whether it is a length varying DNA fragment of the type employed in the marker system of PCT/GB01/03929. A variety of potential manners of deployment exist. The marking system may be a applied to an item or location in the event of certain circumstances arising and will then remain on the item or location during its subsequent life, or at least at a significant time period. The circumstances may be the disturbance of the item or location and/or a container or article associated with the item or location. In one example the marking system may be provided in a container within a case for an amount of money, as a security device. In the event of the case being broken into the container is designed to break and hence bring the marking system into contact with the money. Any subsequent contact of the money with persons, other items or other locations is designed to give partial transfer of the marking system to them. The marking system is thus intended to allow the money stolen, persons handling that money and cars, houses and the like which are linked to the robbery to be detected. The marking system can be applied to an item or location during a stage of that item's or location's production and remain a feature of it during its subsequent life or be added by the purchaser themselves at a later date. In this form the marking system can be used to verify the genuine nature of the item, for instance genuine rather than counterfeit perfume, and/or to identify a feature of the item's production, for instance the particular location of the producer which made the item so as to trace the source of production should a problem arise. The marking system enables these benefits, but without interfering with the item's or location's normal use or appearance. In this form it is desirable for the DNA to be retained by the item or location in the event of contact with another item or another location. In a particularly preferred form the marker is applied to the item (particularly money) by the triggering of a device, but is not subsequently transferred from the item.

When a sample needs to have its DNA fragment mixture decoded to investigate the person, item or location from which that sample arose, a sample of the DNA is recovered. This may involve swabbing the person, item or location with a damp cotton swab. The lifted sample of the DNA fragment type marker system is then subjected to washing and centrifugal micro filtration to obtain the sample for subsequent analysis. As an alternative, where the item or location is suitable, an area of the item or location bearing the marking system, or even the whole item or location, may be washed (sterile water or buffered solution) to remove the DNA fragment type with the sample subsequently being purified using centrifugal micro filtration and / or Qiagen extraction chemistry.

Once obtained, the sample of the DNA fragment type is contacted with primers and analysed as described above.

To enable a marker system to be used in a practical way in the real world, it is likely that a number of separate organisations will want to use the marker system. Whilst it is possible for each of those organisations to co-operate with one another, or a central body such as the marker system manufacturer, to record which of the possible combinations they deploy as a DNA fragment type to mark in a particular case, this places an administrative burden on all parties. Without such administration there is a risk that the same DNA fragment type will be used by different organisations for different marking jobs, but with the potential for confusion. Even with administration controls, there is a risk that inaccurate or incomplete record keeping or accidents could result in the same DNA fragment type being used by different operators to mark unrelated items. These could clash with one another during analysis, leading to a false, or at least ambiguous, result. To avoid this issue, the present invention provides an important control function.

With a marker system which is to be deployed by ten operating organisations, independently of one another, an organisation identifying DNA fragment is assigned to each organisation. That DNA fragment acts as a unique identifier for that organisation being the marking organisation in respect of any DNA fragment types recovered. This DNA fragment forms one of the ten DNA fragments used as a DNA fragment type in each marking which occurs. The other nine DNA fragment types are freely selected so as to provide unique DNA fragment types for marking between different situations. There is no need for the organisations to keep records of the combinations of DNA fragments they use to mark and pass those records on to each other as, when any sample is recovered and analysed, a clear indication as to which organisation that marking arose from is provided by virtue of the organisation identifying DNA fragment that will be present. Thus, even if the same combination of nine DNA fragments is selected out of the ninety possible candidates and used by two organisations to mark items, a sample recovered from one of those items will be clearly linked to the organisation that marked it by virtue of the organisation identifying DNA fragment. By sharing information the very limited information as to which organisation identifying DNA fragment belongs to which organisation, the marking system can be successfully operated by a number of independent organisations with minimum co-operation between one another.

Of course, the number of organisations involved in a marking system could be increased by increasing the number of organisation identifying DNA fragments included in the marking system. Thus, one hundred and ten DNA fragments would allow the selection of nine fragments from ninety to provide the unique identifying sequence, with the twenty other DNA fragments being available to allow twenty independent organisations to operate the marking system. Other permutations on the numbers involved are, of course, possible. In a further development of this concept, with particular emphasis on length varying sequences, for instance, as provide by PCT/GB01/03929, it is preferred that the minimum length variation between unique identifying DNA fragments is at least four base pairs. It is perfectly possible to provide the organisation identifying DNA fragment as an intermediate rung between such fragments, for instance by causing it to differ from two of the DNA fragments present by only two base pairs. Thus a blue yellow indication base pair 70 may be part of the unique identification, a green yellow indication base pair 74 could also be part of the unique identification, whereas a green blue indication base pair 72 would be indicative of a particular organisation who combined and deployed that DNA fragment type. Again, this would enable the detected result to be linked to the records of an organisation which would unambiguously indicate an item, location or other event marked using that DNA fragment type.

Claims

CLAMS:

1. A marking system, the marking system comprising a plurality of different DNA fragment types, each of the plurality of different DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence, wherein the DNA fragments include a 5' end portion and a 3' end portion with an identity variable portion provided between them, the sequence of the 5' end portion being the same for each DNA fragment in the marking system, the sequence of the 3' end portion being the same for each DNA fragment in the marking system, the sequence of the identity variable portion of each DNA fragment in the marking system being different, a DNA fragment type being formed of at least 8 different

DNA fragments from the at least 50 different DNA fragments in the marking system.

2. A marking system, the marking system comprising a plurality of different DNA fragment types, each of the plurality of different DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence.

3. A marking system according to claim 2 in which the DNA fragment types are formed of at least 8 different DNA fragments selected from at least 50 different DNA fragments.

4. A marking system according to claim 2 or claim 3 in which the fragments differ due to the differences in an identity variable portion included within them.

5. A marking system according to any of claims 2 to 4 in which all the fragments in the marking system are of the same length in terms of the number of bases.

6. A marking system according to any of claims 2 to 5 in which a 5' end portion is provided between the identity variable portion and the 5' end of the fragment and a 3' end portion is provided between the identity variable portion and the 3' end of the fragment.

7. A marking system according to any of claims 2 to 6 in which each of the fragments are provided with a portion having a common sequence between fragments on the 5' end side of the identity variable portion and a portion having a common sequence between fragments on the 3* end side of the identity variable portion.

8. A marking system according to claim 4 and any claim depending therefrom in which each DNA fragment of a DNA fragment type has the same a Tm and/or the identity variable portion has a GC base ratio within a predetermined variation range about that ratio, the predetermined variation range being the ratio +/-10% of the ratio value.

9. A marking system according to any preceding claim in which all the fragments of the marking system are of the same length.

10. A marking system according to any preceding claim in which the marking system further includes a plurality of organisation identifying DNA fragments, the organisation identifying DNA fragment of an organisation being present in all DNA fragment types of that organisation.

11. A marking system according to claim 10 in which the marking system includes at least 50 DNA fragment types for use by the organisations and at least one organisation identifying DNA fragment for each organisation using the marking system.

12. A marking system according to claim 10 or claim 11 in which a DNA fragment type is made of 9 DNA fragments and one organisation identifying DNA fragment.

13. A marker, the marker including a DNA fragment type, the DNA fragment type including a plurality of different DNA fragments, the different DNA fragments differing from one another in terms of at least part of their sequence.

14. A marker according to claim 13 in which the DNA fragment type and/or DNA fragments are provided according to any of claims 1 to 12.

15. A method of marking an article, the method including providing a marking system, the marking system including a plurality of different DNA fragment types, each of the plurality of DNA fragment types including a plurality of different DNA fragments, a known one of the different DNA fragment type being applied to the article, the different DNA fragment types differing from one another in terms of at least part of their sequence.

16. A method of marking an article, the method including providing a known DNA fragment type, the DNA fragment type including a plurality of different DNA fragments, the DNA fragment type being applied to the article, the different DNA fragment types differing from one another in terms of at least part of their sequence.

17. A method of detecting the marking of an article by a DNA fragment type from amongst a plurality of different DNA fragment types, each of the plurality of DNA fragment types comprising a plurality of different DNA fragments, the different DNA fragment types differing from one another in terms of at least part of their sequence, the method comprising obtaining a sample of the DNA fragment type from the article; contacting the sample with an amplifying mixture, the amplifying mixture comprising a least one forward primer and at least one reverse primer; amplifying the DNA fragment type; contacting the amplified DNA fragment type with a plurality of different probes, each different probe having a sequence which hybridises to a particular amplified DNA fragment and each of the different probes having a distinct detectable element; hybridising those probes and amplified DNA fragments whose sequences hybridise to produce a hybridisation product; analysing the hybridisation product to identify the detectable elements present.

18. A method of producing a marking system, the method including: selecting a sequence for a DNA fragment; synthesising a DNA fragment having that sequence; incorporating the DNA fragment in a vector; using the vector to replicate the DNA fragment; obtaining the DNA fragment; producing a plurality of different DNA fragments according to this method.