HK1062697A - A marker of objects to be identified that comprises at least one fragment of dna - Google Patents

Description

Marker for an object to be identified, consisting of at least one DNA fragment

Technical Field

The invention relates to a marker for an object to be identified, consisting of at least one DNA fragment; a method for incorporating the tag into an object to be identified and a method for identifying a tagged object.

In order to understand the invention so that it may be readily carried out, the following paragraphs will provide a brief description of the preferred method of practice. The description is intended as an illustrative rather than a restrictive example of the present invention, and the components thereof may be selected from various equivalents without departing from the principles of the invention described herein.

Background

The quest to provide more secure elements, processes and mechanisms for what is commonly done is permanent. From the remote time of bill generation until today, people are looking for ways to prevent robbery, fraud and counterfeiting. Perfecting the printing process, and incorporating security elements, are clear examples of such a search. This approach has surpassed the technological advances being addressed by the general public and the initial quality of the equipment. The use of marked paper is started as an erasure against checks to be written and paid in high cash. Counterfeiting of paper money and documents generally leads to the use of special papers and inks, optical inks, incorporation of security elements in the pulp, protection with translucent films, etc. The inventors recognized the presence of elements such as fingerprints and DNA unique to each person. In fact, each individual possesses a particular biological track that can be almost absolutely identified with today's technology. The inventors also recognize that counterfeiters can easily develop or use the incorporation of human DNA into objects for reliable and accurate identification, since simple samples of human DNA can be obtained as long as they are the owner. It appears to be genuine since it is sufficient to obtain the DNA required to be incorporated into a counterfeit object from the hair, saliva in a glass, a drop of blood, and even epithelial cells. Accordingly, the inventors have considered that the object to be identified will have to be marked with at least one specific DNA fragment, and even better, with a combination of such DNA fragments. Although counterfeiters may be able to obtain the desired DNA and incorporate it into an object, they do not know which fragment or fragments in the millions or their possible combinations to use to achieve their purpose. That is why the object of the invention is: a marker for an object to be identified, which marker consists of at least one DNA fragment, a method for incorporating such a marker into an object to be identified, and a method for identifying an object marked with such a marker.

Deoxyribonucleic acid (DNA) is a large molecule that stores the genetic information of an individual. Some sequences have specific functions, such as genes and their regulators. Others appear to be silent and their function is still neglected, being very abundant in the genome. Some sequences vary from individual to individual. For this reason, comparison of these polymorphic regions forms the basis for molecular recognition. Polymorphic regions are inherited from parents to children (Mendelian inheritance), and therefore they allow people to be identified by methods that compare potentially biologically relevant individuals. The development of DNA classification techniques that allow any individual to recognize has entered adolescence. It has been for fifteen years since the discovery that allows accurate identification of a person by studying his or her DNA and comparing such DNA with that of his or her relative. Although the conceptual aspects on which such recognition processes are based have not changed substantially, the methodical aspects have undergone the evolution of vertigo. The detection of new markers, the automation of some experimental phases, and the generation of computerized systems with enormous storage capacity and data analysis have marked permanent trends in the field of human recognition. The existence of polymorphic sites in the human genome was known from 1980. The first sites to be detected are the regions of the gene that are located close to some genes, such as insulin. However, the discovery of the multiple variable regions (MLP) has made possible the development of individual recognition systems that are not limited to human species, but that encompass a large number of animal and plant species. This application, which contributes greatly to scientific knowledge and has unexpected application value, was implemented in 1985 by Alec Jeffreys, a scientist in the united kingdom, and his collaborators. This variable region is scattered throughout the genome, allowing the display of individual specific characteristics inherited according to Mendelian's law of inheritance. The impossibility of quasi-locating these regions in the genome only allows to obtain a "molecular phenotype". This phenotypic comparison makes individual identification possible. Although this type of comparison is objective, its statistical evaluation is based on a global analysis, the use of which has led to the development of a first sub-system of human recognition with a huge social impact, the widespread use of which did not start until the 90 s. By using probes that enable the generation of a multi-locus pattern to search in human libraries (the entire genetic information of individuals contained in a microbial host), it is possible to detect variable sites previously localized in the genome and to detect markers of Specific Loci (SLPs) that can define the genotype of individuals presenting certain loci. This method allows for ancestral replacement thereof, greatly contributing to the standardization of analytical methods for human identification, particularly those faced by FBI in the united states and the legal scientific administration in the united kingdom. These reliable (robust) genetic markers were not used as recognition tools until the end of 1995. Later, they were gradually replaced by the use of microsatellites or Short Tandem Repeats (STRs) consisting of polymorphic sequences of short repeat units, the analysis of which relied on Polymerase Chain Reaction (PCR) amplification. These markers exhibit properties such as high sensitivity, making them suitable for forensic analysis, since very small amounts of DNA are required for analysis. Because of this sensitivity, speed of analysis, and simplicity of interpretation of results, microsatellites or STRs are favored forensic markers. Despite the very high number of STRs at the time, a panel of effective markers has been selected for incorporation into standard assay systems of commercial kits. The marker panel or kit allows up to 16 polymorphic human markers to be simultaneously analyzed, thirteen of which make up the basic panel designed into an "intelligent database". The inventors are aware of the fact that Single Nucleotide Polymorphisms (SNPs) occur approximately every one thousand pairs of bases, with a total of more than three million results in the human genome. To accomplish the labeling of objects that counterfeiters cannot replicate, the inventors incorporate DNA polymorphic fragments into such objects, such as microsatellites (STRs) and Single Nucleotide Polymorphisms (SNPs). The inventors herein also disclose methods for incorporating these fragments into objects to be identified and methods for identifying tagged objects. Document No. CN1302905 relates to a DNA metal ion-containing security material prepared by mixing an aqueous solution of a soluble metal salt having a high complexing ability with a DNA solution and ethanol to obtain a decanted solution of water-soluble M-DNA containing gelatin, dextrin, an aqueous solution of soluble starch or rubber for labeling or an imprint ink. Document No. CN1306266 relates to a card for genetic identification, a preparation method of such a card including the issue of owner DNA, information processing and imprinting thereof. Document No. DE4446042 relates to cards comprising entities providing the identification of a large number of chemically related different species, for example methods using substance carriers such as polystyrene, nitrocellulose, proteins, polysaccharides or ethanol. Such recognition may be enzymes, antibodies, antigens and DNA. The card may be used to identify bank notes, spices, documents, etc. Document No. US6167518 relates to registrants biological characteristics; for example, a digital certificate is formed from a digital representation of the chromosomal DNA of a registrant. This representation has personal information that is passed to the certificate itself. The identity of the registrar is verified in a remote method. These characteristics may be extracted from the certificate and compared. Document No. US6213391 relates to an identification system that identifies biometric characteristics (e.g. voice analysis, DNA, etc.) that begin with a characteristic. Biometric information is used for various functions such as control and security of transaction processing. An algorithm is provided for generating a key number for use as the second identification code. Document No. US6256737 relates to a system, method and software for enterprise derived user authentication using biometric determination methods. A biometric control decision method by which a user can be authenticated by the system. The operation of which includes the use of at least one biomedical parameter. It uses scientific methods to identify users by comparing unique characteristics, such as DNA. The disclosed method stores identification parameters. Document No. US6312911 relates to a method of concealing information encoded in microparticles using DNA, and a method of identifying an object using a marker having encoded information. Document No. WO0068431 relates to a shorthand method of hiding information encoded in DNA. The method includes the use of sample DNA hidden in the microparticles and the labeling and verification of the object of interest. Document No. WO0165375 relates to a system, method and software for authenticating a source user using measurements. It uses unique individual characteristics obtained by biometric methods and compares with those stored in memory. This unique property is, among other things, finger and hand geometry; facial and retinal image analysis, voice, DNA, etc.

Disclosure of Invention

The invention relates to a marker for objects to be identified, which incorporates at least one DNA fragment, in particular DNA polymorphic fragments of the microsatellite (STR) and Single Nucleotide Polymorphism (SNP) type, into the object to be identified. The invention also relates to a method comprising the following steps: a first step of selecting an organism from which DNA is to be extracted from any one of its cells; secondly, purifying the obtained DNA; thirdly, amplifying polymorphic fragments of microsatellite and single nucleotide polymorphism types; the fourth step, concentration and/or micro-encapsulation of DNA; fifthly, solubilizing the DNA microcapsule; sixthly, determining and/or correcting the fluidity degree and concentration of the solution; and a seventh step of incorporating the solution into a suitable applicator (applicator) and marking the object to be marked. The invention also relates to a method of identifying a marked object. In a first step, suitable detection techniques are used to detect the components incorporated into the solution and to personalize the marked object. In the second step, the key to incorporate the label is obtained. In a third step, the necessary analysis is carried out to complete the verification of the marked object.

Detailed Description

Once the components and sequence of stages of the present invention have been developed to explain the nature of the invention, they are then supplemented by their functions and operational relationships, and the results provided. In order to obtain markers for objects to be identified, which constitute a security method for protecting valuable items, the invention proposes to incorporate markers into these objects. Preferably, the inventors have considered that this label would have to be a compound that can be subsequently detected. The inventors preferentially used deoxyribonucleic acid (DNA) as a marker for the object to be recognized. In particular, a hairIt has been considered by the inventors that due to a large number of various combinations, it is almost impossible to perform marker replication by incorporating polymorphic fragments of DNA of microsatellite and single nucleotide polymorphism types into an object to be identified. This may be conveniently explained by the fact that, when referring to DNA, a particular reference is made to the polymorphic fragment thereof. That is, those that make all living things on earth different. Furthermore, when referring to individual polymorphic fragments, it is to be understood that such reference only relates to short tandem repeats/microsatellites (STRs) and Single Nucleotide Polymorphisms (SNPs). At this stage, an exception must also be mentioned, namely that only univitelian twins will have common polymorphic fragments that do not allow to distinguish them. However, with the present invention and even in univitelialan twins, it will be possible to derive a distinguishing marker based on the combination of polymorphic fragments of the selected individuals. In fact, when using a particular combination, the only person who knows the exact site within the thousands of polymorphic sites of an organism's genome that have been used for object labeling is himself. If desired and once this site is revealed, it will be possible to perform comparative analysis using DNA typing to identify populations in any laboratory in the world. This event has high utility, primarily in those situations where object identification is required in other jurisdictions, and primarily in striving for fairness. The present invention comprises a number of steps with regard to the step of incorporating a robust marker comprising at least one DNA fragment into an object to be identified. In a first step, the organisms are selected and the DNA to be used is subsequently extracted. The inventors propose that at least one fragment of biological DNA is used as a marker. This should be interpreted in a broad sense, that is, anyone who decides to perform their object marking will either choose himself or herself as donor of the DNA fragment or will choose any organism (human, animal or plant). As a result, the micro-probability of a counterfeiter duplicating a marker is reduced even lower. DNA extraction begins with the use of conventional techniques, such as buccal swabs; blood puncture; harvesting of epithelial cells, hair follicles, etc. to extract cells or body fluids. In the second step, the resulting DNA was released into a solution containing "TrisClH 10 mM" -EDTA0.1(mM), SDS to 20% (w/v) and proteinase K10 mg/ml. Followed by purification with phenol/chloroform-10/9 (v/v). In the third step, the United states of America is usedU.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159, which are incorporated herein by reference. The mixture placed in the thermal cycler contains DNA samples at concentrations between 6pg and 0.05. mu.g, PCR buffer 10 ×, dNTP10 ×, primers attached to the polymorphic regions, 10 ×, each, and 5000 units Taq polymerase per ml. In the fourth step, in order to prevent DNA degradation, the step consists of ultracentrifugation concentration using a microconcentrator such as Centricon100, and microencapsulation by the phase inversion technique. In this step, the polymorphic DNA to be microencapsulated is dissolved in a solvent and then the polymer is dissolved in the same solvent to a final concentration of between 0.25% and 10% weight/volume. The polymers used may be of ambiguous choice as to which polymers are biodegradable or which are not biodegradable. Preferred biodegradable polymers are those such as lactic acid and glycolic acid and esters such as polyanhydrides, polyurethanes, butyric acid (butyric acid), valeryl acid (valeric acid), and the like. In addition, among the non-biodegradable polymers, vinylene acetate and acrylic acid (acrylic polymeric acid) are preferable. The use of polyamides and copolymers and mixtures thereof is also acceptable. The polymers utilized may also be selected from natural polymers. In this case, dextran, cellulose, collagen, albumin, casein, and the like are preferably used. The resulting mixture is then introduced into a non-solvent at a solvent/non-solvent ratio of at least 1/40 to 4/200 to achieve spontaneous microcapsule formation. In this step, the solvent is selected from organic solvents of chloroform and dichloromethane, and preferred non-solvents are ethanol and hexane. Alternatively, in the fourth step, the microcapsules are created with polycationic agents such as poly-L-lysine and CINa. In this case, in the first stage, a polymer selected from those already mentioned is dissolved in an organic solvent such as chloroform. In the second stage of the fourth step, the polymorphic DNA is dissolved in water which produces a first aqueous phase. In the third stage of the fourth step, the first aqueous phase is emulsified with the organic phase to obtain a first emulsion. In the fourth stage of the fourth step, CINa is dissolved in polyvinyl alcohol to produce a second aqueous phase. In a fifth stage of the fourth step, emulsifying the second aqueous phase with the first emulsion to produce the emulsionA second emulsion. Finally, in the sixth stage of the fourth step, the organic solvent of the second emulsion is evaporated to produce microcapsules containing polymorphic DNA. Alternatively, in the fourth step, the DNA may be combined with magnetic microspheres or with pigments visible or invisible to the human eye or with liquids or pigments having electrical properties and/or fluorescence emitted by ultraviolet and/or by infrared radiation. In addition, in order to disguise the selected DNA fragments and to make counterfeiting the marker even more difficult, an alternative combination of techniques described in the fourth step may be used; and other DNA fragments different from the one selected may be incorporated. In the fifth step, the DNA microspheres or microencapsulated DNA are solubilized in a solution containing a substance sensitive to ultraviolet radiation, such as fluorescein, tetrametil, rhodamine 3, texas red (texas red), etc., and/or a substance sensitive to infrared radiation, such as gallium oxysulfide (gallimum oxysulfur) of an upconversion phosphor-like substance (upconversion phosphor like) or a lanthanide ion bound to a naphthyl group, etc. The substances sensitive to ultraviolet radiation and to infrared radiation can be added in free form or microencapsulated using any of the techniques previously described. Alternatively, it is proposed to solubilize the DNA microspheres or microencapsulated DNA in a mixture of substances sensitive to infrared and ultraviolet radiation, for example a mixture containing from 0.0001 to 0.02% by weight of ftlaconine having a wavelength of fluctuation between 670 and 720nm and from 0.05 to 0.5% by weight of a fluorochromogen (fluorophore) having a wavelength of fluctuation between 250 and 380nm selected from the group consisting of stilbene, dihydropyrazole, coumarin, carbostilo and pirene. In the sixth step, the degree of fluidity and the concentration of the solution which should be suitable for facilitating its application to the object to be marked are determined and, if necessary, corrected. It is believed that the degree of fluidity should allow the applicator to be used at a rate of each mm²The solution was precipitated at a concentration of between 6pg and 10. mu.g of label on the surface. In a seventh step, the marker is incorporated into an applicator that can be selected from a pen (a pen with a small opening (nip), a ball-point pen or a felt-tip pen), a different type of filter, a drawing tool, a painting brush, a stamp (stamp) or some automatic machine such as an ink-jet printer, etc. Alternatively, a vehicle (intermedia) is used between the solution containing the marker and the object to be marked. In this case, the vehicle is absorbed into the solution. Media (media)The medium may be selected from different substances, such as nitrocellulose, paper, wood, cardboard, plastic materials, charged nylon, cloth, organic substances in the form of droplets or gels, inorganic substances, etc. Finally, the designated object is marked with the selected applicator. The method of identifying an object labeled with at least one DNA fragment comprises the step of detecting the object with a suitable system. To this end, the system may be a filter (filter) facilitating the visualization of the pigments or elements incorporated in the marker or a detector capable of verifying the presence of some type of radiation, such as wavelengths mixed with sensitive substances, magnetic particles or the conductivity properties of the solution. Different types of magnetic detection and electronic verification systems for detecting certain components according to their degree of conductivity are known in the art. The first step is to detect the object to be identified. In the second step, the key to incorporate the label is obtained. The third step is to verify the labeled object and then to perform the analysis required for typing of the DNA polymorphic fragments using polymerase chain reaction. The third step can only be performed if the owner of the labeled object reveals which is the oligonucleotide fragment that has been placed on the object. Since this information is the starting point, any forensic laboratory in the world using polymerase chain reaction technology, as described, can amplify such oligonucleotide fragments of interest and detect STR/SNP polymorphic fragments. The detection of the fragment can be carried out using methods and techniques commonly used in the art. For example, gels according to j.m. robertson (1994); capillary electrophoresis according to McCord (1993); detection of Multihybridization or Multicapillarity proposed by Wang (1995), using a microchip as set forth in Woolley (1996); mass spectrometry according to Becker (1997); and the like. In turn, single nucleotide polymorphisms can be detected using unique strand conformation assays demonstrated by Orita and others (1989); particular allelic oligonucleotides, as taught by Landegren and others (1988); primer hyperextension or other techniques such as chips, mass spectrometry, etc., according to Syvanen and others (1990). Also, in the event of a dispute, the rights of all parties are to be guaranteed, since the tests relating to the identification of the marked objects may be repeated as many times as necessary anywhere in the world, since, as previously mentioned, the markers STR and SNP are routine steps in identifying a population. On the other hand, in the case of a liquid,it is understood that these markers are recommended by the international union of forensic genetics. The use of the markers described herein will make it possible to positively identify a myriad of objects such as paintings, sculptures, sport inputs, art works, handicrafts, video tapes, recorders, televisions, household objects, computers, printers, software, office components and business equipment. Likewise, it would be possible to authenticate fragrances, cloths, wallets, briefcases, boxes, automobile parts, airplanes, bicycles, paper currency, checks, notary documents, identification cards, driver's licenses, passports, visas, credit cards, phone cards, and similar such substances as academic certificates, inventory, lottery tickets, and other games at chance. Thus, one possible sequence of stages embodying the present invention and its method of operation has been described.

Claims (30)

1. A marker of an object to be identified comprising at least one DNA fragment in association with at least one detection system selected from the group consisting of magnetic microspheres, pigments, liquids with electrical properties, fluorescence for ultraviolet radiation and fluorescence for infrared radiation.

2. The marker of claim 1, wherein said at least one DNA fragment is a polymorphic fragment of a plurality of microsatellite (STR) type DNA.

3. The marker of claim 1, wherein the at least one DNA fragment is a Single Nucleotide Polymorphism (SNP).

4. The marker of claim 1, wherein the at least one DNA segment is microencapsulated.

5. The marker of claim 1, wherein the at least one DNA segment is microencapsulated and incorporated into a solution containing at least one substance sensitive to ultraviolet radiation.

6. The marker of claim 1, wherein the at least one substance sensitive to ultraviolet radiation is selected from the group consisting of fluorescein, tetrametil, rhodamine 3 and texas red and mixtures thereof.

7. The marker of claim 1, wherein the at least one DNA segment is microencapsulated and incorporated into a solution containing at least one substance sensitive to infrared radiation.

8. The label of claim 7, wherein the at least one substance is an up-converting phosphor, such as a lanthanide ion in combination with a naphthyl group.

9. The label of claim 1, wherein the at least one DNA segment is microencapsulated and incorporated into a solution containing a substance or pigment sensitive to ultraviolet radiation and/or infrared radiation.

10. The label of claim 1, wherein the at least one DNA segment is microencapsulated and incorporated into a solution of a mixture of substances sensitive to infrared and ultraviolet radiation.

11. The marker of claim 10, wherein said solution contains 0.0001-0.02 wt%, ftlaconine having a wavelength between 670 and 720nm and 0.05-0.5 wt%, a fluorescent chromophore selected from the group consisting of stilbene, dihydropyrazole, coumarin, carbostii 1o and pirene compounds, and said fluorescent chromophore has a wavelength of 250-380 nm.

12. A method of incorporating a tag into an object to be identified, comprising: extracting a sample containing DNA from the selected organism; determining and optionally correcting the degree of fluidity and concentration of the solution containing the extracted DNA and adding the solution to the applicator; and marking the object to be identified.

13. The method of claim 12, further comprising releasing the DNA in a solution comprising Tris-ClH10mM-edta0.1mm, SDS to 20% (w/v) and proteinase K10mg/ml, purifying the DNA with phenol/chloroform 10/9 (v/v); performing a microsatellite and single nucleotide polymorphism amplification step using polymerase chain reaction to obtain a DNA sample having a concentration of 6pg to 0.05. mu.g, PCR buffer 10X, dNTP 10X, primers attached to the polymorphic region, 10X each, and 5000 units of Taq polymerase per ml, and placing the solution and DNA in a thermal cycler; ultracentrifugation is carried out to concentrate DNA; dissolving a DNA-containing solution in a solvent at a polymer concentration of 0.25-10% w/v, introducing the resulting mixture into a non-solvent at a solvent/non-solvent ratio of 1/40 to 4/200 to microencapsulate the DNA, and solubilizing the DNA microcapsules in a solution containing a substance sensitive to ultraviolet and/or infrared radiation.

14. The method of claim 13, further comprising preparing microcapsules containing a polycationic agent selected from the group consisting of polylysine and ClNa, the first aqueous phase being produced by dissolving the polymorphic DNA in water by dissolving the polymer in an organic solvent; emulsifying an organic solvent with a first aqueous phase to obtain a first emulsion; dissolving a polycationic agent in polyvinyl alcohol and water to produce a second aqueous phase; emulsifying the second aqueous phase with the first emulsion to form a second emulsion, and evaporating the organic solvent of the second emulsion and producing microspheres containing polymorphic DNA.

15. The method of claim 13, wherein the polymer used is biodegradable and is selected from the group consisting of lactic acid, glycolic acid and esters.

16. The method of claim 15 wherein said ester is selected from the group consisting of polyanhydrides, polyurethanes, butyric acid polyacids, valeryl polyacids, and mixtures thereof.

17. The method of claim 13 wherein the polymer used is non-biodegradable and is selected from the group consisting of vinyletilene acetate, acrylic polyacids, polyamides, copolymers and mixtures thereof.

18. The method of claim 13, wherein the polymer used is a natural polymer selected from the group consisting of dextran, cellulose, collagen, albumin and casein.

19. The method of claim 13, wherein the DNA is combined with magnetic microspheres or pigments visible or invisible to the human eye.

20. The method of claim 13, wherein the DNA is bound to a liquid having electrical properties and/or fluorescing with ultraviolet and/or infrared radiation.

21. The process of claim 14, wherein the organic solvent is selected from the group consisting of chloroform and dichloromethane, and the non-solvent is selected from the group consisting of ethanol and hexane.

22. The method of claim 13 further comprising adding a mixture of infrared and ultraviolet radiation sensitive materials comprising from 0.0001 to 0.02 weight percent of ftlacocanine having a wavelength of 670-720nm and from 0.05 to 0.5 weight percent of a fluorogenic chromophore selected from the group consisting of stilbene, dihydropyrazole, coumarin, carbostirilo and pirene compounds, said fluorogenic chromophore having a wavelength of 250-380 nm.

23. The method of claim 13, wherein the concentrating step comprises adding a micro-concentrator of the Centricon100 type.

24. The method of claim 13, wherein said determining step comprises providing a volume per mm²A solution with a concentration of label between 6pg and-10 μ g at the surface.

25. The method of claim 13, wherein said incorporating step comprises an applicator selected from the group consisting of a pen with a small opening, a ball point pen, a felt tip pen, a filter, a drawing implement, a paintbrush, a stamp, and an automated machine.

26. The method of claim 13, wherein the incorporating step comprises incorporating a vehicle selected from the group consisting of a droplet-form organic substance, a gel-form organic substance, and an inorganic substance into the solution containing the marker.

27. The method of claim 13, wherein the incorporating step comprises adding a vehicle selected from the group consisting of nitrocellulose, paper, wood, cardboard, plastic materials, charged nylon, and cloth.

28. A method of identifying an object labeled with at least one DNA fragment, comprising detecting the object to be identified, identifying the at least one DNA fragment in the labeled object and verifying the labeled object using a primer for detecting a flanking polymorphic region using polymerase chain reaction.

29. The method of claim 28, wherein the detecting step is performed by means of a filter that visualizes at least one of pigments and elements incorporated in the marker.

30. The method of claim 28, wherein the step of detecting is performed by detecting at least one radiation type, such as wavelength, magnetism and/or conductivity, having conductive properties of at least one radiation sensitive substance, magnetic particles and solution.