Authenticating an object using a nucleic acid marker

Abstract

The present disclosure relates to compositions and methods for identifying / marking objects for recognition. In particular, objects are marked with nucleic acid markers (gene marker-based product authentication).

Description

[0001] Priority declaration

[0002] This application claims the benefit of priority to U.S. Application Serial No. 15 / 972,951, filed May 7, 2018, the entire contents of which are incorporated herein by reference. Background Art

[0003] Counterfeiting has become a major problem for brand names. Counterfeit goods result in trillions in lost revenue and millions of jobs. In addition to lost revenue, some counterfeit products are directly linked to health and safety issues. Counterfeit goods have infiltrated most industries, from textiles to microchips and pharmaceuticals.

[0004] Products including medications, toys, entertainment products, clothing, fashion accessories, currency, electronics and any other product of value have counterfeit copies available to consumers. The problem with counterfeit products is that they not only damage the original brand or manufacturer's name and economy, but because these products are not from a reliable source, the quality and efficacy of these products may be compromised. The impact of counterfeiting on the person distributing the counterfeit goods is usually minimal compared to the impact that could result from a malfunction of a product with counterfeit ingredients. Summary of the invention

[0005] The present disclosure relates to compositions and methods for marking or labeling objects for identification. In particular, the present disclosure relates to labeling objects with nucleic acid markers (eg, commodity authentication based on genetic markers).

[0006] One embodiment provides a method for treating an object with a DNA marker for authentication or tracking, the method comprising: a) obtaining an object containing a DNA marker, wherein the DNA marker includes telomeres; b) treating the object in a) to remove at least a portion of the DNA marker; c) optionally purifying the removed DNA marker in b); d) amplifying the removed DNA marker in b) or the purified DNA marker in c) to produce an amplified DNA marker, and analyzing the amplified DNA marker by size and / or sequence analysis; and e) replicating the removed DNA marker in b), the purified DNA marker in c), or the amplified DNA marker in d), wherein the DNA marker has telomeres, and the replication results in shortening of the telomeres of the DNA marker.

[0007] In some embodiments, the method further comprises f) coating the object with the shortened DNA marker. In some embodiments, the method comprises repeating a) to f) two or more times on the object. In one embodiment, each shortening of the DNA marker in e) indicates a sale of the object, and the size and / or sequence of the DNA marker applied in f) allows determination of the total number of times the item has been sold.

[0008] One embodiment provides a method for treating an object with a DNA marker for authentication or tracking, the method comprising: a) obtaining an object containing a DNA marker, wherein the DNA marker comprises an expression vector encoding a detectable protein marker; b) treating the object in a) to remove the DNA marker; c) optionally purifying the removed DNA marker in b); d) introducing the removed DNA marker in b) or the purified DNA marker in c) into a host cell for transcription and translation to produce a detectable protein marker; and e) detecting the detectable protein marker in d). In some embodiments, the marker is a protein that fluoresces, provides a color, provides an odor, illuminates, and / or is detectable with an antibody.

[0009] The methods provided herein may further comprise at least one of searching a database for the DNA marker, entering one or more subjects treated with the DNA marker into the database, or a combination thereof.

[0010] One embodiment provides a kit comprising: a DNA marker, wherein the DNA marker comprises a telomere or is disposed in an expression vector; a DNA marker coating device; instructions for marking objects, one or more tables for listing marked items; a login ID; a password or unique identification code; one or more containers; instructions for searching a website / database; instructions for submitting information about marked objects to a website / database; or a combination thereof. DETAILED DESCRIPTION

[0011] Many product manufacturers use the appearance quality and clear design that can be identified as "trade dress" to uniquely identify the product manufacturer's high-quality and high-value products, thereby gaining the trust of their customers. Other product manufacturers also add labels for anti-counterfeiting purposes. Traditional anti-counterfeiting labels are usually formed of materials with particularly targeted physical or chemical properties, such as magnetic stripes on checkbooks, laser holograms on credit cards, fluorescent inks on stock certificates, and heat-sensitive inks on confidential documents. Anti-counterfeiting labels are also made by adding specific antigens to the objects that need to be identified, and then antibodies specific to the antigens can be used to detect the antigens. However, both antigens and antibodies are proteins, which are characterized by poor stability under many environmental conditions of temperature and humidity, and are easily denatured or even degraded, thereby losing activity and can be easily destroyed, thereby reducing the accuracy and reliability of identification.

[0012] Another method used is to add fluorophores to products to be able to visually detect which products are legitimate. Fluorophores can be used as dyes or markers for these products, and can be applied during the early production stages of the entire production or applied to the finished product / goods. Although this method works initially, it no longer protects the product because the counterfeiter has copied the product with the fluorophore. Counterfeiters can extract the fluorophore from the product, copy the fluorophore, and add the fluorophore to their counterfeit product so that the product fluoresces as the genuine product would fluoresce.

[0013] Therefore, nucleic acid such as for example deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) has been regarded as an improved substitute for conventional anti-counterfeiting labels and logos. Although composed of relatively simple nucleotide building blocks, nucleic acid can still encode a large amount of information: for example, the human genome encodes all the information required for the synthesis and assembly of all components of the complex structure from the neural network of the brain to bones, tissues and organs of the human body. Nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid sequences can be unique and complex.

[0014] Another advantage of nucleic acid as mark or marker is that, under suitable protection, these molecules can be preserved for a long time. Evidence from specimens preserved in glaciers, ice fields, tar pits and swamps and wetlands shows that DNA can resist degradation for thousands of years and in some cases millions of years. Protected marker DNA can also be stabilized in the polymer for coating high-value articles or objects of interest so that it survives for a long time, and can then be used for identification, authentication and tracking purposes. This long-term ability to persist adds a very sensitive method for detecting low-quantity molecules, such as by using polymerase chain reaction (PCR) to amplify, making nucleic acid, particularly DNA, become the attractive candidate for use as mark. In addition, nucleic acid provides almost unlimited encoding power, because along with the sequence of oligonucleotides or polynucleotides, the number of possible unique sequences just increases four times.

[0015] definition

[0016] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," "the," and similar designators used in the context of describing the disclosed subject matter (especially in the context of the claims above) should be interpreted to cover both the singular and the plural. As used herein, the terms "bound to a substrate" and "immobilized" are interchangeable when applied to DNA binding and immobilization.

[0017] As used herein, the term "marker" refers to a DNA or RNA marker, and optionally, the DNA marker can be combined with a second marker substance. When there is a marker DNA and one or more additional markers, the marker DNA and one or more additional markers are fixed to an object to indicate the characteristics of the object, such as, for example, the manufacturing source of the object.

[0018] The term "PCR" refers to polymerase chain reaction. PCR is an amplification technique that can be used to extend the number of copies of a template nucleic acid sequence using short primer oligonucleotide pairs that are complementary to specific sequences adjacent to the template nucleic acid sequence in the presence of a DNA polymerase, such as a thermostable DNA polymerase, such as the thermostable Taq polymerase originally isolated from a thermophilic bacterium (Thermus aquaticus), via temperature cycling, through cycles of melting, reannealing, and polymerization. PCR includes, but is not limited to, standard PCR methods, in which a DNA strand is replicated to provide millions or more copies of the original DNA strand (e.g., PCR using random primers: see, e.g., PCR with Arbitrary Primers: Approach with Care. WC Black IV, Ins. Mol. Biol. 2: 1-6, December 2007); real-time PCR techniques, in which the amount of PCR product can be monitored at each cycle (real-time quantitative PCR: CA Heid, J. Stevens, KJ Livak and PM Williams, 1996 Genome Research 6: 986-994); reverse transcription PCR, in which RNA is first copied into a DNA strand, and then the DNA strand is amplified by a standard PCR reaction (e.g., see: Quantitative RT-PCR: Pitfalls and Potential: WF Freeman, SJ Walker and KEVrana; BioTechniques 26: 112-125, January 1999).

[0019] As used herein, the term "monomer" refers to any chemical entity that can be covalently linked to one or more other such entities to form an oligomer or polymer. Examples of "monomers" include nucleotides, amino acids, sugars, amino acids, etc.

[0020] The term "nucleic acid" means a polymer composed of nucleotides, which may be deoxyribonucleotides or ribonucleotides. These compounds may be natural or synthetically produced deoxyribonucleotides or ribonucleotides. Synthetically produced nucleic acids may have naturally occurring sequences or non-natural unique sequences.

[0021] The terms "ribonucleic acid" and "RNA" refer to polymers composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" refer to polymers composed of deoxyribonucleotides.

[0022] The term "nucleotide" means a monomeric unit comprising a sugar phosphate, typically ribose-5'-phosphate or 2'-deoxyribose-5'-phosphate, covalently bonded to a nitrogenous base, typically adenine (A), guanine (G), cytosine (C), or thymine (T) in the case of a deoxyribonucleotide, and typically adenine (A), guanine (G), cytosine (C), or uracil (U) in the case of a ribonucleotide.

[0023] The term "oligonucleotide" as used in this specification refers to a single- or double-stranded polymer composed of covalent nucleotide monomers forming a chain of from 2 to about 20 nucleotides in length.

[0024] The term "polynucleotide" as used in this specification refers to a single- or double-stranded polymer composed of covalent nucleotide monomers forming a chain generally greater than about twenty nucleotides in length.

[0025] Nucleic acids with naturally occurring sequences can hybridize with nucleic acids in a sequence-specific manner. That is, they can participate in hybridization reactions in which complementary base pairs A:T (adenine:thymine) and G:C (guanine:cytosine) form intermolecular (or intramolecular) hydrogen bonds and cooperative stacking interactions between planar adjacent bases through Pi electrons in each chain, collectively referred to as Watson-Crick base pairing interactions. The bases of nucleic acid chains can also hybridize to form non-Watson-Crick base pairs through so-called "wobble" interactions, in which G (guanine) pairs with U (uracil), or alternatively, I (inosine) pairs with C (cytosine), U (uracil), or A (adenine), but with lower binding energy than normal Watson-Crick base pairing interactions.

[0026] Exemplary embodiments provide methods for marking an object and recovering the marker from the object without disturbing the appearance of the object.

[0027] Exemplary embodiments of the present disclosure also provide methods for authenticating an object using a marker that has been added to the object or added to a liquid to bind to the activated DNA marker.

[0028] Markers

[0029] Goods / products / objects can be embedded with designer DNA strands, such as custom gene vectors with specific gene sequences that encode proteins or sequences with telomeres that will shorten each time a marker is processed. These vectors can be removed from the object and processed according to molecular biology techniques available to those skilled in the art to produce specific proteins encoded by the vector. This protein can then be detected. These gene sequences can be randomized and customized for each product SKU and also customized according to a specific production date. During certification, the specific protein produced can be processed by a reagent that can be controlled by, for example, a high-end brand, which can react with another protein in a detectable manner, produce a specific smell, emit light, fluoresce, or provide a temporary color. In addition, those customized DNA markers can be designed to degrade within a specific amount of time. Even if one can sequence the vector, one still has to know the specific reagent sites and the detection / marker molecules produced. In addition, given the temporal characteristics of these effective chains, the cost of doing this reverse design will make the cost too high.

[0030] The marker of the present disclosure includes, for example, nucleic acid markers. Nucleic acid is a general term for deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and nucleic acid can be synthetic, or can be obtained from a fragment of any nucleic acid in an animal, plant, bacterium, virus, fungus or synthetic vector or the nucleic acid listed above, etc. It should be noted that synthetic nucleic acids can have the sequence of the naturally occurring nucleic acid of an animal, plant, bacterium, fungus, virus or any other organism or synthetic vector. Alternatively, synthetic nucleic acids can have a unique sequence not found in nature. It should be understood that this unique non-natural sequence can have an extension of a sequence found in nature, but the entire non-natural sequence is unique and is not found in any plant, animal or virus or any other natural organism. In particular, the nucleic acid sequence that encodes the data or elements of the mark encrypted or encoded in the marker of the present disclosure is a unique non-natural sequence, and is therefore suitable for the authentication of an object of interest.

[0031] Thus, the markers of the present disclosure may be recombinant or synthetically prepared. The DNA marker may be natural DNA, whether isolated from a natural source or synthesized; or the DNA marker may be a synthetically produced non-natural sequence. All or part of the DNA may include a recognizable sequence.

[0032] a) Telomeres - Telomeres are regions of repeated nucleotide sequences at each end of chromosomes that protect the ends of chromosomes from degradation or fusion with neighboring chromosomes. For vertebrates, the nucleotide sequence in telomeres is TTAGGG, with the complementary DNA strand being AATCCC, with a single strand of TTAGGG overhanging. This sequence of TTAGGG is repeated about 2500 times in humans. In humans, average telomere length decreases from about 11 kilobases at birth to less than 4 kilobases in old age.

[0033] During chromosome replication, the ends of chromosomes shorten with each replication because the enzymes that copy DNA cannot continue their replication all the way to the end of the chromosome (this is because the synthesis of Okazaki fragments requires the RNA primer to be attached to the lagging strand in advance). Telomeres are one-time buffers at the ends of chromosomes that are truncated during cell division; conversely, the presence of telomeres protects the genes at the front of the chromosome from being truncated. Telomeres themselves are protected by complexes of proteins called complexin and RNA encoded by telomeric DNA (TERRA).

[0034] Over time, with each cell division, the telomere ends become shorter.

[0035] In some embodiments, DNA marker includes telomere sequence at its 5' and 3' ends. In humans, during the aging of various somatic cell types in vitro and in vivo, the amount of terminal (TTAGGG)n, telomere DNA will be reduced. In some embodiments, DNA marker includes double-stranded DNA with specific sequence, with telomere sequence at each end of specific sequence. Specific sequence can be encoded for authentication information described herein. DNA marker useful in the present disclosure can include DNA marker of any suitable length, such as for example, in one example, DNA marker is double-stranded DNA oligomer with length between about 40 base pairs and about 1000 base pairs. In other embodiments, DNA marker is double-stranded DNA oligomer with length between about 80 base pairs and 500 base pairs. In another embodiment, DNA marker is double-stranded DNA oligomer with length between about 100 base pairs and about 250 base pairs. At the 5' and 3' ends of each of the base pairs is the telomeric sequence (TTAGGG)n (where n is from 1 to about 50 or more, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, etc.).

[0036] When such a marker is removed from an object, the size of the marker is determined. The marker is then placed in a somatic eukaryotic cell, such as a fibroblast (by methods available to those skilled in the art) and allowed to replicate. The replication will generate shortened fragments due to telomere shortening. The shorter fragments are analyzed for length and sequence and then applied back to the object from which the original marker was obtained.

[0037] This adds another layer of complexity and a unique identifier for the object. The method can also be used not only to authenticate the object, but also to track, for example, how many times the object has been sold and / or to limit the number of times the object can be sold (each time it is sold, the marker is copied and therefore shortened due to the nature of the telomeres, and a unique shorter DNA marker is applied to the object from which the original marker was obtained). For example, if when transferring the object, the buyer applies a DNA marker authentication process (to verify that it is a genuine object), the DNA strand will shorten / degrade during the authentication. This constitutes the first transfer of ownership. When the item is subsequently resold, the same authentication process is applied (to the now shorter strand applied to the item after the previous authentication), and the DNA strand is shortened again. At a predetermined point, the strand is shortened / degraded so that further authentication is no longer possible.

[0038] In other embodiments, intentional invalidation of markers is provided. For example, this can be used when property is seized by authorities (to prevent theft and resale after police custody). Intentional invalidation of markers can also be used in situations where an item is sold at an unusually high price, because the previous owner wants to destroy the marker, making the new owner the final and ultimate owner. This can be accomplished by oxidation or any number of available processes. In one embodiment, the DNA marker is removed, such as by splitting the telomeres through predetermined nuclease sites, and then reapplying the DNA so that the rest of the coding strand remains intact for authenticity confirmation. In other words, although the absence of any remaining telomeres will end the transfer of ownership, you still need the remaining strand to confirm its authenticity.

[0039] The DNA markers recovered from the object can be amplified by polymerase chain reaction (PCR) and decomposed by gel electrophoresis. Since the sequence of the nucleic acid markers of the present disclosure is unique and specific to the object being marked, the original nucleic acid can be amplified only by the use of primers with a specific sequence complementary to the part of the unique marker sequence. Through this procedure, if the object being inspected carries the original nucleic acid, the PCR procedure will amplify the extracted nucleic acid to produce an amplicon of a predetermined size and a sequence identical to the part of the original nucleic acid sequence of the marker. In contrast, if the sample recovered from the inspection object does not include the unique nucleic acid corresponding to the real object, it is likely that there is no amplified nucleic acid product, or if the primer does amplify the recovered nucleic acid to produce one or more random amplicons, these one or more amplicons cannot have the unique marker nucleic acid sequence from the real object. In addition, the random amplicons obtained from counterfeits also have random lengths, and the possibility of producing an amplicon of the exact length specified by the marker-specific primer is very small. Therefore, by comparing the size of the PCR product, the authenticity of the marked object can be verified, non-real objects can be screened and eliminated, and then the purpose of anti-counterfeiting screening is achieved.

[0040] b) Vectors - Some embodiments of the present disclosure provide expression vectors that include specific nucleic acid sequences and / or encode specific detectable marker proteins, including but not limited to green fluorescent protein (GFP), luciferase-luciferin, β-glucuronidase-β-D-glucuronic acid (GUS assay), β-galactosidase-galactosidase such as X-gal (blue-white screening). An expression vector, also known as an expression construct, is typically a plasmid or virus designed for gene expression in a cell. The vector is used to introduce a specific gene into a target cell, and the cell's protein synthesis machinery can be mobilized to produce the protein encoded by the gene. Expression vectors are basic tools for the production of proteins in biotechnology.

[0041] The vector is designed to contain regulatory sequences that act as enhancer and promoter regions and cause efficient transcription of the genes carried on the expression vector. The goal of a well-designed expression vector is efficient production of proteins, and this can be achieved through the production of large amounts of stable messenger RNA, which is then converted into protein. Escherichia coli is commonly used as a host for protein production, but other cell types can also be used.

[0042] Expression vector has many elements for gene expression.These elements can include promoter, correct conversion initiation sequence such as ribosome binding site and start codon, stop codon and transcription termination sequence.There are differences in the mechanism of protein synthesis between prokaryotes and eukaryotes, so expression vector must have expression elements suitable for selected host.For example, prokaryotic expression vector will have Shine-Dalgamo sequence at its conversion initiation site for combination with ribosome, and eukaryotic expression vector will comprise Kozak consensus sequence.

[0043] The promoter initiates transcription, and is therefore a control point for cloned gene expression. The promoters used in expression vectors are usually inducible, which means that protein synthesis is initiated only when an inducing agent such as IPTG is introduced as needed. However, in some expression vectors, gene expression may also be constitutive (i.e., the protein is constantly expressed).

[0044] After the expression of the gene product, it may be necessary to purify the expressed protein. A purification tag can be added to the cloned gene. The tag can be a histidine (His) tag or a fusion partner, such as glutathione S-transferase or maltose binding protein. Some of these fusion partners can also help increase the solubility of some expressed proteins.

[0045] In one embodiment, the telomere marker and / or the carrier marker can be used alone, in combination, or in combination with any other suitable detectable or traceable marker, such as a chemical marker or a biological marker. In an embodiment of the method of the present disclosure, the marker is selected from UV fluorophores, ceramic IR markers, up-conversion phosphors (UCP) infrared (IR) markers, UV markers, other DNA, amino acids, peptides, proteins, lipids, sugars, polysaccharides, pheromones, odors, trace elements, rare earth elements, or any combination of two or more thereof.

[0046] In embodiments of the present disclosure, the marker comprises a nucleic acid. In one embodiment, the marker consists essentially of DNA (eg, a telomere marker and / or a vector marker) and has no other significant components that can be used for identification or authentication.

[0047] Labeled objects and encoded information

[0048] Any object including a substantially solid surface can be identified with a nucleic acid marker (marker). For example, the ownership of an object can be identified using a DNA marker and optional additional markers according to an exemplary embodiment of the present disclosure. Examples of such objects may include, but are not limited to, ceramic surfaces, plastic films, vinyl sheets, magnetic stripe cards, souvenirs, paper products, currency, bank notes, bonds, checks, guarantee documents, or any other printed matter; jewelry, including but not limited to gemstones, rings, earrings, necklaces, watches, etc.; artwork, including but not limited to sculptures, paintings, etc.; electronic products, such as but not limited to computers, computer peripherals, printers, microchips, disk drives, televisions, radios, DVD players, CD players, sound systems, etc.; furniture, appliances, antiques, fibers or fabrics, clothing or accessories, such as but not limited to wallets (e.g., luxury handbags), shoes, belts, sunglasses, and other personal items, such as but not limited to cameras, cars, bicycles, motorcycles, luggage, etc.; and sports collectibles and other collectibles (e.g., collectible sneakers) or any valuable items.

[0049] It is also contemplated that nucleic acid markers can be used on or in personal identification documents such as, but not limited to, passports, driver's licenses, health cards, identity cards, medical insurance cards, bank cards, credit cards, birth certificates, etc.

[0050] The nucleic acid marker can also be added to a liquid composition. The liquid composition can be any suitable liquid composition, such as, for example, printing ink, dye or spray. The liquid can also be a perfume or cologne.

[0051] The authenticity of these objects can then be verified by identifying the labels bound or covalently bonded thereto by, for example, the methods described in further detail below.

[0052] In one embodiment, the marker is not endogenous to the subject to which it is applied / embedded. In other words, the DNA sequence of the marker is not present in the subject being marked.

[0053] The marker that has been applied to the object provides a traceable nucleic acid marker. The traceable nucleic acid marker can be applied to all or part of an object to be identified, confirmed, authenticated, or if the object is a commercial item, the item can be tracked at any point through the commercial stream.

[0054] The marker may be, for example, specific to the type of company or article (e.g., model), specific to a special batch or lot (batch number) of an article, or specific to an actual article, such as, for example, a unique serial number of an article. In addition, the marker may indicate any one or more of the data of various other useful articles; for example, the marker may encode the name and contact information of the company that manufactures the marked product or article, the date of manufacture, the distributor of the product or article, and / or the data of the expected retailer. The marker may also indicate, for example and not limited to, component data, such as the source of the component incorporated into the article or the identity of the production plant or machine used in the manufacture of the product or article; the date on which the product or article enters the commercial stream, the date on which it is accepted by the distributor and / or the date on which it is delivered to the retailer, the date on which it is sold to the purchaser, and any other useful commercial data, or other data such as, for example, the owner's personal information. Each element of the data or mark may be encrypted or encoded in the marker and may be deciphered from the marker recovered from the object and decoded or decrypted according to the method described herein. The decoded or decrypted data may then be used to verify the properties of the object or to authenticate the object, or to exclude counterfeit articles.

[0055] Nucleic acid markers can exhibit the following properties: after the composition is applied to an object, asset, article, etc., the DNA is stable / the DNA is protected from degradation. For example, the DNA is protected from degradation for at least about 6 months, including a period of about 1 year, such as about 2 years or about 5 years or about 10 years or even about 20 years or longer. In professional terms, "degraded" or "degradation" means that the DNA includes less than 50% of the cleavage of the DNA, including less than about 30% of the cleavage, such as less than about 20% of the cleavage, including less than about 10% of the cleavage, and less than about 1% of the cleavage of the original DNA contained in the composition. In professional terms, "cleavage" means exonuclease cleavage, endonuclease cleavage, or a combination thereof.

[0056] Methods for binding markers to substrates / objects

[0057] Nucleic acid markers can be brushed, sprayed or dipped on with or without an incorporated polymer or polymer coating (see US Published Patent Application 20090253127; the entire contents of which are incorporated herein by reference). Any method can be used as long as the object and the DNA marker are not destroyed.

[0058] In some embodiments, the DNA marker is exposed to alkaline conditions prior to contacting the marker with the substrate / object. Alkali metals are members of Group I of the periodic table, including the elements lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr). These alkali metals of the periodic table are elements that exhibit homologous chemical properties.

[0059] In one embodiment, the DNA marker is exposed to alkaline conditions and then the deoxyribonucleic acid is contacted with the substrate. The DNA bound to the substrate can be used for binding by hybridization probes, PCR amplification, and DNA sequencing methods.

[0060] In one embodiment, the alkaline conditions are generated by mixing the deoxyribonucleic acid with an alkaline solution having a high pH and by contacting the deoxyribonucleic acid that has been exposed to the alkaline conditions with the substrate, for example, the pH of the alkaline solution can be about pH 9.0 or higher; about pH 10.0 or higher; about pH 11.0 or higher, or even about pH 12.0 or higher. In one embodiment, the alkaline solution is a solution of an alkali metal hydroxide.

[0061] Another embodiment of the present disclosure provides a method of binding a deoxyribonucleic acid to a substrate, the method comprising exposing the deoxyribonucleic acid to alkaline conditions, wherein the alkaline conditions are generated by mixing the deoxyribonucleic acid with an alkaline solution and by contacting the deoxyribonucleic acid that has been exposed to the alkaline conditions with the substrate; wherein the alkaline solution is a solution of a hydroxide of an alkali metal, and the alkali metal is selected from lithium (Li), sodium (Na), rubidium (Rb) and cesium (Cs).

[0062] Another embodiment of the present disclosure provides a method for binding a deoxyribonucleic acid to a substrate, the method comprising exposing the deoxyribonucleic acid to alkaline conditions, wherein the alkaline conditions are generated by mixing the deoxyribonucleic acid with an alkaline solution and by contacting the deoxyribonucleic acid that has been exposed to the alkaline conditions with the substrate; wherein the alkaline solution is a solution of an alkali metal hydroxide, wherein the alkali metal hydroxide is selected from lithium hydroxide (LiOH), sodium hydroxide (NaOH) and cesium hydroxide (CsOH). In one embodiment, the alkali metal hydroxide is sodium hydroxide (NaOH).

[0063] These and other base binding methods / compositions are incorporated by reference in US Patent No. 9,790,538.

[0064] Removal and / or analysis of markers on objects

[0065] DNA can be recovered from an object labeled with a marker provided herein by a variety of methods as known in the art. For example, but not intended to be limited in any way, the object can be wiped with a solvent, a solution, or a combination thereof, which removes the nucleic acid marker (e.g., DNA) and dissolves any polymer associated therewith to provide access to the marker (e.g., DNA) contained therein.

[0066] It is also contemplated that the marker may be removed from the object, such as, but not limited to, by gentle rubbing or mild scraping with an appropriate tool. Such tools may include, but are not limited to, files, sandpaper, etc. In some cases, the marker may be applied to a portion of the object designed for removal, such as by cutting or breaking off / tearing off a piece of the object (e.g., an attached fabric flap or piece of a belt).

[0067] The DNA recovered from the object can be extracted, washed, purified, concentrated, amplified, or a combination thereof using methods known in the art. For example, the DNA can be subjected to one or more precipitation, extraction, centrifugation, filtration, microfiltration, chromatographic separation, electrophoretic separation, or a combination thereof. In addition, the separated DNA can be subjected to one or more enzymatic reactions, including but not limited to any cloning steps known in the art, amplification steps such as but not limited to polymerase chain reaction (PCR), etc.

[0068] In embodiments of the present disclosure, which are not meant to limit the present disclosure in any way, preparation of telomerase and protein is added ... DNA isolated from the treated subject is purified and amplified by PCR or similar methods as known in the art. Once amplified, the DNA can be sequenced, restriction fragment length polymorphism, microsatellite, short tandem repeat (STR) analysis, etc. as a means for providing information about the owner of the subject. This information can be used to search a database or data structure to identify the subject and / or its details.

[0069] database

[0070] The method of the present disclosure may also include the step of recording or registering objects that have been marked in a database, data structure, etc., which may be, but not limited to, an electronic database. The electronic database may be online or offline. In one embodiment, the electronic database is online, and information may be transmitted to the database via the Internet. Without intending to be limited in any way, in such an embodiment, the subject may be able to list and optionally describe the objects that have been marked. The information that may be recorded or registered may include, but is not limited to, the name, address, telephone number, fax number, email address, description of the marked object, the location of the marked object, the date of purchase or acquisition of the object, the location of the purchase or acquisition of the object, serial number or other identification number, etc. of the owner or manufacturer.

[0071] Methods described herein may optionally include searching a database or data structure to confirm or deny the step of object authenticity. For example, one embodiment of the present disclosure provides a method for authenticating an object, the method includes providing an object that is combined or covalently bound to a marker, sampling the object for identifying, tracking or verifying the authenticity of the object by identifying a unique traceable deoxyribonucleic acid (DNA) marker. In one embodiment, a unique marker is a DNA marker that may or may not have a unique DNA sequence, but has a unique length based on the number of times that telomerase and the marker have been replicated. Unique DNA sequence and / or length are stored in a database, and the database matches unique DNA sequence and / or length with a data element corresponding to an object that is combined or covalently bound to a unique marker. The database can be located on an accessible computer accordingly, to locate, track, authenticate and verify the identity of the labeled object from which the marker is detected.

[0072] Reagent test kit

[0073] The present disclosure also provides a kit for identifying an object with a nucleic acid marker. The kit may include a DNA marker, a marker application device such as a brush, instructions for identifying an object with the marker, one or more tables that can be used to list items identified with the marker of the present disclosure, a login ID and password or unique identification code or number or a combination thereof for a system, database or data structure that allows a subject to record items that have been identified with the marker in a database.

[0074] In embodiments of the present disclosure, which are not meant to be considered limiting in any way, the unique registration code / number can provide the owner with private access to a web-based security application, wherein the individual records or registers information about their tagged physical objects and assets, thereby linking the owner to their assets or physical property. The web-based security application can record information about the individual, such as, but not limited to, the individual's name or code name and address and answers to one or more general questions about the individual.

[0075] The markers may be provided in one or more containers, such as, but not limited to, one or more vials, screw-top tubes, Eppendorf tubes, etc. It is also contemplated that the markers may be provided as a single-use container that may be used to identify one or more objects.

[0076] Certain embodiments are described herein. Of course, after reading the foregoing description, variations of the embodiments described will become apparent to those of ordinary skill in the art. Those skilled in the art will know how to appropriately adopt such variations, and the embodiments disclosed herein can be practiced in a manner different from that specifically described. Therefore, all modifications and equivalents of the subject matter described in the claims appended hereto are included within the scope of this disclosure. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, this disclosure includes any combination of the above-mentioned elements in all possible variations thereof.

[0077] Throughout the specification, references have been made to publications, patents, and / or patent applications (collectively referred to as "references"). Each of the cited references is individually incorporated herein by reference for the teachings specifically cited and for the entirety of their disclosure.

Claims

1. A method of treating an object with a DNA marker to authenticate and track the object, the method include: a) obtaining an object containing a DNA marker, wherein the DNA marker comprises a double-stranded DNA having a specific sequence, and a telomere sequence at each end of the specific sequence; b) treating the subject in a) to remove at least a portion of the DNA markers; c) amplifying the removed DNA markers in b) to produce amplified DNA markers, and analyzing the amplified DNA markers by size and / or sequence analysis; d) placing the removed DNA marker in b) or the amplified DNA marker in c) in a somatic eukaryotic cell for replication, wherein the DNA marker has telomeres, and the replication results in shortening of the telomeres of the DNA marker; and e) applying the shortened DNA marker back to the subject to track the subject; wherein each shortening of the DNA marker in d) represents a sale of the object or an action taken on the object, and wherein the size and / or sequence of the applied DNA marker in e) allows determination of the total number of times the object has been sold or the total number of times actions have been taken on the object.

2. The method according to claim 1, in, Repeat a) to e) two or more times for the subject.

3. The method according to claim 1, in, The object is selected from the group consisting of ceramic, cotton, wool, leather, nylon, plastic, paper, stone, metal, glass, liquid or medicine.

4. The method according to claim 1, in, The object is selected from the group consisting of currency, bank notes, bonds or security documents.

5. The method according to claim 1, in, The object is selected from the group consisting of a gemstone, a ring, earrings, a necklace or a watch.

6. The method according to claim 1, in, The object is selected from the group consisting of a sculpture or a painting.

7. The method according to claim 1, in, The object is selected from the group consisting of a computer, a computer peripheral, a microchip, a disk drive, a television, a radio, a DVD player, a CD player, or a stereo system.

8. The method according to claim 1, in, The object is selected from the group consisting of furniture, electrical appliances, antiques, clothing, handbags, shoes, belts or sunglasses.

9. The method according to claim 1, in, The object is selected from the group consisting of a camera, a car, a bicycle, a motorcycle, luggage, or a sports collectible.

10. The method of claim 1, further comprising at least one of searching a database for the DNA marker, entering one or more subjects treated with the DNA marker into a database, or a combination thereof.

11. The method according to claim 1, further comprising purifying the removed DNA markers in b) before c).

12. The method according to claim 1, in, The object is vinyl resin.

13. The method according to claim 1, in, The object is a check.

14. The method according to claim 1, in, The object is a printer.