Methods and Systems for the Generation of Plurality of Security Markers and the Detection Therof

• US 20100285985A1
• Filed: 01/20/2010
• Published: 11/11/2010
• Est. Priority Date: 04/15/2003
• Status: Abandoned Application

• Current Assignee: APDN (B.V.I.) Inc. (Applied DNA Sciences Incorporated)
• Original Assignee: Applied DNA Sciences Incorporated
• Inventors: Jung, Lawrence, Hayward, James A., Liang, Minghwa
• Application Number:
  US12/690,799
• Publication Number:
  US 20100285985A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C08L 89/00 Compositions of proteins; Compositions of derivatives thereof foodstuff preparations A23J3/00
• CPC Class Codes: C09D 11/00 Inks
• CPC Class Codes: C09D 7/65 macromolecular C09D7/41-C09D7/48 take precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: G07D 7/14 using chemical means

Abstract

This invention pertains to methods for generating large quantities of DNA security markers by combinatorial variation techniques using polymorphic fragment length DNA for unique identification security marker applications such as explosive ink used in dye/smoke pack and cash carrying boxes.

Claims

1. A method of producing a plurality of security markers, the method comprising:

providing a single DNA template;

providing a pool of rtDNA oligonucleotides complementary to the template;

grouping primers in said pool of rtDNA oligonucleotides into a plurality of smaller subsets using combinatorial variation techniques; and

generating a plurality of security markers from said plurality of smaller subsets of rtDNA oligonucleotides in the pool of rtDNA oligonucleotides, each of the smaller subsets defining a distinct security marker.

2. The method of claim 1, wherein each of the plurality of smaller subsets comprise at least two sequencably distinct rtDNA oligonucleotides.

3. The method of claim 1, wherein the DNA template is from about 50 bases to about 90,000,000,000 bases in length.

4. The method of claim 1, wherein the grouping of primers in said pool of rtDNA oligonucleotides into the plurality of smaller subsets of rtDNA oligonucleotides is carried out according to the equation;

n!/(Y!(n−

Y)!).

wherein;

n is the number of amplicons that can be generated by said pool of rtDNA oligonucleotides with a detection primer and the single DNA template; and

Y is the number of amplicons generated by each of the plurality of smaller subsets of rtDNA oligonucleotides with a detection primer and the single DNA template.

5. The method of claim 1, wherein the DNA template is selected from the group consisting of artificially synthesized DNA, biosynthesized DNA from living organisms, extracted DNA from living organism, or a PCR product.

26. The method of claim 1, wherein the sequences of the pool of rtDNA oligonucleotides have lengths ranging from about 5 bp to about 100 bp in length.

6. A method of generating security markers comprising:

providing a first DNA fragment as a template,

providing a pool of oligonucleotides having corresponding sequences to the first DNA fragment template; and

generating, by combinatorial variations, a plurality of security markers each comprising a different grouping of oligonucleotides from the pool of oligonucleotides.

7. The method of claim 6, wherein the pool of oligonucleotides comprises a plurality of non-repeat rtDNA oligonucleotides having sequences complementary to the first DNA fragment template.

8. The method of claim 6, wherein the sequences of the pool of oligonucleotides have lengths ranging from about 5 bp to about 100 bp in length.

9. The method of claim 6, further comprising providing at least one fluorescent labeling dye for signal detection of at least one of the security markers.

10. The method of claim 6, further comprising providing a second DNA fragment as a template, the second DNA fragment template having a pool of oligonucleotides with sequences corresponding to the second DNA fragment template.

11. The method of claim 7, further comprising providing a detection primer, wherein the rtDNA oligonucleotides and the detection primer produce a plurality of different-sized amplicons during PCR amplification.

12. The method of claim 9, wherein the fluorescent dyes are terminal oligonucleotide labeling dyes.

13. The method of claim 6, wherein the combinatorial variations are generated using the equation

n!/(Y!(n−

Y)!)

wherein;

n is the number of oligonucleotides in the pool of oligonucleotides; and

Y is the number of oligonucleotides in each grouping used to form an individual security marker.

14. The method of claim 13, wherein the number of said groupings range from 1 to n, where n is the number of oligonucleotides in the pool of oligonucleotides.

27. The method of claim 6, wherein the DNA template can be of any length greater than the length sum of one of the rtDNA oligonucleotides and a detection primer, preferably from about 50 bases to about 90,000,000,000 base pairs.

15. A security marker comprising:

a plurality of oligonucleotides, said oligonucleotides complementary to a DNA template;

wherein said oligonucleotides are chosen by a combinatorial variation technique from a pool of oligonucleotides complementary to the DNA template.

16. The security marker of claim 15, wherein said pool of oligonucleotides are non-repeat rtDNA oligonucleotides to said DNA template.

17. The security marker of claim 16, wherein the rtDNA oligonucleotides are labeled with a fluorescent dye.

18. The security marker of claim 15, wherein the security marker is a covert marker for individual product identification.

19. The security marker of claim 15, wherein the combinatorial variation technique comprises grouping the pool of oligonucleotides by the equation

n!/((Y!(n−

Y)!)

where n is the number of possible amplicons that can be generated during PCR by the pool of oligonucleotides and a detection primer, and Y is the number of oligonucleotides in each grouping within n.

20. The security marker of claim 19, wherein the detection primer is fluorescently labeled.

21. The security marker of claim 19, wherein the detection primer is included in the security markers.

22. A method for authenticating an article, comprising:

selecting a security marker comprising oligonucleotides for the article to be authenticated, said oligonucleotides derived from a pool of rtDNA oligonucleotides;

applying said security marker to the article;

collecting a sample of the security marker from the article;

analyzing the oligonucleotides in the security marker using PCR techniques with one DNA template complementary to the oligonucleotides in the security marker and a detection primer;

generating an amplicon length profile corresponding to the oligonucleotides in the security marker;

comparing the amplicon length profile to a security marker profile database; and

determining if the amplicon length profile generated corresponds to the security marker associated with the article.

23. The method of claim 22, wherein the generating an amplicon length profile utilizes capillary electrophoresis.

24. The method of claim 22, wherein the pool of rtDNA oligonucleotides ranges from about 5 to about 100 unique rtDNA oligonucleotides.

25. The method of claim 22, the oligonucleotides are selected from the pool of rtDNA oligonucleotides using combinatorial variation techniques.