Nucleic acid-based authentication and identification codes
The present disclosure relates to nucleic-acid based product authentication and identification by determining authentication codes comprising target nucleic acids using oligonucleotide probes associated with samples. The presence of the authentication code is determined using detection methods, such as flow cytometric methods, capable of particle discrimination based on the light scattering or fluorescence properties of the particle. Target-correlated fluorescence signal, originating from a target nucleic acid hybridized to labeled complementary oligonucleotides is determined as an indicator of the presence of the authentication code. In some embodiments, an intercalating dye is used to determine the presence of target nucleotide/oligonucleotide heterodimers and identify an authentication code.
1. A method for authenticating a sample by determining the presence of at least one target nucleic acid, the method comprising:
a) receiving a sample to be authenticated, wherein the sample comprises an associated authentication code comprising at least two target nucleic acids having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16;
b) contacting the target nucleic acids with:
a dye that fluoresces when the dye intercalates in dsDNA, and
a microparticle comprising an oligonucleotide probe complementary to at least a portion of the target nucleic acid, wherein each microparticle comprises one or both of at least one unique first reporter compound and at least one discretely measurable electromagnetic radiation property;
c) determining fluorescence associated with the microparticle, wherein the fluorescence is caused by the dye intercalating in the dsDNA formed by a duplex heterodimer of the target nucleic acid and the complementary oligonucleotide probe; and
d) identifying the sample as being authenticated by determining the presence of the authentication code based on the fluorescence thereby associated with the microparticle.
2. The method of claim 1 , wherein the target nucleic acids are incorporated into a formulation of the sample.
3. The method of claim 1 , wherein the sample is selected from the group consisting of a biological specimen, a pharmaceutical, a food, and a nutraceutical.
4. The method of claim 1 , wherein the microparticle further comprises a fluorescent compound capable of fluorescing at a different wavelength than the dye.
5. The method of claim 1 , wherein the at least one unique first reporter compound comprises a fluorescence signal.
6. The method of claim 1 , wherein the authentication code further comprises a known ratio other than 1:1 of the duplex heterodimers formed by a plurality of target nucleic acids and complementary oligonucleotide probes.
7. A method for authenticating a sample by determining the presence of at least one target nucleic acid, the method comprising:
receiving a sample to be authenticated, wherein the sample comprises an associated authentication code comprising one or more target nucleic acids having a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16;
contacting the target nucleic acid with a substrate comprising an immobilized oligonucleotide probe complementary to at least a portion of the target nucleic acid and a second reporter compound capable of producing a detectable signal upon formation of a duplex heterodimer of the target nucleic acid and the complementary oligonucleotide probe;
detecting the detectable signal associated with the substrate; and
identifying the sample as being authenticated by determining the presence of the authentication code based on the detectable signal associated with the substrate.
8. The method of claim 7 , wherein the second reporter compound comprises an optical label, a fluorescent dye, a radionucleotide, or an enzyme capable of producing the detectable signal upon intercalation in the duplex heterodimer.
9. The method of claim 7 , wherein the second reporter compound comprises a fluorophore covalently attached to either the oligonucleotide probe or the substrate such that the detectable signal is a fluorescence signal detectable upon formation of the duplex heterodimer.
10. The method of claim 7 , wherein the substrate comprises a microparticle.
11. The method of claim 10 , wherein the authentication code comprises at least two target nucleic acids, and wherein each microparticle comprises one or both of at least one unique first reporter compound and at least one discretely measurable electromagnetic radiation property.
12. The method of claim 11 , wherein detecting the detectable signal associated with each of the uniquely identifiable microparticles is carried out by laser flow cytometry, laser scanning, or fluorescence microscopy, and combinations thereof.
13. The method of claim 7 , wherein the authentication code comprises two or more target nucleic acids, and wherein the target nucleic acids are present at a known ratio other than 1:1.
14. The method of claim 7 , wherein the substrate comprises an array having the oligonucleotide probe attached to a solid support at a known location.