Detection of nucleic acids and proteins
The invention generally relates to methods for detecting a target nucleic acid and a target protein in a single assay.
1. A method of detecting multiple analytes in a sample, the method comprising the steps of:
obtaining a sample comprising at least one target nucleic acid and at least one target protein;
introducing an aptamer that binds to the target protein in the sample;
removing unbound aptamer; and
conducting a single assay, wherein the assay detects both the target nucleic acid and the target protein, the assay comprising: performing a sequencing reaction on the target nucleic acid and the aptamer, wherein sequencing comprises: hybridizing a primer to each of the nucleic acid and the aptamer to form each of a nucleic acid/primer duplex and an aptamer/primer duplex; contacting each of the duplexes with a polymerase enzyme in the presence of at least one detectably labeled nucleotide under conditions that permit the polymerase to add nucleotides to the primers in a template-dependent manner; detecting a signal from the incorporated labeled nucleotide; and sequentially repeating the contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of each of the nucleic acid and the aptamer thereby detecting the nucleic acid and the aptamer in the sample.
2. The method according to claim 1 , further comprising quantifying the target nucleic acid.
3. The method according to claim 1 , further comprising quantifying the target protein.
4. The method according to claim 1 , further comprising quantifying the target nucleic acid and the target protein.
5. The method according to claim 1 , wherein prior to sequencing, the nucleic acid and the aptamer are attached to a substrate.
6. The method according to claim 5 , wherein the nucleic acid and the aptamer are directly attached to the substrate.
7. The method according to claim 5 , wherein the nucleic acid and the aptamer are indirectly attached to the substrate.
8. The method according to claim 1 , wherein prior to sequencing, the method further comprises amplifying the nucleic acid.
9. The method according to claim 1 , wherein sequencing is sequencing by synthesis.
10. The method according to claim 9 , wherein the sequencing by synthesis is single molecule sequencing by synthesis.
11. The method according to claim 1 , wherein the detectably labeled nucleotide is an optically labeled nucleotide.
12. The method according to claim 11 , wherein the optically labeled nucleotide is a fluorescently labeled nucleotides.
13. The method according to claim 1 , wherein the target nucleic acid is associated with a disease.
14. The method according to claim 13 , wherein the disease is cancer.
15. The method according to claim 13 , wherein the target nucleic acid is selected from the group consisting of: FGFR3, K-ras, K-ras2, APC, DCC, TP53, PRC1, NUSAPI1, CAPZ, PFKP, EVER1, FLT1, ESPL1, AKAP2, CDC45L, RAMP, SYNGR2, NDRG1, ZNF533, and hypermethylated nucleic acid.
16. The method according to claim 1 , wherein the target protein is associated with a disease.
17. The method according to claim 16 , wherein the disease is cancer.
18. The method according to claim 17 , wherein the target protein is selected from the group consisting of: MMP-2, MMP-9, MMP9/NGAL complex, MMP/TIMP complex, MMP/TIMP1 complex, ADAMTS-7, ADAM-12, thymosin 15, thymosin 16, profilin, prohibitin, ubiquitin, tropomyosin, Cyr61, cystatin B, and chaperonin 10.
19. A method for diagnosing a disease, the method comprising:
obtaining a sample comprising at least one target nucleic acid and at least one target protein, each of which is suspected of being associated with a disease;
introducing an aptamer that binds to the target protein in the sample;
removing unbound aptamer;
conducting a single assay, wherein the assay detects both the target nucleic acid and the target protein, the assay comprising: performing a sequencing reaction on the target nucleic acid and the aptamer; wherein sequencing comprises: hybridizing a primer to each of the nucleic acid and the aptamer to form each of a nucleic acid/primer duplex and an aptamer/primer duplex; contacting each of the duplexes with a polymerase enzyme in the presence of at least one detectably labeled nucleotide under conditions that permit the polymerase to add nucleotides to the primers in a template-dependent manner; detecting a signal from the incorporated labeled nucleotide; and sequentially repeating the contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of each of the nucleic acid and the aptamer; and
diagnosing the disease based on results of the assay.
20. A method for diagnosing a cancer, the method comprising:
obtaining a sample comprising at least one target nucleic acid and at least one target protein, each of which is suspected of being associated with a cancer;
introducing an aptamer that binds to the target protein in the sample;
removing unbound aptamer;
conducting a single assay, wherein the assay detects both the target nucleic acid and the target protein, the assay comprising: performing a sequencing reaction on the target nucleic acid and the aptamer; wherein sequencing comprises: hybridizing a primer to each of the nucleic acid and the aptamer to form each of a nucleic acid/primer duplex and an aptamer/primer duplex; contacting each of the duplexes with a polymerase enzyme in the presence of at least one detectably labeled nucleotide under conditions that permit the polymerase to add nucleotides to the primers in a template-dependent manner; detecting a signal from the incorporated labeled nucleotide; and sequentially repeating the contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of each of the nucleic acid and the aptamer; and
diagnosing the cancer based on results of the assay.
21. The method according to claim 20 , wherein the cancer is bladder or colorectal cancer.