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- Nachgewiesen in: USPTO Patent Applications
- Sprachen: English
- Document Number: 20150167060
- Publication Date: June 18, 2015
- Appl. No: 14/394780
- Application Filed: April 16, 2013
- Assignees: SEEGENE, INC. (Seoul, KR)
- Claim: 1. A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SC (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Cleavage) assay, comprising: (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a PTO (Probing and Tagging Oligonucleotide); said upstream oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; said PTO comprising (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; wherein the 3′-targeting portion is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence; the upstream oligonucleotide is located upstream of the PTO; (b) contacting the resultant of the step (a) to an enzyme having a 5′-nuclease activity under conditions for cleavage of the PTO; wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the enzyme having a 5′-nuclease activity such that the cleavage releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO; (c) hybridizing the fragment released from the PTO with a CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a template portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO; (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase, wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended strand comprising an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex; (e) hybridizing the extended strand with SO (Signaling Oligonucleotide) to form an extended strand/SO hybrid; wherein the SO comprises a hybridizing nucleotide sequence complementary to the extended strand and at least one label; (f) cleaving the SO of the extended strand/SO hybrid using a nucleolytic enzyme to generate a cleaved fragment of the SO; and (g) detecting the occurrence of the cleavage reaction in the step (f); wherein the detection is performed by measuring a signal provided from the label linked to the SO, whereby the occurrence of the cleavage reaction of the SO of the extended strand/SO hybrid indicates the presence of the target nucleic acid sequence.
- Claim: 2. The method according to claim 1, said SO comprising a hybridizable sequence to the extended sequence.
- Claim: 3. The method according to claim 1, wherein the nucleolytic enzyme is a 5′ nuclease and the formation of the extended strand/SO hybrid in the step (e) produces a cleavage site for the 5′ nuclease, whereby the SO of the extended strand/SO hybrid is cleaved in a 5′ to 3′ direction by the 5′ nuclease.
- Claim: 4. The method according to claim 1, wherein the nucleolytic enzyme is a 5′ nuclease and the step (f) is performed in the presence of an upstream oligonucleotide located upstream of the SO, such that the SO of the extended strand/SO hybrid is cleaved by the nucleolytic activity of the 5′ nuclease dependent on the upstream oligonucleotide or its extended strand.
- Claim: 5. (canceled)
- Claim: 6. The method according to claim 1, wherein the nucleolytic enzyme is a ribonuclease, said SO comprising a RNA sequence and the formation of the extended strand/SO hybrid in the step (e) produces a DNA-RNA hybrid duplex, whereby the SO of the extended strand/SO hybrid is cleaved by the ribonuclease.
- Claim: 7. The method according to claim 1, wherein the nucleolytic enzyme is a restriction enzyme, said SO comprising a sequence recognized by the restriction enzyme and the formation of the extended strand/SO hybrid in the step (e) produces a cleavage site for the restriction enzyme, whereby the SO of the extended strand/SO hybrid is cleaved by the restriction enzyme.
- Claim: 8. The method according to claim 1, wherein the formation of the extended strand/SO hybrid in the step (e) produces a cleavage site for a nucleolytic enzyme capable of cleaving a DNA duplex, a RNA duplex or a DNA-RNA hybrid duplex.
- Claim: 9. The method according to claim 1, wherein the nucleolytic enzyme in the step (f) is a 5′ nuclease and the 5′ nuclease is a template-dependent DNA polymerase having a 5′ nuclease activity or FEN nuclease.
- Claim: 10. The method according to claim 1, wherein the SO has an interactive dual label comprising a reporter molecule and a quencher molecule, the cleavage site for the nucleolytic enzyme is positioned between the reporter molecule and the quencher molecule linked to the SO, the cleavage of the SO of the extended strand/SO hybrid separates the reporter molecule and the quencher molecule from each other and the occurrence of the cleavage reaction of the SO of the extended strand/SO hybrid is detected by measuring a signal from the label.
- Claim: 11. The method according to claim 1, wherein the SO has a single label, the cleavage of the SO of the extended strand/SO hybrid produces a fragment having the single label, and the occurrence of the cleavage of the SO of the extended strand/SO hybrid is detected by detecting the release of the single-labeled fragment.
- Claim: 12. (canceled)
- Claim: 13. The method according to claim 1, said SO comprising a 5′-tagging portion, said 5′-tagging portion comprising in its 5′-direction a non-complementary sequence to the extended strand.
- Claim: 14. The method according to claim 13, wherein the cleavage of the SO of the extended strand/SO hybrid releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the SO and the fragment released from the SO is capable of hybridization with the CTO and extension.
- Claim: 15. The method according to claim 1, wherein the PTO, CTO and/or SO is blocked at its 3′-end to prohibit its extension.
- Claim: 16-20. (canceled)
- Claim: 21. The method according to claim 1, wherein the SO is immobilized on the surface of a solid substrate via its 3′-end or 5′-end, the SO has a single label, the cleavage of the SO of the extended strand/SO hybrid produces a fragment having the single label, and the fragment is released on the solid substrate, whereby a signal change occurs on the solid substrate to detect the occurrence of the cleavage of the SO of the extended strand/SO hybrid.
- Claim: 22. The method according to claim 1, wherein the method further comprises a denaturation step between the steps (d) and (e).
- Claim: 23. The method according to claim 1, wherein the method further comprises repeating all or some of the steps (a)-(g) with denaturation between repeating cycles.
- Claim: 24. The method according to claim 1, wherein the steps (a)-(g) are performed in a single reaction vessel or some of the steps (a)-(g) are performed in separate vessels.
- Claim: 25. The method according to claim 1, wherein the method is performed to detect at least two types of target nucleic acid sequences; wherein the upstream oligonucleotide comprises at least two types of oligonucleotides, the PTO comprises at least two types of the PTOs, the CTO comprises at least two types of the CTOs and the SO comprises at least two types of the SOs.
- Claim: 26. (canceled)
- Claim: 27. The method according to claim 1, wherein the target nucleic acid sequence comprises a nucleotide variation.
- Claim: 28. The method according to claim 1, wherein the method is performed in the presence of a downstream primer.
- Claim: 29. A method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SC (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Cleavage) assay, comprising: (a) hybridizing the target nucleic acid sequence with a primer pair comprising an upstream primer and a downstream primer and a PTO (Probing and Tagging Oligonucleotide); each of said upstream primer and said downstream primer comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequences; said PTO comprising (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; wherein the 3′-targeting portion is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence; the PTO is located between the upstream primer and the downstream primer; wherein the PTO is blocked at its 3′-end to prohibit its extension; (b) contacting the resultant of the step (a) to an enzyme having a 5′-nuclease activity under conditions for cleavage of the PTO; wherein the upstream primer or its extended strand induces cleavage of the PTO by the enzyme having a 5′-nuclease activity such that the cleavage releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO; (c) hybridizing the fragment released from the PTO with a CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a template portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO; (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase, wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended strand comprising an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex; (e) hybridizing the extended strand with SO (Signaling Oligonucleotide) to form an extended strand/SO hybrid; wherein the SO comprises a complementary sequence to the extended strand and at least one label; (f) cleaving the SO of the extended strand/SO hybrid using a nucleolytic enzyme to generate a cleaved fragment of the SO; and (g) detecting the occurrence of the cleavage reaction in the step (f); wherein the detection is performed by measuring a signal provided from the label linked to the SO, whereby the occurrence of the cleavage reaction of the SO of the extended strand/SO hybrid indicates the presence of the target nucleic acid sequence.
- Claim: 30. The method according to claim 29, said wherein the method further comprising repeating all or some of the steps (a)-(g) with denaturation between repeating cycles.
- Claim: 31. A kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SC (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Cleavage) assay, comprising: (a) a probing and targeting oligonucleotide (PTO); wherein the PTO comprises (i) a 3′-targeting portion comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5′-tagging portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence; wherein the 3′-targeting portion of the PTO is hybridized with the target nucleic acid sequence and the 5′-tagging portion is not hybridized with the target nucleic acid sequence; (b) an upstream oligonucleotide; wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the upstream oligonucleotide is located upstream of the PTO; wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by an enzyme having a 5′ nuclease activity such that the cleavage releases a fragment comprising the 5′-tagging portion or a part of the 5′-tagging portion of the PTO; (c) a CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) a template portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; wherein the fragment released from the PTO is hybridized with the capturing portion of the CTO and extended to generate an extended strand comprising an extended sequence complementary to the template portion of the CTO, whereby an extended duplex is formed; (d) a SO (Signaling Oligonucleotide) having at least one label; the SO comprises a complementary sequence to the extended strand; wherein the SO is hybridized with the extended strand to form an extended strand/SO hybrid; and (e) a nucleolytic enzyme; wherein the nucleolytic enzyme cleaves the SO of the extended strand/SO hybrid.
- Claim: 32. The kit according to claim 31, said SO comprising a hybridizable sequence to the extended sequence.
- Claim: 33. The kit according to claim 31, wherein the nucleolytic enzyme is a 5′ nuclease and the formation of the extended strand/SO hybrid produces a cleavage site for the 5′ nuclease, whereby the SO of the extended strand/SO hybrid is cleaved in a 5′ to 3′ direction by the 5′ nuclease.
- Claim: 34. The kit according to claim 31, wherein the nucleolytic enzyme is a 5′ nuclease, said kit further comprising an upstream oligonucleotide located upstream of the SO, such that the SO of the extended strand/SO hybrid is cleaved by the nucleolytic activity of the 5′ nuclease dependent on the upstream oligonucleotide or its extended strand.
- Claim: 35. (canceled)
- Claim: 36. The kit according to claim 31, wherein the nucleolytic enzyme is a ribonuclease, said SO comprising a RNA sequence and the formation of the extended strand/SO hybrid produces a DNA-RNA hybrid duplex, whereby the SO of the extended strand/SO hybrid is cleaved by the ribonuclease.
- Claim: 37. The kit according to claim 31, wherein the nucleolytic enzyme is a restriction enzyme, said SO comprising a sequence recognized by the restriction enzyme and the SO of the extended strand/SO hybrid is cleaved by the restriction enzyme.
- Claim: 38. The kit according to claim 31, wherein the formation of the extended strand/SO hybrid produces a cleavage site for a nucleolytic enzyme capable of cleaving a DNA duplex, a RNA duplex or a DNA-RNA hybrid duplex.
- Claim: 39. The kit according to claim 31, wherein the nucleolytic enzyme is a 5′ nuclease and the 5′ nuclease is a template-dependent DNA polymerase having a 5′ nuclease activity or FEN nuclease.
- Claim: 40. The kit according to claim 31, wherein the SO has an interactive dual label comprising a reporter molecule and a quencher molecule, the cleavage site for the nucleolytic enzyme is positioned between the reporter molecule and the quencher molecule linked to the SO, the cleavage of the SO of the extended strand/SO hybrid separates the reporter molecule and the quencher molecule from each other.
- Claim: 41. The kit according to claim 31, wherein the SO has a single label, the cleavage of the SO of the extended strand/SO hybrid produces a fragment having the single label, and the occurrence of the cleavage of the SO of the extended strand/SO hybrid is detected by detecting the release of the single-labeled fragment.
- Claim: 42. (canceled)
- Claim: 43. The kit according to claim 31, said SO comprising a 5′-tagging portion comprising in its 5′-direction a non-complementary sequence to the extended strand.
- Claim: 44. (canceled)
- Claim: 45. The kit according to claim 31, wherein the PTO, CTO and/or SO is blocked at its 3′-end to prohibit its extension.
- Claim: 46. (canceled)
- Claim: 47. The kit according to claim 31, said kit further comprising an enzyme having a 5′ nuclease activity for cleaved the PTO hybridized with the target nucleic acid sequence.
- Claim: 48. The kit according to claim 31, wherein the SO is immobilized on the surface of a solid substrate via its 3′-end or 5′-end and the SO has a single label.
- Claim: 49. The kit according to claim 31, wherein the kit is used to detect at least two types of target nucleic acid sequences; said upstream oligonucleotide comprising at least two types of oligonucleotides, said PTO comprising at least two types of the PTOs, said CTO comprising at least two types of the CTOs and said SO comprising at least two types of the SOs.
- Claim: 50. The kit according to claim 31, wherein the upstream oligonucleotide is an upstream primer and said kit further comprising a template-dependent nucleic acid polymerase for the extension of the upstream primer.
- Claim: 51. The kit according to claim 31, said kit further comprising a downstream primer.
- Current International Class: 12
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