Detection of target nucleic acid sequence by PTO cleavage and extension-dependent signaling oligonucleotide cleavage

Patent metadata

Field	Value
Publication number	US-9683259-B2
Application number	US-201314394780-A
Country	US
Kind code	B2
Filing date	Apr 16, 2013
Priority date	Apr 19, 2012
Publication date	Jun 20, 2017
Grant date	Jun 20, 2017

Abstract

The present invention relates to the detection of a target nucleic acid sequence by a PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Cleavage assay (PCE-SC assay). The present invention is carried out in such a manner that the extended strand is produced on the CTO having arbitrary sequences as templates depending on the presence of target nucleic acid sequences and in turn the SO as probes is hybridized with the extended strand to give signal. The present invention employs a series of reactions including PTO hybridization and cleavage, CTO hybridization and extension, and SO hybridization and cleavage, which is responsible for the highly enhanced specificity of the present invention.

First claim

What is claimed is:

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.

2. The method according to claim 1 , said SO comprising a hybridizable sequence to the extended sequence.

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.

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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. The method according to claim 11 , 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.

13. The method according to claim 1 , wherein the PTO, CTO and/or SO is blocked at its 3′-end to prohibit its extension.

14. 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.

15. The method according to claim 1 , wherein the method further comprises a denaturation step between the steps (d) and (e).

16. 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.

17. 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.

18. 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.

19. The method according to claim 1 , where

Assignees

• Chun Jong Yoon
• Lee Young Jo
• Seegene Inc

Inventors

• Chun Jong Yoon
• Lee Young Jo

Classifications

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6823 Primary

  Release of bound markers · CPC title

• C12Q1/34

  involving hydrolase · CPC title

• C12Q2537/149

  Sequential reactions · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title