Detection of target nucleic acid sequence by pto cleavage and extension-dependent non-hybridization assay

Abstract

The present invention relates to the detection of a target nucleic acid sequence by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay. The present invention adopts the occurrence of the inhibition of the hybridization between the HO with the CTO by the formation of the target-dependent extended duplex. Therefore, the present invention may detect target sequences even when the HO is not cleaved. In this regard, the design of the 5′-tagging portion of PTO, CTO and HO sequences may be readily performed and the conditions for reactions may be also easily established. In addition, the detection of the hybrid between the CTO and the HO may be performed in a different vessel from that for the extension of the CTO.

What is claimed is: 
     
         1 . A kit for detecting a target nucleic acid sequence in a nucleic acid sample by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay, comprising:
 (a) an upstream oligonucleotide; wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid sequence;   (b) a PTO (Probing and Tagging Oligonucleotide); 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; the upstream oligonucleotide is located upstream of the PTO; the 5′-tagging portion, the 3′-targeting portion or a junction site between the 5′-tagging portion and the 3′-targeting portion has a cleavage site for an enzyme having a 5′ nuclease activity;   (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 templating portion comprising a nucleotide sequence non-complementary to the 5′-tagging portion and the 3′-targeting portion of the PTO; and   (d) a HO (hybridizing oligonucleotide) comprising a hybridizing nucleotide sequence complementary to the CTO;   wherein the kit further comprises (i) a label linked to the HO, (ii) a label linked to the CTO, (iii) a label linked to the HO and a label linked to the CTO, or (iv) an intercalating label.   
     
     
         2 . The kit according to  claim 1 , wherein the HO or the CTO has an interactive dual label comprising a reporter molecule and a quencher molecule; wherein the interactive dual label is positioned at a site such that a signal from the interactive dual label in the case of the formation of a hybrid between the CTO and the HO is different from a signal from the interactive dual label in the case of no formation of the hybrid between the CTO and the HO. 
     
     
         3 . The kit according to  claim 1 , wherein the HO has one of an interactive dual label comprising a reporter molecule and a quencher molecule and the CTO has the other of the interactive dual label; wherein the interactive dual label is positioned at a site such that a signal from the interactive dual label in the case of the formation of a hybrid between the CTO and the HO is different from a signal from the interactive dual label in the case of no formation of the hybrid between the CTO and the HO. 
     
     
         4 . The kit according to  claim 1 , wherein the kit further comprises one additional HO comprising a hybridizing nucleotide sequence complementary to the CTO and the two HOs are hybridized with the CTO in an adjacent manner to each other; wherein one of the two HOs has one of an interactive dual label comprising a reporter molecule and a quencher molecule and the other of the two HOs has the other of the interactive dual label; wherein the interactive dual label is positioned at a site such that a signal from the interactive dual label in the case of the formation of a hybrid between the CTO and the two HOs is different from a signal from the interactive dual label in the case of no formation of the hybrid between the CTO and the two HOs. 
     
     
         5 . The kit according to  claim 1 , wherein the HO or the CTO has a single label; wherein the single label is positioned at a site such that a signal from the single label in the case of the formation of a hybrid between the CTO and the HO is different from a signal from the single label in the case of no formation of the hybrid between the CTO and the HO. 
     
     
         6 . The kit according to  claim 1 , wherein one of the CTO and HO is immobilized on a solid substrate or to become immobilized on a solid substrate before the detection of a signal. 
     
     
         7 . The kit according to  claim 1 , wherein one of the CTO and the HO is labeled with a single label and the other unlabeled is immobilized on a solid substrate or is to be immobilized on a solid substrate before the detection of a signal. 
     
     
         8 . The kit according to  claim 1 , wherein the HO comprises a nucleotide sequence being competitive with the 5′-tagging portion of the PTO or its part in terms of hybridization with the CTO. 
     
     
         9 . The kit according to  claim 1 , wherein the PTO, the CTO and/or the HO is blocked at its 3′-end to prohibit its extension. 
     
     
         10 . The kit according to  claim 1 , wherein the upstream oligonucleotide is an upstream primer or an upstream probe. 
     
     
         11 . The kit according to  claim 1 , wherein the capturing portion of the CTO comprises at its 5′-end part a nucleotide sequence complementary to a 5′-end part of the 3′-targeting portion of the PTO. 
     
     
         12 . The kit according to  claim 1 , wherein the kit is for detection of 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 HO comprises at least two types of the HOs. 
     
     
         13 . The kit according to  claim 1 , wherein the kit further comprises an enzyme having a 5′ nuclease activity. 
     
     
         14 . The kit according to  claim 1 , wherein the kit further comprises a template-dependent nucleic acid polymerase. 
     
     
         15 . The kit according to  claim 1 , wherein the kit further comprise a downstream primer. 
     
     
         16 . The kit according to  claim 1 , wherein the kit is used for a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay, comprising the steps of:
 (a) hybridizing the target nucleic acid sequence with the upstream oligonucleotide and the PTO (Probing and Tagging Oligonucleotide); wherein the upstream oligonucleotide comprises the hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) the 3′-targeting portion comprising the hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) the 5′-tagging portion comprising the 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 the enzyme having the 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 the 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 the CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) the capturing portion comprising the nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) the templating portion comprising the 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 produce an extended strand complementary to the templating portion of the CTO and an extended duplex is formed; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed;   (e) performing a melting analysis or a hybridization analysis for the resultant of the step (d) over a range of temperatures with the HO (hybridizing oligonucleotide) comprising the hybridizing nucleotide sequence complementary to the CTO; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed and the CTO and the HO form a hybrid, thereby providing a signal indicative of the presence of the hybrid between the CTO and the HO; wherein when the target nucleic acid sequence is present in the nucleic acid sample, the extended duplex is formed to prevent the formation of the hybrid between the CTO and the HO, thereby not providing the signal; wherein the signal is provided by (i) the label linked to the HO, (ii) the label linked to the CTO, (iii) the label linked to the HO and the label linked to the CTO, or (iv) the intercalating label; and   (f) detecting the signal indicative of the presence of the hybrid between the CTO and the HO; wherein the presence of the hybrid between the CTO and the HO indicates the absence of the target nucleic acid sequence; wherein the absence of the hybrid between the CTO and the HO indicates the presence of the target nucleic acid sequence.   
     
     
         17 . The kit according to  claim 1 , wherein the kit is used for a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay on a solid phase, comprising the steps of:
 (a) hybridizing the target nucleic acid sequence with the upstream oligonucleotide and the PTO (Probing and Tagging Oligonucleotide); wherein the upstream oligonucleotide comprises the hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) the 3′-targeting portion comprising the hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) the 5′-tagging portion comprising the 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 the enzyme having the 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 the 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 the CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) the capturing portion comprising the nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) the templating portion comprising the 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 produce an extended strand complementary to the templating portion of the CTO and an extended duplex is formed; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed;   (e) hybridizing the resultant of the step (d) with the HO (hybridizing oligonucleotide) comprising the hybridizing nucleotide sequence complementary to the CTO under conditions suitable for hybridization between the CTO and the HO; wherein one of the CTO and the HO is labeled with a single label and the other unlabeled is immobilized on a solid substrate or is to become immobilized on a solid substrate before the detection of the signal in the step (f); wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed and the CTO and the HO form the hybrid, thereby providing a signal from the single label on the solid substrate; wherein when the target nucleic acid sequence is present in the nucleic acid sample, the extended duplex is formed to prevent the formation of the hybrid between the CTO and the HO, thereby providing no signal from the single label on the solid substrate; and   (f) detecting the signal on the solid substrate to detect the hybrid between the CTO and the HO on the solid substrate; wherein the presence of the hybrid between the CTO and the HO indicates the absence of the target nucleic acid sequence; wherein the absence of the hybrid between the CTO and the HO indicates the presence of the target nucleic acid sequence.   
     
     
         18 . The kit according to  claim 1 , wherein the kit is used for a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay, comprising the steps of:
 (a) hybridizing the target nucleic acid sequence with the upstream oligonucleotide and the PTO (Probing and Tagging Oligonucleotide); wherein the upstream oligonucleotide comprises the hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) the 3′-targeting portion comprising the hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) the 5′-tagging portion comprising the 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 the enzyme having the 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 the 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 the CTO (Capturing and Templating Oligonucleotide); wherein the CTO comprises in a 3′ to 5′ direction (i) the capturing portion comprising the nucleotide sequence complementary to the 5′-tagging portion or a part of the 5′-tagging portion of the PTO and (ii) the templating portion comprising the 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 produce an extended strand complementary to the templating portion of the CTO and an extended duplex is formed; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed;   (e) hybridizing the resultant of the step (d) with the HO (hybridizing oligonucleotide) comprising the hybridizing nucleotide sequence complementary to the CTO under conditions suitable for hybridization between the CTO and the HO; wherein when the target nucleic acid sequence is not present in the nucleic acid sample, the extended duplex is not formed and the CTO and the HO form a hybrid, thereby providing a first signal indicative of the presence of the hybrid between the CTO and the HO; wherein when the target nucleic acid sequence is present in the nucleic acid sample, the extended duplex inhibits the hybridization of the HO with the CTO, thereby providing a second signal indicative of the presence of HO unhybridized with CTO; wherein the signals are provided by (i) the label linked to the HO, (ii) the label linked to the CTO, (iii) the label linked to the HO and the label linked to the CTO, or (iv) the intercalating label; and   (f) detecting the first signal or the second signal at a predetermined temperature at which the hybrid between the CTO and the HO maintains its double-stranded form; wherein the presence of the hybrid between the CTO and the HO indicates the absence of the target nucleic acid sequence; wherein the absence of the hybrid between the CTO and the HO indicates the presence of the target nucleic acid sequence; wherein the difference in the first signal and the second signal allows to determine the presence or absence of the hybrid between the CTO and the HO to indicate the presence or absence of the target nucleic acid sequence in the nucleic acid sample.