NZ627919B2

NZ627919B2 - Detection of target nucleic acid sequence by pto cleavage and extension-dependent signaling oligonucleotide hybridization assay

Info

Publication number: NZ627919B2
Application number: NZ627919A
Authority: NZ
Inventors: Joug Yoon Chun; Young Jo Lee
Original assignee: Seegene Inc
Priority date: 2012-02-02
Filing date: 2012-07-03
Publication date: 2017-02-28

Abstract

Disclosed is method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signalling Oligonucleotide Hybridisation) assay, comprising: (a) hybridising the target nucleic acid sequence with an upstream primer and a probing and targeting oligonucleotide (PTO); wherein the upstream oligonucleotide comprises a hybridising nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) a 3 - targeting portion comprising a hybridising 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 hybridised with the target nucleic acid sequence and the 5'-tagging portion is not hybridised with the target nucleic acid sequence; the upstream primeris 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 extended strand of the upstream primer 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) hybridising the fragment released from the PTO with a capturing and templating oligonucleotide (CTO); 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; wherein the fragment released from the PTO is hybridised 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 hybridised 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) hybridising the extended strand with a signalling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridisation with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence. probing and targeting oligonucleotide (PTO); wherein the upstream oligonucleotide comprises a hybridising nucleotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) a 3 - targeting portion comprising a hybridising 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 hybridised with the target nucleic acid sequence and the 5'-tagging portion is not hybridised with the target nucleic acid sequence; the upstream primeris 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 extended strand of the upstream primer 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) hybridising the fragment released from the PTO with a capturing and templating oligonucleotide (CTO); 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; wherein the fragment released from the PTO is hybridised 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 hybridised 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) hybridising the extended strand with a signalling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridisation with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

Description

DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO-CLEAVAGE AND EXTENSION-DEPENDENT SIGNALING OLIGONUCLEOTIDE HYBRIDIZATION ASSAY BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to the/detection of a target nucleic acid sequence by a PCE-SH (PTO Cleavage and Extension~Dependent Signaling Oligonucleotide Hybridization) assay.

PTION OF THE RELATED ART DNA hybridization is a fundamental s in molecular biology and is affected by ionic strength, base ition, length of fragment to which the nucleic acid has been reduced, the degree of mismatching, and the presence of denaturing agents.

DNA hybridization—based logies would be a very useful tool in specific nucleic acid sequence determination and clearly be valuable in clinical sis, genetic ch, and forensic laboratory analysis.

However, the conventional methods and processes depending mostly on hybridization are very likely to produte false positive results due to non-specific hybridization between probes and non—target sequences. Therefore, there remain problems to be solved for improving their reliability.

Besides probe hybridization ses, several approaches using additional enzymatic reactions, for example, TaqManTM probe method, have been suggested.

In TaqManTM probe method, the labeled probe hybridized with a target nucleic acid sequence is cleaved by a 5’ nuclease activity of an upstream —dependent DNA polymerase, ting a signal indicating the presence of a target sequence (us. Pat. Nos. 5,210,015, 5,538,848 and 145). The TaqManTM probe method suggests two approaches for signal generation: polymerization—dependent cleavage and polymerization-independent cleavage. In polymerization-dependent cleavage, " ion of the upstream primer must occur before a nucleic acid polymerase encounters the 5’-end of the labeled probe. As the extension reaction continues, the polymerase progressively cleaves the 5’-end of the labeled probe. In rization- independent cleavage, the upstream primer and the labeled probe are hybridized with a target nucleic acid sequence in close ity such that binding of the nucleic acid polymerase to the 3’—end of the upstream primer puts it in contact with the 5’-end of the labeled probe to release the label. In addition, the TaqManTM probe method discloses that the labeled probe at its 5’-end having a 5’—tail region not-hybridizable with a target ce is also d to form a fragment sing the 5’—tail region.

There have been reported some methods in which a probe having a 5’-tail region non-complementary to a target sequence is cleaved by 5’ nuclease to release a fragment comprising the 5’-tail regiOn.

For. instance, US. Pat. No. 5,691,142 discloses a cleavage structure to be digested by 5’ nuclease activity of DNA rase. The cleavage structure is exemplified in which an oligonucleotide comprising a 5’ portion non-complementary to and a 3’ portion complementary to a template is ized with the template and an upstream oligonucleotide is hybridized with the template in close proximity. The cleavage structure is cleaved by DNA polymerase having 5’ nuclease activity or modified DNA polymerase with d synthetic activity to release the 5’ n non-complementary to the template. The released 5’ portion is then hybridized with an oligonucleotide having a hairpin structure to form a cleavage structure, thereby inducing progressive cleavage reactions to detect a target ce.

US. Pat. No. 7,381,532 ses a process in which the cleavage structure having the upstream oligonucleotide with blocked 3’~end is cleaved by DNA polymerase having 5’ nuclease activity or FEN nuclease to release non-complementary ’ ﬂap region and the released 5’ ﬂap region is detected by size analysis or interactive dual label. US. Pat. No 819 discloses that detectable released ﬂaps are produced by a c acid synthesis dependent, ﬂap-mediated sequential _-——-,Vv~vv.. amplification method. In this method, a released ﬂap from a first cleavage structure . cleaves, in a nucleic acid synthesis dependent manner, a second cleavage structure to release a ﬂap from the second cleavage structure and the release ﬂaps are ed.

By hybridization of ﬂuorescence-labeled probes in a liquid phase, a plurality of target c acid sequences may be simultaneously detected using even a single type of a fluorescent label by melting curve analysis. However, the conventional logies for detection of target sequences by 5’ nuclease-mediated cleavage of interactive-dual d probes require different types of ﬂuorescent labels for different target sequences in multiplex target detection, which limits the number of target sequences to be ed due to limitation of the number of types of ﬂuorescent labels.

US. Pat. Appln. Pub. 2008-0241838 discloses a target detection method using cleavage of a probe having a 5’ portion non-complementary to a target c acid sequence and hybridization of a capture probe. A label is oned on the non- complementary 5’ portion. The labeled probe ized with the target sequence is cleaved to release a fragment, after which the fragment is then hybridized with the capture probe to detect the presence of the target sequence. In this method, it is necessary that an uncleaved/intact probe is not hybridized with the capture probe. For that, the capture probe having a shorter length has to be immobilized onto a solid substrate. However, such a limitation s in lower efficiency of hybridization on a solid substrate and also in difficulties in optimization of reaction conditions.

Therefore, there remain long-felt needs in the art to develop novel approaches for detection of a target sequence, preferably multiple target sequences, in a liquid phase and on a solid phase by not only hybridization but also enzymatic reactions such as 5’ nucleolytic reaction in a more ient, reliable and reproducible manner.

Furthermore, a novel target detection method not limited by the number of types of labels (particularly, ﬂuorescent labels) is also needed in the art.

Therefore, there remain long-felt needs in the art to p novel approaches for detection of a target nucleic acid sequence in a more convenient, reliable and reproducible manner, which is capable of being free from shortcomings of the conventional logies. hout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention ns.

SUMMARY OF THE INVENTION The present inventors have made intensive researches to develop novel ches to detect target ces with more improved accuracy and convenience, inter alia, in a multiplex manner. As a result, we have ished novel protocols for detection of target sequences, in which target detection is accomplished by tic reactions such as 5’ nucleolytic reaction and ion and extension- dependent hybridization as well as probe hybridization. The present protocols are well adopted to liquid phase reactions as well as solid phase ons, and ensure detection of multiple target sequences with more improved accuracy and ience.

Accordingly, the invention provides methods for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling ucleotide Hybridization) assay.

The invention also provides kits for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH assay.

Advantages of the present invention will become apparent from the detailed description to follow in view the appended claims and drawings.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the schematic structures of PTO (Probing and Tagging Oligonucleotide), CTO (Capturing and Templating Oligonucleotide) and SO (Signaling Oligonucleotide) used in a PCE-SH (PTO Cleavage and Extension-Dependent ing Oligonucleotide Hybridization) assay. Preferably, the 3'-ends of the PTO, CTO and SO are blocked to prohibit their extension.

Fig. 2 represents schematically PCE-SH assay using an intrastrand interactive dual label. The SO has a reporter molecule and a quencher molecule.

Fig. 3 represents schematically PCE-SH assay using a single label. The SO has a reporter molecule as a single label. The reporter molecule is required to show different signal ity depending on its presence on a -stranded form or a double-stranded form.

Fig. 4 represents schematically PCE-SH assay using an interstrand interactive dual label and two SOs. The two SOs each comprises one label among a er molecule and a quencher molecule of an ctive dual label.

Fig. 5 represents schematically PCE-SH assay using an interstrand interactive dual label. The SO comprises a reporter molecule and the extended strand comprises a quencher molecule.

Fig. 6 represents schematically PCE-SH assay using an interstrand interactive dual label. The SO comprises a er molecule and the extended strand comprises quencher-iso-dG residue incorporated during the extension reaction.

Fig. 7 represents schematically PCE-SH assay using an interstrand interactive dual label. The SO comprises a reporter molecule and the extended strand comprises er-dA residues incorporated during the extension reaction.

Fig. 8 represents schematically PCE-SH assay using intercalating dyes. The SO comprises an acceptor. SYBR green is used as donors.

Fig. 9 represents tically PCE-SH assay for detection of a tide variation.

Fig. 10A shows the results of the real-time detection of Neisseria gonorrhoeae gene by PCE-SH assay. The SO has a reporter molecule and a quencher molecule.

Template1) is a synthetic oligonucleotide for Neisseria gonorrhoeae gene. PTO2) (Probing and Tagging ucleotide) is blocked with a carbon spacer at its .

CTO3) (Capturing and Templating Oligonucleotide) is blocked with a carbon spacer at its 3’-end. SO4) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a quencher molecule at its 3’-end.

Fig. 10B shows the results of the detection of Neisseria hoeae gene by PCE-SH assay comprising steps for a g analysis. The SO has a reporter molecule and a quencher molecule. Template1) is a synthetic oligonucleotide for Neisseria gonorrhoeae gene. PTO2) (Probing and Tagging Oligonucleotide) is blocked with a carbon spacer at its 3’-end. CTO3) (Capturing and Templating Oligonucleotide) is blocked with a carbon spacer at its 3’-end. SO4) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a quencher molecule at its 3’-end. Tm5) represents melting temperature of the extended strand-SO hybrid.

Fig. 11A shows the results of the real-time detection of Neisseria gonorrhoeae gene by PCE-SH assay with PCR amplification. The SO has a reporter molecule and a quencher le. te1) is a genomic DNA of Neisseria gonorrhoeae. Primers2) are a pair of primers for PCR. PTO3) (Probing and Tagging Oligonucleotide) is blocked with a carbon spacer at its 3’-end. CTO4) (Capturing and ting ucleotide) is d with a carbon spacer at its 3’-end. SO5) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a quencher molecule at its 3’-end.

Fig. 11B shows the results of the detection of Neisseria gonorrhoeae gene by PCE-SH assay comprising steps for post-PCR melting analysis. The SO has a reporter molecule and a quencher molecule. Template1) is a genomic DNA of Neisseria gonorrhoeae. Primers 2) are a pair of primers for PCR. PTO3) (Probing and Tagging Oligonucleotide) is blocked with a carbon spacer at its 3’-end. CTO4) (Capturing and Templating Oligonucleotide) is blocked with a carbon spacer at its 3’-end. SO5) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a er molecule at its 3’-end. Tm6) represents g temperature of the extended - SO hybrid.

Fig. 12 shows the results of the detection of a single nucleotide variation of a target nucleic acid sequence by PCE-SH assay with comprising steps for post-PCR melting analysis. The C677T mutation on the MTHFR (Methylenetetrahydrofolate reductase) gene was detected. Template1) is MTHFR(C677T) ype (C), mutanttype (T), or hetero-type (C/T) human genomic DNA. Primers2) are a pair of primers for PCR. PTOs3) (Probing and Tagging Oligonucleotide) include a PTO for detecting wildtype template and a PTO for mutant-type and are blocked with a carbon spacer at its 3’-end. CTOs4) (Capturing and ting ucleotide) include a CTO for detecting wild-type template and a CTO for mutant-type and are blocked with a carbon spacer at its 3’-end. SO5) (Signaling Oligonucleotide) has a quencher molecule at its 5’-end and a fluorescent molecule at its 3’-end. Tm6) represents melting temperature of the extended strand-SO hybrid.

Fig. 13A shows the s of the real-time ion of Neisseria hoeae gene by PCE-SH assay using am oligonucleotide-independent 5’ nuclease activity.

Template1) is a synthetic oligonucleotide for Neisseria gonorrhoeae gene. PTO2) (Probing and Tagging Oligonucleotide) is blocked with a carbon spacer at its 3’-end.

CTO3) ring and Templating Oligonucleotide) is blocked with a carbon spacer at its 3’-end. SO4) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a quencher molecule at its 3’-end.

Fig. 13B shows the results of the detection of Neisseria gonorrhoeae gene by PCE-SH assay comprising steps for a melting analysis using am oligonucleotideindependent ’ nuclease activity. Template1) is a synthetic oligonucleotide for Neisseria gonorrhoeae gene. PTO2) (Probing and Tagging Oligonucleotide) is blocked with a carbon spacer at its . CTO3) ring and Templating Oligonucleotide) is blocked with a carbon spacer at its 3’-end. SO4) (Signaling Oligonucleotide) has a fluorescent molecule at its 5’-end and a quencher molecule at its 3’-end. Tm5) represents melting temperature of the extended strand-SO hybrid.

DETAILED DESCRIPTION OF THIS ION In one aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay, sing: (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a probing and ing oligonucleotide (PTO); wherein the upstream oligonucleotide comprises a hybridizing nuCIeotide sequence complementary to the target nucleic acid sequence; the PTO comprises (i) a 3'—targeting portion comprising a hybridizing nucleotide sequence mentary 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; the upstream ucleotide 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 the 5’ se activity such that the cleavage releases a fragment comprising the 5’—tagging n or a part of the 5’-tagging n of the PTO; (c) hybridizing the fragment released from the PTO with a capturing and templating oligonucleotide (CT0); wherein the CT0 comprises in a 3’ to 5’ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5’- tagging n or a part of the 5’-tagging portion of the PTO and (ii) a templating n comprising a tide 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 CT0; (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 CT0 is ed to form an ed strand comprising an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex; (e) hybridizing the extended strand with a signaling oligonucleotide (SO); n the SO comprises a complementary sequence to the ed strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

In another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay, comprising: (a) hybridizing the target nucleic acid ce with an upstream primer and a probing and targeting oligonucleotide (PTO); wherein the upstream primer comprises a hybridizing nucleotide sequence complementary to the target c acid sequence; the PTO comprises (i) a 3’-targeting n comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5’- tagging portion sing 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; the upstream primer is d upstream of the PTO; (b) contacting the resultant of the step (a) to an enzyme having a 5’ se activity under conditions for cleavage of the PTO; wherein the ed strand of the upstream primer induces cleavage of the PTO by the enzyme having the ’ nuclease activity such that the ge 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 capturing and templating oligonucleotide (CTO); wherein the CTO comprises in a 3’ to 5’ direction (i) a capturing n 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 ce mplementary to the ging 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 on 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 sing an extended sequence complementary to the templating portion of the CTO, thereby forming an extended duplex; (e) izing the extended strand with a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

The present inventors have made intensive researches to develop novel approaches to detect target sequences with more ed accuracy and convenience, inter alia, in a multiplex . As a result, we have established novel protocols for detection of target sequences, in which target detection is accomplished by enzymatic reactions such as 5’ nucleolytic reaction and extension and extensiondependent hybridization as well as probe hybridization. The present ols are well adopted to liquid phase reactions as well as solid phase reactions, and ensure detection of multiple target sequences with more improved cy and convenience.

The present invention employs successive events followed by probe hybridization; cleavage and extension of PTO (Probing and Tagging Oligonucleotide); and extension-dependent signaling oligonnucleotide hybridization. Therefore, it is named as a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide ization) assay.

The PCE-SH assay will be described in more detail as follows: Step (a): Hybridization of an upstream ucleotide and a PTO with a target nucleic acid sequence According to the t invention, a target nucleic acid sequence is first hybridized with an am oligonucleotide and a PTO (Probing and Tagging Oligonucleotide).

The term used herein t nucleic acid”, “target c acid sequence” or “target sequence” refers to a nucleic acid sequence of interest for detection, which is annealed to or hybridized with a probe or primer under hybridization, annealing or amplifying conditions.

The term used herein “probe” refers to a single—stranded nucleic acid molecule comprising a portion or portions that are substantially complementary to a target c acid sequence.

The term r” as used herein refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer ion t which is complementary, to a nucleic‘acid strand (template) is induced, Ila, in the presence of tides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH.

Preferably, the probe and primer are single-stranded deoxyribonucleotide molecules. The probes or primers used in this invention may be comprised of naturally occurring dNMP (i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide, or non- natural nucleotide. The probes or primers may also include ribonucleotides.

The primer must be sufficiently long to prime the synthesis of ion products in the presence of the agent for polymerization. The exact length of the primers will depend on many factors, including ature, application, and source of primer. The term “annealing” or “priming" as used herein refers to the tion of an eoxynucleotide or nucleic acid to a template nucleic acid, whereby the apposition enables the polymerase to rize nucleotides into a nucleic acid molecule which is complementary to the template nucleic acid or a portion thereof.

The term used “hybridizing” used herein refers to the formation of a double- stranded nucleic acid from complementary single stranded nucleic acids. The hybridization may occur between two nucleic acid strands perfectly matched or substantially matched with some mismatches. The complementarity for ization may depend on ization conditions, particularly temperature.

The hybridization of a target nucleic acid sequence with the upstream oligonucleotide and the PTO may be carried out under suitable hybridization conditions routinely determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied depending on various WO 15442 factors, including the “length and GC content of ucleotide (upstream oligonucleotide and PTO) and the target nucleotide sequence. For instance, when a relatively short oligonucleotide is used, it is preferable that low stringent conditions are adopted. The detailed conditions for hybridization can be found in Joseph Sambrook, et al., Mo/ecu/ar C/on/ng, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M.L.M. Anderson, Nucleic Acid Hybr/U/zat/‘on, Springer-Verlag New York Inc. N.Y.(1999).

There is no intended distinction between the terms “annealing” and “hybridizing”, and these terms will be used interchangeably.

The am oligonucleotide and PTO have hybridizing nucleotide sequences mentary to the target nucleic acid sequence. The term “complementary” is used herein to mean that primers or probes are sufficiently complementary to hybridize ively to a target c acid ce under the ated annealing conditions or stringent conditions, encompassing the terms “substantially complementary” and “perfectly complementary”, preferably perfectly complementary.

The 5’-tagging portion of the PTO has a nucleotide sequence non- complementary to the target nucleic acid ce. The templating portion of the CTO (Capturing and Templating Oligonucleotide) has a nucleotide sequence non- complementary to the ging portion and the 3’-targeting portion of the PTO. The term “non-complementary” is used herein to mean that primers or probes are sufficiently non-complementary not to hybridize selectively to a target nucleic acid sequence under the designated annealing conditions or stringent ions, encompassing the terms “substantially non-complementary” and “perfectly non— complementary”, preferably perfectly non-complementary.

For example, the term “non-complementary” in conjunction with the 5’—tagging portion of the PTO means that the 5’—tagging portion is sufficiently non- complementary not to hybridize selectively to a target nucleic acid sequence under the designated annealing conditions or stringent conditions, encompassing the terms “substantially non-complementary” and “perfectly mplementary”, preferably perfectly non-complementary.

The term used herein “PTO (Probing and Tagging Oligonucleotide)” means an oligonucleotide comprising (i) a 3’-targeting portion serving as a probe and (ii) a 5’- tagging portion with a tide sequence non-complementary to the target nucleic acid sequence, which is nucleolytically released from the PTO after ization with the target nucleic acid ce. The 5’-tagging portion and the 3’-targeting portion in the PTO have to be positioned in a 5’ to 3’ order. The PTO is schematically rated in Fig. 1.

Preferably, the hybridization in step (a) is preformed under stringent conditions that the geting portion is hybridized with the target nucleic acid sequence and the 5’-tagging portion is not hybridized with the target c acid sequence.

The PTO does not require any specific lengths. For example, the length of the PTO may be 15-150 nucleotides, 15—100 nucleotides, 15-80 nucleotides, 15-60 nucleotides, 15—40 nucleotides, 20—150 nucleotides, '20-100 nucleotides, 20-80 nucleotides, 20-60 nucleotides, 20-50 nucleotides, 30-150 nucleotides, 30-100 nucleotides, 30-80 nucleotides, 30—60 nucleotides, 30-50 nucleotides, 35-100 nucleotides, 35-80 nucleotides, 35-60 nucleotides, or 35-50 nucleotides. The 3’- targeting portion of the PTO may be in any lengths so long as it is ically hybridized with target nucleic acid sequences. For e, the 3’-targeting portion of the PTO may be 10-100 nucleotides, 10-80 nucleotides, 10-50 nucleotides, 10—40 nucleotides, 10—30 nucleotides, 15—100 nucleotides, 15-80 nucleotides, 15-50 nucleotides, 15-40 nucleotides, 15—30 nucleotides, 20-100 nucleotides, 20-80 nucleotides, 20—50 nucleotides, 20—40 nucleotides or 20-30 nucleotides in length. The ’—tagging portion may be in any lengths so long as it is specifically hybridized with the templating portion of the CTO and then extended. For ce, the S’-tagging portion of the PTO may be 5-50 tides, 5-40 nucleotides, 5-30 tides, 5—20 tides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 15—50 nucleotides, 15—40 nucleotides, 15-30 nucleotides or 15-20 nucleotides in length.

WO 15442 The 3’-end of the PTO may have a 3’-OH terminal. ably, the 3'-end of the PTO is "blocked" to prohibit its extension.

The blocking may be achieved in accordance with conventional methods. For instance, the blocking may be med by adding to the 3'4hydroxyl group of the last nucleotide a chemical moiety such as biotin, labels, a phosphate group, alkyl group, non—nucleotide linker, orothioate or alkane-diol. Alternatively, the blocking may be d out by removing the 3’-hydroxyl group of the last nucleotide or using a nucleotide with no 3'-hydroxyl group such as dideoxynucleotide.

Alternatively, the PTO may be designed to have a hairpin structure.

The non-hybridization between the 5’-tagging portion of the PTO and the target nucleic acid sequence refers to non-formation of a stable double-strand between them under n hybridization conditions. According to a preferred embodiment, the ging portion'of the PTO not involved in the hybridization with the target nucleic acid sequence forms a -strand.

The upstream oligonucleotide is located upstream of the PTO.

In addition, the upstream oligonucleotide or its extended strand hybridized with the target'nucleic acid sequence induces cleavage of the PTO by an enzyme having a ’ nuclease activity.

The induction of the PTO cleavage by the upstream ucleotide may be accomplished by two fashions: (i) upstream oligonucleotide extension-independent cleavage induction; and (ii) upstream oligonucleotide ion-dependent cleavage induction.

Where the upstream oligonucleotide is positioned adjacently to the PTO sufficient to induce the PTO cleavage by an enzyme having a 5’ nuclease activity, the enzyme bound to the upstream oligonucleotide digests the PTO with no extension reaction. In st, where the upstream oligonucleotide is positioned distantly to the PTO, an enzyme having a polymerase activity (e.g., template-dependent polymerase) catalyzes extension of the am oligonucleotide (e.g., upstream primer) and an enzyme having a 5’ nuclease activity bound to the extended product digests the PTO.

WO 15442 Therefore, the upstream oligonucleotide may be d relatively to the PTO in two fashions. The upstream oligonucleotide may be located adjacently to the PTO sufficient to induce the PTO cleavage in an extension-independent manner.

Alternatively, the upstream oligonucleotide may be located distantly to the PTO sufficient to induce the PTO cleavage in an extension-dependent manner.

The term used herein “adjacent” with referring to positions or locations means that the am oligonucleotide is located adjacently to the 3’-targeting portion of the PTO to form a nick. Also, the term means that the am oligonucleotide is located 1-30 nucleotides, 1—20 nucleotides or 1-15 nucleotides apart from the 3’— targeting portion of the PTO.

The term used herein “distant” with referring to positions or locations includes any positions or locations sufficient to ensure extension reactions.

According to a preferred embodiment, the am oligonucleotide is d distantly to the PTO sufficient to induce the PTO cleavage in an extension—dependent . ing to a preferred embodiment, the upstream oligonucleotide is an upstream primer or an upstream probe. The upstream primer is suitable in an extension—independent cleavage induction or an extension-dependent cleavage, and the upstream probe is suitable in an extension~independent cleavage induction.

Alternatively, the upstream oligonucleotide may have a l-overlapped sequence with the rt of the 3’-targeting portion of' the PTO. Preferably, the overlapped sequence is 1—10 nucleotides, more preferably 1-5 nucleotides, still more preferably 1-3 nucleotides in length. Where the am oligonucleotide has a partial-overlapped sequence with the 5’-part of the 3’-targeting portion of the PTO, the 3’-targeting portion is partially ed along with the gging portion in the cleavage reaction of the step (b). In addition, the overlapped sequence permits to cleave a desired site of the 3’—targeting portion.

According to a preferred embodiment, the upstream primer induces through its extended strand the cleavage of the PTO by the enzyme having the 5’ nuclease activity.

The conventional technologies for cleavage reactions by upstream oligonucleotides may be applied to the present invention, so long as the upstream oligonucleotide induces cleavage of the PTO hybridized with the target nucleic acid sequence to release a fragment comprising the 5’-tagging portion or a part of the 5’— g portion of the PTO. For example, US. Pat. Nos. 5,210,015, 5,487,972, ,691,142, 5,994,069 and 532 and US. Appln. Pub. No. 2008-0241838 may be d to the present invention.

According to a preferred embodiment, the method is performed in the presence of a downstream primer. The downstream primer generates additionally a target nucleic acid sequence to be hybridized with the PTO, enhancing sensitivity in a target detection.

According to a preferred embodiment, when the upstream primer and the " downstream primer are used, a template-dependent nucleic acid polymerase is additionally employed for extension of the primers.

According to a red embodiment, the upstream oligonucleotide (upstream primer or upstream , the downstream primer and/or 5’-tagging portion of the PTO have a dual priming oligonucleotide (DPO) structure developed by the present inventor. The oligonucleotides having the DPO structure show significantly improved target specificity compared with conventional primers and probes (see WO 2006/095981; Chun et al., Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene, Nucleic Acid Research, 35: 6e40(2007)). ing to a preferred embodiment, the 3’-targeting n of the PTO has a ed dual specificity oligonucleotide (mDSO) structure developed by the present inventor. The modified dual specificity oligonucleotide (mDSO) structure shows icantly improved target specificity compared with conventional probes (see WO 2011/028041) 2012/005281 Step (b): Release of a fragment from the PTO Afterwards, the resultant of the step (a) is contacted to an enzyme having a 5’ nuclease activity under conditions for cleavage of the PTO. The PTO hybridized with the target nucleic acid sequence is digested by the enzyme having the 5’ nuclease activity to e a nt sing the ging portion or a part of the 5’- tagging portion of the PTO.

The term used herein “conditions for cleavage of the PTO” means conditions sufficient to digest the PTO hybridized with the target nucleic acid sequence by the enzyme having the 5’ nuclease activity, such as temperature, pH, ionic strength, buffer, length and sequence of oligonucleotides and enzymes. For e, when 7397 DNA polymerase is used as the enzyme having the 5’ nuclease activity, the conditions for cleavage of the PTO include Tris-HCl buffer, KCl, M90; and temperature.

When the PTO is hybridized with the target nucleic acid sequence, its 3’- targeting portion is involved in the hybridization and the 5’-tagging portion forms a single-strand with no hybridization with the target nucleic acid sequence (see Fig. 2).

As such, an oligonucleotide comprising both single-stranded and double-stranded structures may be digested using an enzyme having a 5’ nuclease activity by a variety of technologies known to one of skill in the art.

The cleavage sites of the PTO are varied ing on the type of upstream oligonucleotides (upstream probe or upstream primer), hybridization sites of upstream oligonucleotides and cleavage conditions (see US. Pat. Nos. 015, 5,487,972, ,691,142, 5,994,069 and 7,381,532 and US. Appln. Pub. No. 2008-0241838).

A multitude of conventional technologies may be employed for the cleavage reaction of the PTO, releasing a ntcomprising the 5’-tagging portion or a part of the 5’-tagging portion.

Brieﬂy, there may be three sites of cleavage in the step (b). Firstly, the cleavage site is a junction site between a hybridization portion of the PTO (3’— targeting portion) and a bridization portion (5’-tagging portion). The second cleavage site is a site located several nucleotides in a 3’-direction apart from the 3’- end of the 5’-tagging portion of the PTO. The second cleavage site is located at the ’-end part of the 3’-targeting portion of the PTO. The third ge site is a site located several nucleotides in a 5’-direction apart from the 3’-end of the 5’-tagging n of the PTO.

According to a preferred embodiment, the l site for the cleavage of the PTO by the te—dependent polymerase having the 5’ nuclease activity upon extension of the upstream primer is a starting point of the double strand n the PTO and the target nucleic acid sequence or a site 1-3 nucleotides apart from the ng point.

In this regard, the term used herein “a fragment comprising the 5’-tagging portion or a part of the 5’-tagging portion of the PTO” in conjunction with cleavage of the PTO by the enzyme having the 5’ nuclease activity is used to encompass (i) the ’—tagging portion, (ii) the 5’-tagging portion and the 5’—end part of the 3’—targeting portion and (iii) a part of the 5’—tagging portion. In this application, the term “a 1‘5 fragment comprising the 5’—tagging portion or a part of the 5’—tagging portion of the PTO" may be also bed as “PTO fragment”.

The term “part” used in conjunction with the PTO or Cl‘O such as the part of the 5’-tagging portion of the PTO, the 5’-end part of the 3’-targeting portion of the PTO and the 5’-end part of the capturing portion of the CTO refers to a nucleotide sequence composed of 1-40, 1~30, 1-20, 1-15, 1-10 or 1-5 nucleotides, preferably 1, 2, 3 or 4 nucleotides.

According to a preferred embodiment, the enzyme having the 5’ nuclease activity is DNA polymerase having a 5’ nuclease activity or FEN nuclease, more preferably a thermostable DNA polymerase having a 5’ nuclease activity or FEN nuclease.

A suitable DNA polymerase having a 5’ nuclease activity in this invention is a thermostable DNA polymerase obtained from a variety of bacterial species, ing Thermus aquaﬁcus (Taq), s thermophi/us (Tth), Thermus rm/Ls, Therm/'5 ﬂavus, Thermococcus //'tera//'s, s antran/k/an/i, Thermus caldop/v/Yus, Thermus ch/Iaroph/Yus Thermus ﬂavus, Thermus lyn/Terrae, Thermus lacteus, Thermus ash/mad Thermos rubel; Thermus rubens, Thermus scotoductus, s $/'/I/anus, s speC/ES 205, Thermus spec/es 5,05 17, Thermus thermophi/us, Thermoz‘oga mark/ma, Thermotoga neapo/I’tana, ThermOS/pho afr/t‘anus, Thermococcus /'5, Thermococcus barossL Thermococcus gorgonar/us, Thermotoga mar/07776, Thermoz‘oga neapo/itana, ThermOS/phoafr/ranus, Pyrococcus woeseI; Pyrococcus hon'koshﬂ} Pyrococcus abyss; Pyrod/ct/‘um occu/tum, x pyroph/lus and Aquifex aeo/ieus. Most preferably, the thermostable DNA rase is 72m polymerase.

I Alternatively, the t invention may employ DNA polymerases having a 5’ nuclease activity modified to have less polymerase activities.

The FEN (flap endonuclease) nuclease used is a 5’ ﬂap-specific nuclease.

The FEN nuclease le in the present invention comprises FEN nucleases obtained from a variety of bacterial species, including SU/fo/obus so/faz‘ar/cus, Pyrobacu/um aeroph/Yum, Thermococcus Ili‘ora/is, Archaeag/obus veneﬂ'cus, ag/obus profundus, Add/anus br/er/yl, us ambivalens, Desu/furococcus amy/o/ytl'cus, Desu/furococcus mob/Wis“, Wrod/ct/Um brock/I; Thermococcus gorgonar/Us, Thermococcus zil/I'g/i, Methanopyrus kand/eri} Methanococcus I'gneus, Pyrococcus shli} Aeropyrum pern/X, and ag/obus veneﬁcus.

Where the upstream primer is used in the step (a), it is preferable that the conditions for cleavage of the PTO comprise extension on of the upstream According to a preferred embodiment, the upstream primer is used in the step (a), a template-dependent polymerase is used for extension of the upstream .

The template-dependent polymerase may be identical to or different from the enzyme having the 5’ nuclease activity.

Optionally, the upstream primer is used in the step (a), a template-dependent polymerase is used for extension of the upstream primer and the template-dependent polymerase is different from the enzyme having the 5’ nuclease activity.

Step (c): Hybridization of the fragment released from the PTO with CT0 The fragment released from the PTO is hybridized with a CT0 (Capturing and Templating Oligonucleotide).

The CT0 comprises in a 3’ to 5’ direction (i) a capturing n 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.

The CTO is acted as a template for ion of the fragment released from the PTO. The fragment serving as a primer is hybridized with the CT0 and extended to form an'extended .

The templating portion may comprise any sequence so long as it is non- complementary to the 5’-tagging portion and the 3’-targeting portion of the PTO.

Furthermore, the templating portion may se any sequence so long as it can be acted as a template for extension of the fragment released from the PTO.

As described above, when the fragment having the 5’—tagging portion of the PTO is released, it is red that the capturing portion of the CT0 is designed to comprise a nucleotide sequence complementary to the 5’-tagging portion. When the fragment having the 5’-tagging portion and a 5’-end part of the 3’-targeting portion is ed, it is preferred that the capturing n of the CT0 is designed to comprise a nucleotide sequence complementary to the 5’-tagging portion and the 5’-end part of the 3’-targeting portion. When the fragment having a part of the 5’—tagging portion of the PTO is ed, it is preferred that the capturing portion of the CTO is designed to comprise a nucleotide sequence complementary to the part of the 5'—tagging portion. er, it is possible to design the capturing portion of the CTO with anticipating cleavage sites of the PTO. For e, where the capturing portion of , the CT0 is designed to comprise a nucleotide sequence complementary to the 5’- tagging portion, either the fragment having a part of the 5’-tagging portion or the fragment having the 5’—tagging portion can be hybridized with the capturing portion and then ed. Where the fragment comprising the Sl-tagging n and a 5’- end part of the 3’-targeting portion is released, it may be hybridized with the capturing n of the CT0 designed to comprise a nucleotide sequence complementary to the S’-tagging portion and then successfully extended although mismatch nucleotides are present at the 3’—end n of the fragment. That is because primers can be extended depending on reaction conditions although its 3’- end contains some mismatch nucleotides (e.g. 1—3 mismatch nucleotides).

When the nt comprising the 5’-tagging portion and-a 5’-end part of the 3’-targeting portion is released, the 5’-end part of the capturing portion of the CT0 may be designed to have a nucleotide sequence complementary to the cleaved 5’-end part of the 3’-targeting portion, overcoming problems associated with mismatch : nucleotides (see Fig. 1).

Preferably, the nucleotide sequence of the 5’-end part of the capturing portion ' of the CT0 mentary to the cleaved 5’—end part of the 3’-targeting portion may be selected depending on anticipated cleavage sites on the 3’-targeting portion of the PTO. It is preferable that the nucleotide sequence of the 5’-end part of the capturing portion of the CT0 complementary to the cleaved 5’—end part of the 3’-targeting portion is 1-10 nucleotides, more preferably 1—5 nucleotides, still more ably 1-3 nucleotides.

The 3’—end of the CT0 may comprise additional nucleotides not involved in hybridization with the fragment. Moreover, the capturing n of the CT0 may comprise a nucleotide sequence mentary only to a part of the nt (8.9., a part of the fragment containing its 3’-end portion) so long as it is stably hybridized with the fragment.

The term used “capturing portion comprising a nucleotide sequence complementary to the 5’—tagging portion or a part of the 5’—tagging n” is described herein to encompass various designs and compositions of the capturing portion of the CT0 as discussed above.

The CT0 may be designed to have a hairpin structure.

The length of the CTO may be widely varied. For example, the CTO is 7-1000 nucleotides, 7-500 tides, 7-300 nucleotides, 7-100 nucleotides, 7-80 nucleotides, 7-60 nucleotides, 7-40 nucleotides, 0 nucleotides, 15-500 nucleotides, 15-300 nucleotides, 15—100 nucleotides, 1580 nucleotides, 15-60 nucleotides, 15-40 nucleotides, 20-1000 tides, 20—500 nucleotides, 20—300 nucleotides, 20-100 nucleotides, 20—80 nucleotides, 20—60 nucleotides, 20-40 nucleotides, 30-1000 nucleotides, 30-500 nucleotides, 30—300 tides, 30—100 nucleotides, 30-80 nucleotides, 30-60 nucleotides or 30-40 nucleotides in length. The ing portion of the CTO may have any length so long as it» is specifically hybridized 'with the fragment released from the PTO. For example, the capturing portion of the CT0 is 5—100 nucleotides, 5-60 nucleotides, 5-40 nucleotides, 5-30 tides, 5-20 nucleotides, 10-100 nucleotides, 10-60 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10—20 nucleotides,' 15-100 nucleotides, 15-60 nucleotides, 15-40 nucleotides, 15—30 nucleotides or 15-20 nucleotides in length. The templating portion of the CTO may have any length so long as it can act as a template in extension of the fragment ed from the PTO. For example, the templating portion of the CTO is 1—900 nucleotides, 1—400 nucleotides, 1-300 nucleotides, 1-100 nucleotides, 1-80 nucleotides, 1-60 tides, 1—40 nucleotides, .. ‘20 1-20 nucleotides, 2-900 nucleotides, 2-400 tides, 2—300 nucleotides, 2-100 nucleotides, 2-80 nucleotides, 2—60 nucleotides, 2—40 nucleotides, 2-20 tides, 5— 900 nucleotides, 5-400 nucleotides, 5-300 nucleotides, 5—100 nucleotides, 5-80 nucleotides, 5-60 nucleotides, 5-40 nucleotides, 5-30 nucleotides, 10-900 nucleotides, -400 nucleotides, 10—300 nucleotides, 15—900 nucleotides, 15-100 nucleotides, 15- 80 nucleotides, 15-60 nucleotides, 15-40 nucleotides or 15-20 nucleotides in length.

The 3’-end of the CT0 may have a 3’~OH terminal. Preferably, the 3'—end of the CT0 is d to prohibit its extension. The non-extendible blocking of the CT0 may be achieved in accordance with conventional s. For instance, the blocking may be performed by adding to the 3'-hydroxyl group of the last nucleotide of the CT0 a al moiety such as biotin, labels, a phosphate group, alkyl group, non-nucleotide linker, phosphorothioate or alkane-diol. Alternatively, the blocking may be carried out by removing the 3'-hydroxyl group of the last nucleotide or using a nucleotide with no 3'-hydr0xyl group such as dideoxynucleotide.

The fragment released from the PTO is ized with the CT0, providing a form suitable in extension of the fragment. Although an undigested PTO is also hybridized with the capturing portion of the CT0 through its 5’-tagging portion, its 3’- targeting portion is not ized to the CT0 which prohibits the formation of an extended duplex.

The hybridization in the step (c) can be described in detail with ing to descriptions in the step (a).

Step (d): Extension of the fragment The extension reaction is carried out using the resultant of the step (c) and a template-dependent nucleic acid polymerase. The nt hybridized with the capturing portion of the CT0 is extended to form an extended strand comprising an ed sequence complementary to the templating portion of the CTO, thereby forming an extended duplex. In contrast, ved PTO hybridized with the capturing portion of the CT0 is not extended such that no extended strand is formed.

The term used herein “extended duplex” means a duplex formed by extension reaction in which the fragment hybridized with the capturing portion of the CT0 is extended using the ting portion of the CT0 as a template and the template- dependent nucleic acid polymerase.

The term 'used herein “extended strand" in conjunction with the fragment means a sequence ed of the fragment and its ed sequence.

The term used hereinl“extended sequence” in conjunction with the fragment means only a newly extended sequence which is a portion of the extended strand except the fragment.

The te-dependent nucleic acid polymerase used in the step (d) may include any nucleic acid polymerases, for example, Klenow fragment of E. C0//' DNA rase I, a thermostable DNA rase and bacteriophage “[7 DNA polymerase.

Preferably, the polymerase is a thermostable DNA polymerase which may be obtained from a y of bacterial species, ing Thermus aquat/CUS (Taq), Thermus thermopM/us (Tth), Thermus ﬁY/‘I‘orm/Ls Therm/'5 ﬂan/us, Thermococcus litera/is, Thermus antran/k/an/I} 777€rmus ca/a’op/v/lug s ch/Iaroph/lus, Thermus ﬂaw/5, Thermus fgnl'terrae, s /acteu5, Thar/nus ash/Ma; Thermus rube; Thermus rubens, Thermus scotoductus, Thermus 5/‘/vanu5, Thermus spec/es 205, Thermus speC/es 5,05 17, s thermophi/us, Thermoz‘oga mar/277773, 7779rm0toga neapo/itana, ThermOS/pho nus, Thermococcus //'t0ra//'s, Thermococcus barossc Thermococcus ar/us, Thermotoga merit/Ma, Thermotoga itana, Therm05/p/70am'canus, Pyrococcus fur/osus(Pfu), Pyrococcus woesel, Pyrococcus hor/kos/wi} Pyrococcus abyss; cﬁum occu/z‘um, Aquifex /lus and Aqw'fex aeol/eus. Most preferably, the template-dependent nucleic acid polymerase is 723:7 polymerase.

According to a preferred embodiment, the template—dependent nucleic acid polymerase includes a reverse transcriptase. ing to a preferred embodiment, the enzyme having the 5’ nuclease activity used in the step (b) is identical to the template-dependent nucleic acid polymerase used in the step (d). More preferably, the enzyme having the 5’ nuclease activity used in the step (b), the template-dependent nucleic acid polymerase used for extension of the upstream primer and the template-dependent nucleic acid - polymerase used in the step (d) are identical to one another.

Step (e): Signal Generation by hybridization between the extended strand and SO Following the extension reaction, the extended strand is hybridized with a signaling oligonucleotide ($0). The signal indicative of the presence of the target nucleic acid ce is provided. The signal includes a signal generation or extinguishment, or signal change l increase or decrease).

The SO to be hybridized with the extended strand comprises a mentary sequence to the ed strand.

Where the 50 comprises a complementary sequence only to the PTO fragment, a non—target signal may not be generated due to hybridization of undigested PTO and the SO in some of signaling systems described hereinbelow.

Where the-position of incorporated labels in the extended strand as illustrated in Pig. 6 is suitably adjusted, a non-target signal may not be generated even using the SO comprising a mentary sequence only to the PTO nt.

In the meantime, where the SO comprises a mentary ce only to the PTO fragment, a non-target signal may be generated due to hybridization of undigested PTO and the SO in some of signaling systems described hereinbelow (6.9., the signaling system of Fig. 2).

Where the non-target signal becomes problematic, a portion of the SO should be designed to se a complementary sequence to a portion of the extended sequence newly synthesized.

According to a preferred embodiment, the SO comprises a complementary sequence to the extended sequence. ing to a preferred embodiment, at least a portion of the 50 comprises a complementary sequence to the extended sequence. The portion of the SO comprising a complementary sequence to the extended sequence is at least one, two, three, four, five or ten nucleotides in length.

When a portion of the SO is designed to comprise a complementary sequence to a portion of the extended sequence newly synthesized, the Tm value of the 725 hybridization resultant of the SO and the extended strand s different from that of the hybridization resultant of the SO and the undigested PTO. The ence in the Tm values ensures to differentiate signals from the two hybridization resultants. For example, non-target signals may be excluded in a real-time ion by adjusting temperature for detection in considering Tm values, or in a melting curve analysis by melting peaks.

Preferably, the SO may se hout its whole sequence a complementary sequence to the extended sequence. Alternatively, the SO may comprise a portion having a complementary sequence to the extended sequence. For instance, one n of the SO may comprise a complementary sequence to the extended sequence and the other portion may comprise a complementary ce to the fragment.

Preferably, the SO comprises throughout its whole sequence a complementary sequence to the extended sequence.

The SO may have any length, for e, 5-100 nucleotides, 5-80 nucleotides, 5-60 nucleotides, 5—40 nucleotides, 5-20 tides, 5-10 nucleotides, —100 nucleotides, 10-80 nucleotides, 10—60 nucleotides, 10-40 nucleotides, 10-30» nucleotides, 10-20 tides, 15—100 nucleotides, 15-80 nucleotides, 15—60" nucleotides, 15-40 nucleotides, 15-30 nucleotides, 15-20 nucleotides, 20-100 nucleotides, 20-80 nucleotides, 20-60 nucleotides, 20—40 nucleotides or 20-30 tides.

The SO may have a hairpin structure.

Preferably, the 3’—end of the $0 is blocked to prohibit its ion.

Alternatively, the SO having a non-blocked 3’-OH end may be extended.

The signaling system adopted in the present invention is featured by association of signal generation with hybridization of the SO. In other words, upon hybridization of the SO with the extended strand, a able signal is provided. The hybridization of the 50 with the extended strand occurs only when the target nucleic acid sequence is present and the PTO is cleaved. Therefore, the detectable signal is indicative of the presence of the target nucleic acid sequence. In this regard, if desired, the present invention may be carried out in a real—time manner.

To directly associate the ization of the SO with signals, the present invention uses at least one label linked to the SO.

According to a preferred embodiment, the detectable signal indicative of the presence of the target nucleic acid sequence is provided by (i) the label linked to the 80, (ii) a combination of the label linked to the SO and a label linked to the fragment from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the extended strand during the extension reaction of the step (d), or (iv) a combination of the label linked to the SO and an intercalating dye.

. The labeling systems useful in this invention will be described in detail as (i) Single label linked to the SO The present invention may provide signal for formation of the extended strand indicating the presence of the target nucleic acid sequence using a single label (see Fig. 3).

According to a preferred embodiment, the $0 is labeled with a single label and the hybridization between the SO and the extended strand in the step (e) induces change in signal from the single label to e the able signal.

The single label used herein has to be capable of providing a different signal depending on its ce on a double strand or single strand. The single label includes a fluorescent label, a luminescent label, a chemiluminescent label, an electrochemical label and a metal label.

Preferably, the single label includes a ﬂuorescent label which provides different- intensity signals depending on whether it is linked to a double-strandedvor single- strand nucleic acid.

Fig. 3 illustrates a preferable embodiment of the present invention using a single label. As illustrated in Fig. 3, the single ﬂuorescent label linked to the SO hybridized with the extended strand exhibits more intense ﬂuorescence that that linked to the so nOt hybridized.

The s (increase or decrease) in fluorescent intensity of single ﬂuorescent labels are measured to detect the ce of "the target nucleic acid sequence.

The types and preferable g sites of single ﬂuorescent labels used in this invention are disclosed U.S. Pat. Nos. 7,537,886 and 7,348,141, the ngs of which are orated herein by reference in their entity. Preferably, the single ﬂuorescent label includes JOE, FAM, TAMRA, ROX and ﬂuorescein-based label. The d nucleotide residue is ably positioned at internal nucleotide residue within the oligonucleotide rather than at the 5’—end or the 3’—end.

According to a preferred embodiment, the single label on the $0 is located at 1-15 nucleotide, 1-10 nucleotide or 1-5 nucleotide apart from its 5’—end or its 3’-end.

More preferably, the single label is located at the middle portion of $0.

The single ﬂuorescent label useful in the present invention may be described with reference to descriptions for er and quencher molecules as indicated below. (ii) Intrastrand interactive—dual label linked to 50 The interactive label system is a signal generating system in which energy is passed non—radioactively between a donor molecule and an acceptor molecule. As a representative of the interactive label system, the FRET (ﬂuorescence resonance energy transfer) label system includes a ﬂuorescent reporter le (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor is ﬂuorescent, but the energy acceptor may be ﬂuorescent or non-ﬂuorescent. In another form of interactive label systems, the energy donor is non—ﬂuorescent, e.g., a . chromophore, and the energy acceptor is ﬂuorescent. In yet another form of interactive label systems, the energy donor is luminescent, e.g. bioluminescent, chemiluminescent, ochemiluminescent, and the acceptor is ﬂuorescent. The donor molecule and the acceptor molecule may be described as a er lar and a quencher molecule in the present invention, respectively.

Preferably, the signal indicative of the formation of the extended strand (/Ze., the presence of the target c acid sequence) is generated by interactive label systems, more preferably the FRET label system (/13, ctive dual label system).

According to a preferred embodiment, the SO is labeled with an interactive dual label sing a reporter molecule and a quencher molecule and the 2012/005281 hybridization n the SO and the extended strand in the step (e) induces change in signal from the interactive dual label to e the able signal. Prior to hybridization of the 50, the reporter molecule and the quencher molecule on the SO are conformationally adjacent to each other to allow the quencher molecule to quench the signal from the reporter molecule. Upon hybridization, the reporter molecule and the quencher molecule on the SO are conformationally separated to allow the quencher molecule to unquench the signal from the reporter molecule, causing changes in signals from the interactive dual label.

Fig. 2 represents a able embodiment of the present invention using an interactive dual label. The fragment released from the PTO hybridized with the target nucleic acid sequence is hybridized with the capturing portion of the CT0 and ed to form the extended strand. Upon hybridization of the extended strand with the SO, the reporter molecule and the quencher molecule on theSO are conformationally separated to allow the quencher molecule to unquench the signal from the reporter molecule, giving rise to changes in signals from the interactive dual label (6.9., increase in signal from reporter les). In contrast, where the target nucleic acid ce is not present, the cleavage of the PTO does not occur. The undigested PTO is not extended while it is hybridized with the capturing portion of the CT0. The reporter le and the quencher molecule on the SO not involved in the hybridization are conformationally adjacent to each other to allow. the er molecule to quench the signal from the reporter molecule.

The expression used herein “the reporter molecule and the quencher molecule are conformationally adjacent” means that the reporter molecule and the quencher le are three-dimensionally adjacent to each other by a conformational structure of the fragment or SO such as random coil and hairpin structure.

The expression used herein “the reporter molecule and the quencher molecule are conformationally separated” means that the reporter molecule and the quencher molecule are three-dimensionally separated by change of a conformational structure of the SO upon the ion of a double strand by hybridization with the extended strand.

According to a preferred embodiment, the reporter molecule and the quencher molecule are positioned at the 5’-end (or 3’-end) and 3’-end (or 5’-end) of the SO.

According to a preferred embodiment, one of the reporter molecule and the quencher molecule on the S0 is located at its 5’-end or at 1-5 nucleotides apart from its 5’-end and the other is_located to quench and ch the signal from the er molecule ing on conformation of 50 According to the preferred embodiment, one of the er molecule and the er molecule on the SO is located at its 3’-end or at 1-5 nucleotides apart from its, 3’-end and the other is located to quench and unquench the signal from the reporter molecule depending on conformation of the SO.

According to a preferred embodiment, the reporter molecule and the quencher molecule are positioned at no more than 80 tides, more preferably no more than 60 nucleotides, still more preferably no more than 30 tides, still much more preferably no more than 25 nucleotides apart from each other. ing to a preferred embodiment, the reporter molecule and the quencher molecule are separated by at least 4 nucleotides, more preferably at least 6 nucleotides, still more preferably at least 10 nucleotides, still much more preferably at least 15 tides.

The reporter molecule and the quencher molecule useful in the present invention may include any molecules known in the art. Examples of those are: Cy2TM (506), YO~PRO”"-1 (509), YOYOTM-l (509), n (517), FITC (518), Fluor TM (519), M (520), Rhodamine 110 (520), Oregon GreenTM 500 (522), Oregon GreenTM 488 (524), RiboGreenTM (525), Rhodamine GreenTM (527), Rhodamine 123 (529‘), Magnesium GreenT”(531), Calcium GreenTM (533), TO-PRO’M-l (533), TOTOl (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/57O (570), Cy3TM (570), AlexaTM 546 (570), TRITC (572), Magnesium OrangeTM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange‘”(576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine RedTM (590), Cy3.5TM (596), ROX (608), Calcium CrimsonTM (615), AlexaTM 594 (615), Texas Red(615), Nile Red (628), YO-PROTM-3 (631), YOYOTM-3' (631), R- phycocyanin (642), C-Phycocyanin (648), TO-PROTM—3 (660), TOTO3 (660), DiD Di|C(5) (665), Cy5TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), rch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL HuorRed 590(591L CAL HuorRed 610(610) CAL HuorRed 635(637) FAM (520), scein (520), Fluorescein—C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The numeric in hesis is a maximum emission wavelength in nanometer. ably, the reporter molecule and the quencher molecule include JOE, FAM, TAMRA, ROX and ﬂuorescein-based label.

Suitable pairs of reporter-quencher are disclosed in a variety of publications as s: Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel , New .

York, 1970); n, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd .

Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of ' Organic Molecules (Academic Press, New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals’(Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, R. P., Handbook of Fluorescent Probes and Research als, 6th Edition (Molecular Probes, Eugene, Oreg., 1996) US. Pat. Nos. 3,996,345 and 4,351,760.

It is noteworthy that a non-ﬂuorescent black quencher le (or dark quencher molecule) capable of quenching a cence of a wide range of wavelengths or a ic wavelength may be used in the present invention. Examples of those are BHQ and DABCYL.

In the FRET label adopted to the SO, the reporter encompasses a donor of FRET and the quencher encon1passes the other parhwer (acceptor) of FRETZ For exanuﬁe, a ﬂuorescehi dye is used as the reponer and a rhodanﬂne dye as the quenchen (iii) Interstrand interactive-dual label In the ment using the interstrand interactive-dual label, the extended strand has one of an interactive dual label comprising a reporter molecule and a quencher molecule and the SO has the other of the ctive dual label.

The embodiment using the interstrand interactive-dual label may be conducted in accordance with the following three fashions: According to the first n, the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, the fragment from the PTO comprises the other label among the er molecule and the quencher molecule; the extended strand ses the label originated from the fragment from the PTO, and wherein the hybridization between the SO and the extended strand induces change in signal from the interactive dual label to e the detectable signal (see Fig. 5).

A label linked to the SO may be either a reporter molecule or a quencher molecule, and a label to the fragment may be either a quencher molecule or a reporter molecule.

The labeling site on the PTO is determined in considering its cleavage site, so that the PTO fragment may have the label.

The label may be linked to any site (6.9., the tagging portion of the PTO) on 2O the PTO fragment, so long as it interacts with the label to the SO upon hybridization with the $0 to induce change in signals. The label may be linked to any site (e.g., the ’-end of the SO) on the SO, so long as it interacts with the label on the PTO fragment upon hybridization with the PTO fragment to induce change in signals.

According to the second fashion, the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, and the templating n of the CT0 comprises a nucleotide having a first non—natural base; n the ion on in the step (d) is performed in the presence of a nucleotide having both a second non-natural base with a specific binding affinity to the first non- natural base and the other among the reporter molecule and the quencher molecule, y orating the label into the extended strand; 'wherein the hybridization between the SO and the extended strand induces change in signal from the interactive dual label to provide the detectable signal (see Fig. 6).

The term used herein “non-natural base” refers to derivatives of natural bases such as adenine (A), guanine (G), e (T), cytosine (C) and uracil (U), which are capable of forming hydrogen-bonding base pairs. The term used herein “non—natural base” includes bases having different base pairing patterns from natural bases as mother compounds, as described, for example, in U.S. Pat. Nos. 272, ,965,364, 6,001,983, and 6,037,120. The base pairing between non-natural bases involves two or three hydrogen bonds as natural bases. The base pairing n non-natural bases is also formed in a specific manner.

Specific examples of non-natural bases include the following bases in base pair " combinations: iso-C/iso-G, iso-dC/iso-dG, K/X, H/J, and M/N (see U.S. Pat. No. 7,422,850).

The label incorporated during the extension is preferably linked to a nucleotide, more preferably to a nucleoside triphosphate. Preferably, the label is bound to a base of a nucleoside triphosphate.

The exemplified embodiment is described with nce to Fig. 6. The fragment is hybridized with the CT0 with a nucleotide having a non-natural base (e.g., iso-dC) with a specific binding affinity to a non—natural base (8.9., iso-dG). The extension is carried out in the presence of a nucleotide having the iso-dG labeled with a quencher to form the extended strand. In the extension reaction, the tide having iso-dG with a quencher is incorporated at an opposition site to the tide having iso—dC. Following the hybridization of the extended strand containing the quencher-iso-dG with the SO labeled with a reporter, the er on the extended strand quenches signal from the reporter on the SO to induce changes in signal, ing the detectable signal.

One of the interactive dual label is linked to the SO and the other is incorporated into the extended strand from a reaction solution during the extension reaction.

A label linked to the SO may be either a reporter molecule or a quencher molecule, and a label incorporated into the extended strand may be either a quencher molecule or a reporter molecule.

The label incorporated into the extended strand may be linked to any site on the extended strand (e.g., the 3’-end of the extended strand), so long as it interacts with the label to the SO upon hybridization with the SO to induce change in signals.

The label may be linked to any site (e.g., the 5’-end of the $0) on the SO, so long as it interacts with the label incorporated into the extended strand upon hybridization with the extended strand to induce change in s.

According to the third fashion, the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, and the extension: reaction in the step (d) is performed in the ce of a nucleotide having the other‘ among the reporter molecule and the quencher molecule, y incorporating the label into the extended strand; wherein the hybridization between the SO and the , extended strand induces change in signal from the interactive dual label to provide the able signal (see Fig. 7).

A label linked to the SO may be either a reporter molecule or a quencher molecule rably reporter molecule), and a label incorporated into the extended strand may be either a quencher molecule or a reporter molecule (preferably er molecule). (iv) Interactive-dual label using two $05 In the embodiment of the interactive-dual label using two $05, the method of the present invention uses an additional SO comprising a complementary sequence to the extended strand, the two 505 are ized with the extended strand in an adjacent manner, the two $05 each comprises one label among a er le and a quencher molecule of an interactive dual label; and the hybridization between the two $05 and the extended strand induces change in signal from the interactive dual label to provide the detectable signal (see Fig. 4).

Preferably, at least one of the two 505 comprises a portion hybridized to a ~ newly extended sequence in the extension reaction.

The principle underlying the performance of the embodiment of the interactive- dual label using two 505 are as follows: The fragment released from the PTO hybridized with the target nucleic acid sequence is hybridized with the capturing portion of the CT0 and ed to form the extended strand. ards, the two 805 are hybridized with the extended strand. In the ization, since the two 805 are adjacently hybridized with the extended strand, the reporter molecule and the quencher molecule on the two 505 are adjacent to each other to allow the quencher molecule to quench the signal from the reporter molecule, resulting in change in signals from the interactive dual label (9.9., increase in signal from er molecules). In contrast, where the target nucleic acid sequence is not present, the cleavage of the PTO does not occur. The undigested-PTO is not extended while it is hybridized with the capturing portion of the CT0. The reporter molecule and the quencher molecule on the two 505 not involved in the hybridization are separated to each other to generate signal from the er molecule.

According to a preferred embodiment, the two 505 may be hybridized with any sites of the extended strand so long as their hybridization with the extended strand permits the quencher molecule to quench the signal from‘the reporter molecule.

Preferably, the two 505 are positioned in an immediately adjacent manner or 1-5 nucleotides apart from each other.

According to a red embodiment, where the two 505 may be adjacently hybridized with the extended strand, the er molecule and the quencher molecule may be linked to any sites of the two $05 so long as the quencher molecule quenches the signal from the er molecule. For example, the reporter molecule or the quencher le is linked to the 5’—end of one $0 or 1—5 tides apart from its 5’—end, and the quencher le or the reporter molecule to the 3’-end of the other SO or 1-5 nucleotides apart from its 3’-end. (v) FRET label using intercalating dyes According to the present invention, a FRET (ﬂuorescence resonance energy transfer) signaling becomes practical using alating dyes. ing to a preferred embodiment, the 50 comprises an acceptor of a FRET and the hybridization in the step (e) is preformed in the presence of an intercalating dye; wherein the hybridization between the SO and the ed strand induces change in signal from the acceptor of the $0 to provide the detectable signal (see Fig.

Exemplified intercalating dyes useful in this invention include SYBRTM Green I, PO-PROTM-l, BO-PROTM—l, SYTOTM43, SYTOTM44, 45, SYTOXTMBlue, POPOTM-l, POPOTM-3, BOBOTM—l, BOBOTM-3, LO~PROT”—1, JO-PROTM-l, YO-PROTMl, TO-PROTMl, SYTOTMll, SYTOTM13, SYTOTM15, SYToTMls, SYTOTMZO, SYToTM23, TOTOTM-3, YOYOTM3, GelStarTM and thiazole . The alating dyes intercalate specifically into double-stranded nucleic acid molecules to generate signals.

The principle underlying the performance of the embodiment of the FRET label using intercalating dyes are as s: The fragment released from the PTO hybridized with the target nucleic acid sequence is hybridized with the ing portion of the CT0 and extended to form the extended strand. Afterwards, the SO labeled with the acceptor is hybridized with the extended strand to form a double- stranded nucleic acid molecule and then the intercalating dyes are bound to the double-stranded nucleic acid molecule. The energy transfer occurs from the intercalating dyes serving as a donor molecule to the acceptor by illumination for donor excitation and induces change in signal from the acceptor to provide the ‘25 detectable signal. In contrast, the FRET phenomenon does not occur in the absence of the target nucleic acid ce, resulting in no signal change.

According to a red embodiment, the acceptor linked to the SO includes various single cent labels described above, but not limited to.

A label may be linked to the SO or the PTO by conventional methods.

Preferably, it is linked to the S0 or PTO through a spacer containing at least three carbon atoms (e.g., 3-carbon , 6-carbon spacer or 12—carbon spacer).

The so useful in the present invention includes any probes capable of providing signals dependent on ization, for example, Molecular beaconTM (US Pat. No. 5,925,517), HybeaconsTM (D. J. French, et al., Molecular and Cellular Probes (2001) 13, 363—374 and US Pat. No. 7,348,141), Dual—labeled, self-quenched probe (us Pat. No. 5,876,930), LuxTM (I. A. Nazarenko, et al. Nucleic Acids Res 2002, 9-2095. and US Pat. No. 7,537,886) and Hybridization probe (Bernard PS, et al., Clin Chem 2000, 46, 147-148 and Deepti Parashar et al., Indian J Med Res 124, review article r 2006 385-398).

Step (f): Detection of target signal Finally, the detectable signal provided in the step (e) is detected, whereby the detection of the signal tes the presence of the extended strand and the presence of the target nucleic acid sequence.

As discussed above, the hybridization event of the SO is synchronized. with the signaling event from labels of the hybridization resultant to e s indicative of the target nucleic acid sequence. In this , the present invention may be carried out in a real—time manner using labels proving signals detectable in a real-time fashion.

Alternatively, the detection of the target signal may be carried out by a melting analysis e the labels used in the present invention are capable of providing detectable signals during melting of the hybridization resultant or melting and hybridization of the hybridization resultant.

The term used herein “melting analysis” means a method in which a target signal indicative of the presence of the extended duplex is obtained by melting of the extended duplex, including a method to measure signals at two ent temperatures, melting curve analysis, melting pattern analysis and melting peak analysis. Preferably, the g analysis is a melting curve analysis.

For instance, when the duplex between the SO and the ed strand is , the reporter molecule and the quenchermolecule on the single-stranded 50 are conformationally nt to each other to allow the quencher molecule to quench- the signal from the reporter molecule, such that change in s is induced to give the detectable signal. Furthermore, where the SO and the extended strand is re- hybridized to form a duplex, the reporter molecule and the quencher molecule on the SO are conformationally separated to allow the quencher molecule to unquench the signal from the reporter molecule, such that change in signals is induced to give the detectable signal (see Fig. 2).

According to a preferred embodiment, the presence of the ed strand of the PTO fragment is detected by a melting curve analysis using Tm values of the duplex between the SO and the extended strand.

Where Tm values of the duplex between the SO and the extended strand are used for analysis, it is preferable to use labels (e.g., ﬂuorescent labels) allowing for neous assay with no tion of the hybridization resultant between the SO and the extended .

According to a preferred embodiment, the hybridization resultant between the SO and the extended strand has Tm values that are adjustable by sequence and/or length of the PTO fragment, sequence and/or length of the CT0, sequence and/or length of the SO and their combination.

For instance, Tm, values of the hybridization resultant may be ed by adjusting mismatch extent of the sequence of the SO. Furthermore, by adjusting lengths of the SO, Tm values of the hybridization resultant may be also adjusted.

Preferably, the t method further comprises the step of providing a detectable signal between the steps (e) and (f) by melting the hybridization resultant of the step (e) or by melting and hybridizing the hybridization resultant of the step (e); wherein the step (f) is performed by detecting the signal to determine the presence of the extended strand.

Alternatively, the present method further comprises the step of providing and ing a detectable signal after the step (f) by melting the hybridization resultant of the step (e) or by melting and hybridizing the hybridization resultant of the step (e), whereby the presence of the ed strand is determined one more time. ing to a preferred embodiment, the presence of the extended strand of the PTO fragment is detected by a hybridization curve analysis.

The term used herein “Tm” refers to a melting temperature at which half a population of double stranded nucleic acid molecules are dissociated to single- ed molecules. The Tm value is determined by length and G/C content of nucleotides hybridized. The Tm value may be calculated by conventional methods such as Wallace rule (R.B. e, et al., Nucleic Acids Research, 6:3543—3547(1979)) and nearest-neighbor method (SantaLucia J. Jr., et al., Biochemistry, 3523555— 3562(1996)); Sugimoto N., et‘al., Nuc/eicAc/ds Res, 24:4501—4505(1996)). ing to a preferred embodiment, the Tm value refers to actual TVrn values under reaction conditions actually practiced.

The g curve or hybridization curve may be obtained by conventional technologies, for e, as described in U.S. Pat Nos. 6,174,670 and 5,789,167, Drobyshev et al, Gene 188: 45(1997); Kochinsky and Mirzabekov Human Mutation 19:343(2002); Livehits et al J, Biomoi. Structure Dynam. 11:783(1994); and Howell et al Nature Biotechnology 17:87(1999). For example, a melting curve or hybridization curve may consist of a c plot or display of the variation of the output signal with the parameter of hybridization stringency. Output signal maybe plotted directly against the hybridization parameter. Typically, a melting. curve or hybridization curve will have the output signal, for example ﬂuorescence, which indicates the degree of duplex structure (i.e. the extent of hybridization), plotted on the Y-axis and the hybridization parameter on the X axis.

A plot of the first derivative of the ﬂuorescence vs. temperature, Le, a plot of the rate of change in ﬂuorescence vs. temperature (dF/dT vs. T) or T vs. T) provides melting peak.

The formation of the ed strand may be detected by the size of the extended strand. The 50 hybridized with the extended strand provides a detectable signal for the ion of the extended strand by the size of the extended strand. For example, where the formation of the extended strand is ed by various electrophoresis s such as gel electrophoresis and polyacrylamide gel ophoresis, the SO hybridized with the extended strand provides a signal on a gel matrix indicating the presence of the extended strand. Preferably, the SO with a single ﬂuorescent label is used.

The PTO, CT0 and SO may be sed of naturally occurring dNMPs.

Alternatively, the PTO, CT0 and SO may be comprised of modified nucleotide or non- natural‘ nucleotide such as PNA (peptide c acid, see PCT Publication No. WO 92/20702) and LNA (locked nucleic acid, see PCT Publication Nos. WO 98/22489, WO 98/39352 and WO 99/14226). The PTO, CFO and SO may se sal bases such as deoxyinosine, inosine, 1-(2’-deoxy-beta-_D-ribofuranosyl)nitropyrrole and 5— nitroindole. The term “universal base” refers to one capable of forming base pairs with each of the natural DNA/RNA bases with little discrimination between them.

As described above, the PTO may be cleaved at a site located in a 3’-direction apart from the 3’-end of the 5’—tagging portion of the PTO. The cleavage site may be located at the 5’-end part of the geting portion of the PTO. Where the PTO fragment comprises the 5’-end part of the 3’-targeting portion of the PTO, a site of the CT0 hybridized with the 5’-end part of the 3’-targeting portion may se a universal base, degenerate sequence or their combination. For instance, if the PTO is cleaved at a site located one tide in a 3’-direction apart from the 3’-end of the ’-tagging portion of the PTO, it is advantageous that the 5’-end part of the capturing portion of the CT0 comprises a universal base for hybridization with the nucleotide. If the PTO is cleaved at a site located two nucleotides .in a 3’-direction apart from the 3’- end of the 5’»tagging portion of the PTO, it is advantageous that the 5’-end of the ing portion of the CT0 comprises a degenerate sequence and its 3’-direction- adjacent nucleotide comprises a universal base. As such, where the cleavage of the PTO occurs at various sites of the» 5’-end part of the 3’-targeting portion, the utilization of universal bases and degenerate sequences in the CTO is useful. In addition, where the PTOs having the same 5’-tagging portion are used for screening multiple target nucleic acid ces under upstream primer extension-dependent cleavage induction, the PTO fragments having different 5’—end parts of the 3’- targeting portion may be ted. In such cases, universal bases and degenerate ces are usefully employed in the CT0. The strategies using universal bases and degenerate sequences in the CT0 ensure to use one type or minimal types of the CT0 for screening multiple target nucleic acid sequences.

According to a preferred ment, the present method further comprises the step of denaturation between the steps (d) and (e). The ed duplex formed in the step (d) is denatured to a single strand form and then hybridized with the SO. .

According to a preferred embodiment, the method further comprises repeating all or some of the steps (a)—(f) with denaturation between repeating cycles. For instance, the method further comprises repeating the steps (a)-(b), ) or (a)—(f) with denaturation between repeating cycles. This repetition permits to amplify the- target nucleic acid sequence and/or the target signal.

According to a preferred embodiment, the present invention further comprises repeating the steps (a)—(e) with denaturation between repeating cycles, and melting the hybridization resultant of the step (e) or melting and hybridizing the hybridization resultant of the step (e) to provide a able signal; wherein the step (f) is performed by detecting the signal to determine the presence of the extended strand.

The denaturation may be d out by conventional technologies, including, but not d to, g, alkali, formamide, urea and glycoxal treatment, enzymatic s (e.g., helicase action), and binding proteins. For instance, the melting can be achieved by heating at temperature ranging from 80°C to 105°C. General methods for accomplishing this treatment are provided by Joseph Sambrook, et al., /ar C/on/ng, A Laboratoxy Manua/, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

According to a preferred embodiment, the present invention may be carried out by a series of melting analyses to qualitatively or quantitatively detect the target nucleic acidsequence.

More preferably, the present invention comprises (i) repeating the steps (a)-(d) with denaturation between repeating cycles to form the extended strand, (ii) performing a melting analysis of hybridization resultant of the SO and the extended strand and (iii) repeating the steps (i) and (ii) at least twice. In such approach, the g analysis is repeatedly carried out at least twice in a certain interval.

According to a preferred ment, the number of repetition of the steps . (a)—(d) may be optionally controlled. In ming a series of g analyses, the number of repetition of the steps (a)-(d) for a run of a melting is may be the same as'or ent from that of tion of the steps (a)-(d) for another run of a melting analysis.

It would be understood by one of skill in the art that the repetition of the steps (a)~(d) is an illustrative example for the formation of the extended strand. For instance, the present invention may be carried out by repeating the steps (a)-(b) and performing the steps (c) and (d) to form the extended strand followed by performing a melting analysis.

According to a preferred ment, the steps (a)—(f) are performed in a reaction vessel or in separate reaction vessels. For example, the steps (a)-(b), (c)-(d) or (e)-(f) may be med in separate reaction vessels.

According to a preferred embodiment, the steps (a)-(b) and (c)—(f) may be simultaneously or separately even in a reaction vessel depending on reaction conditions (particularly, temperature). ing to a preferred ment, the steps (a)-(b) are repeated with ration.

Where the upstream primer is used as the upstream oligonucleotide in the repetition process, the present method is preferably performed in the presence of a downstream primer, preferably, by PCR. ing to a preferred ment, at least two melting analyses in the present invention permit to tatively detect the target nucleic acid sequence.

The area and height of a melting peak ed by a melting analysis are dependent on the amount of the extended duplex, providing information on the initial amount of the target nucleic acid sequence.

According to a preferred embodiment, the present invention comprises (i) increasing the number of the extended strand by repetition of the steps (a)-(d) with denaturation between repeating cycles, (ii) performing a melting analysis for the hybridization resultant between the SO and the extended strand and (iii) repeating the steps (i) and (ii) at least twice. The amount of the target nucleic acid sequence may be measured by determining a cycle number of the melting analyses at which a predetermined threshold value over the areas and/or the heights of g peaks obtained is reached.

Alternatively, the fication of the target nucleic acid sequence may be accomplished by ng melting‘analysis information (6.9. area or height of peaks) against the cycle number of the repetition for increase in the amount of the extended ‘ strand.

The present invention does not require that target nucleic acid sequences to be detected and/or amplified have any particular sequenceor , including any DNA (gDNA and cDNA) and RNA molecules.

Where a mRNA is employed as starting material, a reverse ription step is necessary prior to performing annealing step, details of which are found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N..Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 66 (1988). For reverse transcription, a random hexamer or an oligonucleotide dT primer hybridizable to mRNA can be used.

The target nucleic acid sequences which may ected and/or ied include any naturally occurring prokaryotic, eukaryotic (for example, protozoans and parasites, fungi, yeast, higher plants, lower and higher animals, including mammals and humans) or viral (for example, Herpes viruses, HIV, inﬂuenza virus, Epstein—Barr virus, hepatitis virus, polio virus, etc.) or viroid nucleic acid.

The target nucleic acid sequence to be detected by the present invention includes a wide variety of nucleic acid sequences, e.g., sequences in a , artificially isolated or fragmented sequences and synthesized sequences (6.9., cDNA sequences and barcode ces). For instance, the target nucleic acid sequence includes nucleic acid marker sequences for Immuno-PCR (IPCR). IPCR employs conjugates between nucleic acid marker sequences and antibodies together with PCR, which is widely applied for ing various types of targets including proteins (see Sano et al., Science 258 pp:120-122(1992), US. Pat. No. 5,665,539, er et al., Trends in hnology 23 pp:208-216(2005), US. Pat. Pub. No. 2005/0239108 and Ye et al., Journal of Environmental Science 22 pp:796~800(2010)).

The present invention is also useful in detection of a nucleotide ion.

Preferably, the target nucleic acid sequence comprises a nucleotide variation. The term “nucleotide variation” used herein refers to any single or multiple nucleotide substitutions, deletions or insertions in a DNA sequence at a particular on among. contiguous DNA segments that are othenNise similar in sequence. Such contiguous DNA segments include a gene or any other n of a chromosome. These nucleotide variations may be mutant or polymorphic allele variations. For example, the tide variation detected in the present invention includes SNP e nucleotide polymorphism), mutation, deletion, insertion, substitution and ocation. iﬁed nucleotide variation includes numerous variations in a human genome (6.9., variations in the MTHFR (methylenetetrahydrofolate reductase) gene), variations involved in drug resistance of pathogens and tumorigenesis-causing variations. The term nucleotide variation used herein includes any variation at a particular location in a DNA molecule. In other words, the term nucleotide variation includes a wild type and its any mutant type at a particular location in a DNA le.

In the present invention for detection of a nucleotide variation in a target nucleic acid sequence, where primers or probes used have a complementary sequence to the nucleotide variation in the target nucleic acid sequence, the target nucleic acid sequence containing the nucleotide variation is described herein as a matching template. Where primers or probes used have a non-complementary sequence to the nucleotide variation in the target nucleic acid sequence, the target nucleic acid sequence containing the nucleotide ion is described herein as a mismatching template.

For ion of nucleotide variations, the 3’-end of the upstream primer may be designed to be te to a site of a nucleotide variation in a target c acid sequence. According to a preferred ment, the 3’-end of the upstream primer has a complementary sequence to the nucleotide variation in a target nucleic acid sequence. The 3’-end of the upstream primer having a complementary ce to the nucleotide ion in the target nucleic acid sequence is annealed to the matching template and ed to induce cleavage of the PTO. The resultant PTO fragment is hybridized with the CT0, extended and hybridized with the $0 to provide the target signal. In st, where the 3’-end of the upstream primer is mismatched to a nucleotide variation in a mismatching template, it is not extended under conditions that annealing of the 3’-end of primers is essential for extension even when the upstream primer is hybridized with the mismatching template, y ing in no generation of the target signal.

Alternatively, it is possible to use PTO cleavage depending on the hybridization of PTO having a complementary sequence to a nucleotide variation in a target c acid sequence. For example, under controlled conditions, a PTO having a complementary sequence to the nucleotide variation in the target nucleic acid sequence is ,hybridized with the matching te and then cleaved. The resultant PTO fragment is hybridized with the CT0, extended and hybridized with the SO to provide the target signal. While, under the controlled conditions, the PTO is not hybridized with a mismatching template having non-complementary sequence in the nucleotide variation position and not cleaved. Preferably, in this case, the complementary sequence to the nucleotide variation in the PTO is positioned at its middle of the 3’-targeting portion of the PTO.

Alternatively, it is preferable that the 5’-end part of the geting portion of the PTO is positioned to a nucleotide variation in a target nucleic acid sequence for the ion of the nucleotide variation and the 5’-end part of the 3’-targeting portion of the PTO has a mentary sequence to the nucleotide variation in a target nucleic acid sequence (see Fig. 9).

Where a probe having at its 5'-end portion a nucleotide ion discrimination portion is hybridized with a mismatch temple, its 5’—end portion may form a single strand under a certain condition. The probe may correspond to a PTO. The signal may be generated by the t method. This approach may be useful in ion of a target nucleic acid sequence having a nucleotide variation non-complementary to the nucleotide variation discrimination site of probes.

According to a preferred embodiment, the target nucleic acid sequence used in the present invention is a pre-amplified nucleic acid sequence. The utilization of the pre—amplified nucleic acid sequence permits to significantly increase the sensitivity ~ and specificity of target detection of the present invention.

According to a preferred ment, the method is performed in the ce of a downstream primer.

The advantages of the present invention may be highlighted in the simultaneous (multiplex) detection of at least two target nucleic acid sequences.

According to a preferred embodiment, the method is performed to detect at least two types (more preferably, at least three types, still more preferably at least five types) of target nucleic acid sequences.

AcCording to a preferred embodiment, the method is performed to detect at least two types (more preferably, at least three types, still more preferably at least five types) of target nucleic acid sequences; n the upstream oligonucleotide comprises at least two types (more ably at least three \types, still more preferably at least five types) of oligonucleotides, the PTO comprises at least two types (more preferably at least three types, still more preferably at least five types) of the PTOs, the CT0 comprises at least two types (preferably at least three types, more preferably at least five types) of the CT0, and the SO comprises at least two types (preferably at least three types, more preferably at least five types) of the. 50; wherein when at least two types of the target nucleic acid sequences are present, the method provides at least two types of the target signals (the detectable signals) corresponding to the at least two types of the target c acid sequences.

The 5’-tagging portions of the at least two PTOs may have an identical sequence to each other. For ce, where the present invention is carried out for screening target nucleic acid sequences, the 5’-tagging portions of PTOs may have the identical sequence.

Furthermore, a single type of the CT0 may used for detection of a plurality of target nucleic acid sequences. For e, where the PTOs having an identical sequence in their 5’-tagging portions are employed for screening target nucleic acid sequences, a single type of the CTO may used.

Where the present invention is performed to aneously detect at least two types of the target nucleic acid sequences by g curve analysis and the hybridization resultant in the,step (e) corresponding to the at least two types of the target nucleic acid sequences have different Tm values from each other, it is possible to detect at least two types of the target nucleic acid sequences even using a single type of a label (e.g. FAM).

According to a preferred ment, Tm value of the hybrid of SO/extended strand may be adjusted by the sequence and/or length of the 50, the sequence and/or length of a portion of the extended strand to be ized with the $0, or combination thereof. Particularly, where the extended strands formed in the present multiplex detection are hybridized with a .single type of the SO, Tm values of the hybrids between the extended strands and the 505 are different from each other if 2012/005281 the portions of the extended strands to be hybridized with the 505 are designed to have different sequences from each other. Therefore, the multiplex detection may become practical even using a single-typed SO.

The present invention may be performed on a solid phase such as microarray.

According to a preferred embodiment, the present invention is performed on the solid phase and the CT0 or the 50 is immobilized through its 5’—end or 3’—end ' onto a solid substrate.

For the solid phase reaction, the CT0 or the SO is immobilized directly or indirectly (preferably indirectly) through its 5’-end or 3’-end (preferably the ) onto the surface of the solid substrate. Furthermore, the CT0 or the '50 may be immobilized on the surface of the solid substrate in a covalent or non-covalent manner.

Where the immobilized CTOs or 505 are lized indirectly onto the surface of the solid substrate, suitable linkers are used. The linkers useful in this invention may include any linkers utilized for probe immobilization on the surface of the solid substrate. For example, alkyl or aryl compounds with amine onality, or alkyl or aryl nds with thiol onality serve as linkers for CFO or SO immobilization.

In addition, poly (T) tail orpoly (A) tail may serve as linkers.

According to a preferred embodiment, the solid substrate used in the present invention is a microarray. The rray to provide a reaction nment in this invention may include any those known to one of skill in the art. All processes of the present invention, i.e., hybridization to target c acid sequences, ge, extension, melting and ﬂuorescence detection, are carried out on the rray. The immobilized CT05 or $05 on the microarray serve as hybridizable array elements. The solid substrate to fabricate microarray includes, but not limited to, metals (9.57., gold, alloy of gold and copper, aluminum), metal oxide, glass, ceramic, quartz, silicon, semiconductor, Si/SiOz wafer, germanium, gallium arsenide, carbon, carbon nanotube, polymers (e.g., polystyrene, polyethylene, polypropylene and polyacrylamide), sepharose, agarose and ds. A plurality of immobilized CTOs or $05 in this invention may be immobilized on an addressable region or two or more addressable regions on a solid substrate that may comprise 2—1,000,000 addressable regions.

Immobilized CTOs or 505 may be fabricated to produce array or arrays for a given application by conventional fabrication technologies such as photolithography, ink— jetting, mechanical microspotting, and derivatives thereof.

According to a preferred embodiment, a $0 immobilized onto the surface of the solid substrate has an ctive dual label.

In the present invention, a PTO fragment is produced by cleavage of the PTO hybridized with the target nucleic acid and it is annealed to and extended on the CT0, resulting in the formation of an extended strand.

It is also possible to provide additional fragments extendible on the CT0 for enhancing the number of the extended strands by an additional 5’ nuclease ge reaction using an additional PTO which comprises (i) a 3’~targeting portion comprising a hybridizing nucleotide sequence mentary to the extended strand and (ii) a 5’— tagging portion comprising a nucleotide sequence mplementary to the extended strand but complementary to the capturing portion of the CT0.

Preferably, the additional PTO is d downstream of the SO hybridizing to the extended strand. The SO induces cleavage of the onal PTO by an enzyme having a 5’ nuclease activity. When 3'-end of $0 is extensible, SO's extended strand induces cleavage of the additional PTO.

The above preferable ment has the feature that the formation of the additional fragments is ent on the ion of an extended .

Alternatively, the additional fragments may be provided by using an additional PTO which comprises (i) a 3’-targeting portion comprising a hybridizing nucleotide sequence complementary to the templating portion of CTO and (ii) a 5’-tagging portion comprising a nucleotide sequence non—complementary to the ting portion of CT0 but complementary to the capturing portion of the CT0.

Preferab/e Embodiment with AmQ/I'ﬂ'cat/bn ofa Target Nude/c Acid Seguence Preferably, the present invention is d out simultaneously with amplification of a target c acid ce using a primer pair composed of an upstream primer and a downstream primer capable of synthesizing the target nucleic acid ce.

In another aspect of this invention, there is provided a method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE—SH (PTO Cleavage and Extension—Dependent Signaling Oligonucleotide Hybridization) assay, comprising: (a) izing the target nucleic acid sequence with a primer pair comprising an upstream primer and a downstream primer and a PTO (Probing and Tagging Oligonucleotide); wherein each of the upstream primer and the ream primer comprise a izing nucleotide sequence complementary to the target nucleic acid sequences; the PTO comprises (i) a 3’—targeting n comprising a hybridizing nucleotide sequence mentary to the target nucleic acid sequence and (ii) a 5’— tagging n 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 it its extension; (b) contacting the resultant of the step (a) to a template-dependent nucleic acid polymerase having a 5’ nuclease activity under conditions for extension of the primers and for ge of the PTO; wherein when the PTO is hybridized with the target nucleic acid sequence, the upstream primer is extended and the extended strand induces cleavage of the PTO by the template-dependent c acid polymerase having the 5’ nuclease activity such that the cleavage releases a fragment comprising the 5’-tagging portion or a part of the 5’—tagging n of the PTO; (c) hybridizing the fragment released from the PTO with a capturing and templating Oligonucleotide (CT0); wherein the CT0 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; wherein the fragment released from the PTO is hybridized with the capturing portion of the CT0; (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 CT0 is extended to form an extended strand sing an extended sequence complementary to the templating n of the CT0, thereby forming an extended ; (e) hybridizing the extended strand with a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

Since the preferable embodiment of the t invention follows the steps of the present method bed above, the common ptions between them are omitted in order to avoid undue ancy leading to the complexity of this specification.

According to a red embodiment, the method further comprises repeating all or some of the steps (a)-(f) with ration between repeating cycles. For instance, the method further comprises repeating the steps (a)—(b), (a)-(d) or (a)—(f) with denaturation between repeating cycles. The reaction repetition is accompanied 'with amplification of the target nucleic acid sequence. Preferably, the amplification is performed in accordance with PCR (polymerase chain reaction) which is disclosed in U..S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.

According to a preferred embodiment, the method is performed to detect at least two types of target c acid sequences.

Target Detect/0n s by PCE-SH Assaz Based on Ugstream Oligonuc/eot/de— indegendent 5’ﬂue/ease act/'V/Zy The present invention may be carried out with no use of upstream oligonucleotides.

In a still another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE—SH (PTO Cleavage and Extension-Dependent ing Oligonucleotide Hybridization) assay, comprising: (a) hybridizing the target nucleic acid sequence with 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-' mentary to the target c acid ce; wherein the 3’-targeting portion of the PTO is ized with the target nucleic acid sequence and the 5’—tagging portion is not hybridized with the target nucleic acid sequence; (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 PTO is cleaved 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 ging portion of the PTO; (c) hybridizing the fragment released from the PTO with a capturing and templating oligonucleotide (CTO); wherein the CT0 comprises in a 3’ to) 5’ direction (i) a ing portion comprising a nucleotide ce 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; wherein the fragment released from the PTO is hybridized with the ing portion of the CT0; (d) performing an extension reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase; wherein the nt ized 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 CT0, thereby forming an extended duplex; (e) hybridizing the extended strand with a signaling oligonucleotide (SO); n the 50 comprises a complementary sequence to the extended strand and at least one label; the SO es a detectable signal by hybridization with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the ce of the extended strand and the presence of the target nucleic acid sequence.

Considering amplification of target nucleic acid sequences and cleavage efficiency of the PTO, the PCE-SH assay of the present invention is preferably med using am oligonucleotides.

Nucleotide Variation Detection Process by a PCE-SH assay In a further aspect of the present invention, there is provided a method for detecting a nucleotide variation on a target c acid sequence by a PCE-SH (PTO Cleavage and Extension—Dependent Signaling ucleotide Hybridization) assay, comprising: (a) hybridizing the target nucleic acid sequence with an upstream oligonucleotide and a probing and targeting oligonucleotide (PTO); wherein the upstream oligonucleotide comprises a hybridizing nucleotide sequence complementary to the target nucleic acid ce; the PTO comprises (i) a 3’—targeting portion comprising a hybridizing nucleotide sequence mentary to the target nucleic acid sequence, (ii) a 5'—tagging portion comprising a nucleotide sequence non— mentary to the target nucleic acid sequence, and (iii) a nucleotide variation discrimination site, comprising a complementary sequence to the nucleotide variation on the target nucleic acid, positioned on a 5’—end part of the 3’-targeting portion; wherein the 3'~targeting portion is hybridized with the target c acid sequence and the 5’—tagging portion is not ized with the target nucleic acid sequence; the upstream oligonucleotide is located upstream of the PTO; the upstream oligonucleotide or its extended strand induces ge of the PTO by an enzyme having a 5’ nuclease activity; (b) contacting the resultant of the step (a) to an enzyme having a 5’ nuclease activity under conditions for cleavage of the PTO; n when the PTO is hybridized with the target nucleic acid sequence having the nucleotide variation complementary to the tide variation discrimination site, and the 5’-end part of the 3’-targeting portion forms a double strand with the target nucleic acid sequence to induce cleavage from a ﬁrst initial cleavage site, a ﬁrst fragment is released; wherein when the PTO is ized with a target nucleic acid sequence having a nucleotide ion non—complementary to the nucleotide variation discrimination site, and the ’-end part of the 3'-targeting portion does not form a double strand with the target nucleic acid sequence to induce cleavage from a second initial cleavage site located downstream of the ﬁrst initial cleavage site, a second nt is released; wherein the second fragment comprises an additional 3’-end portion allowing the second fragment different from the ﬁrst fragment; (c) hybridizing the fragment released from the PTO with a capturing and templating oligonucleotide (CT0); wherein the CT0 comprises in a 3’ to 5’ direction (i) a capturing portion sing a nucleotide sequence complementary to the 5’- g 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; wherein the ﬁrst fragment or the second fragment released from the PTO is hybridized with the capturing portion of the CTO; (d) performing an ion reaction using the resultant of the step (c) and a template-dependent nucleic acid polymerase; n when the ﬁrst fragment is hybridized with the capturing portion of the CT0, it is extended to form an extended strand sing a extended sequence complementary to the templating portion of the CT0; wherein when the second nt is hybridized with the capturing portion of the CFO, it is not extended; (e) hybridizing the extended strand with a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the 50 provides a able signal by hybridization with the ed strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the nucleotide variation complementary to the nucleotide discrimination site of the PTO.

The present inventors have found that the probe ge site is adjustable ing on the presence and absence of tide variations of interest and the fragments released by cleavage in different sites are distinguished by the ability of extension on an artificial template.

The present invention employs sive events followed by probe hybridization; cleavage of the PTO and extension; formation of a nucleotide variation- dependent extended strand; and detection of the extended strand using a signaling oligonucleotide. Therefore, it is named as VD—PCE-SH (Variation Detection by PTO Cleavage and ion-Dependent Signaling Oligonucleotide Hybridization) assay.

According to a preferred embodiment, the nucleotide variation detected by the present invention is a variation by a single nucleotide such as SNP.

In the present application, a target nucleic acid sequence having a nucleotide variation complementary to the nucleotide variation discrimination site of the PTO is also described as “match template”. A target nucleic acid sequence having a nucleotide variation non-complementary to the nucleotide variation discrimination site of the PTO is also described as tch template”.

According to a preferred embodiment, the term “non-complementary” in ction with a nucleotide variation non-complementary to the nucleotide variation discrimination site is used herein to ass mplementarity due to insertion or deletion.

The VD-PCE-SH assay of the present invention uses the PTO having the nucleotide variation discrimination site positioned on the 5’-end part of the 3’- targeting n for selectivity of the PTO to a specific nucleotide variation. Where the PTO is hybridized with the target nucleic acid sequence (/le., match template) having the nucleotide ion complementary to the nucleotide variation discrimination site, the 5’-end part of the 3’-targeting portion forms a double strand with the match template; r, where the PTO is hybridized with a target nucleic acid ce (/le., mismatch template) having a nucleotide variation non- complementary to the nucleotide variation discrimination site, the 5’-end part of the 3’-targeting portion does not form a double strand with the mismatch template.

It is rthy that such distinct hybridization patterns on the nucleotide variation of interest are responsible for differences in initial cleavage sites of the PTO, thereby ing two types of PTO nts to give signal differentiation depending on the presence of the nucleotide variation of st.

A first fragment is generated by cleavage of hybrid between the PTO and matching template and a second fragment is generate by cleavage of hybrid between the PTO and mismatching template, respectively. The second fragment comprises an additional 3’-end portion rendering the second fragment to be different from the first fragment.

The production of either the first fragment or the second fragment may be distinctly detected by an extension reaction on the CT0.

Generally, the hybridization between a 3’-end part of primers and a template is very crucial to extension of primers in a stringent ion. In the present invention, the first fragment and the second fragment each is hybridized with the same site of the CT0. As described above, the second nt comprises the onal 3’-end portion compared with the first fragment. By adjusting ization conditions and a sequence of the CT0 opposed to the additional 3’-end portion of the second fragment, only the first fragment may be permitted to extend.

WO 15442 2012/005281 According to a preferred ment, the CT0 has a sequence selected such that the CT0 is not hybridized with the additional 3’—end portion of the second fragment to prevent the second fragment from extension when the second fragment is hybridized with the capturing n of the CT0.

According to a preferred embodiment, the ce of the CT0 opposed to the additional 3’-end portion of the second fragment is non-complementary to the onal 3’-end portion.

The production of the extended strand by extension of the first fragment may be detected by using SO as the t invention described above.

According to conventional technologies using 5’ nuclease activities for detection of tide variations, hybridization of probes used is determined or affected by a whole sequence of a probe. In such conventional technologies, probe design and construction, and optimization of reaction conditions are very troublesome as hybridization of probes dependent on the presence of tide variations is compelled to be mainly determined by difference by one nucleotide.

According to the VD-PCE-SH assay, a nucleotide variation discrimination site is positioned on a S’-end part of a hybridization-involving portion of probes, enabling optimization of hybridization conditions to be convenient. In on, the VD-PCE-SH assay differentially s a nucleotide variation by a local portion of probes rather than a whole sequence of probes, such that the difference by even one nucleotide such as SNPs may be accurately detected.

It has been known to one of skill in the art that a probe sequence adjacent to a sequence opposed to a SNP extremely affects probe hybridization. The conventional probes have a sequence opposed to a SNP generally in their middle portion. In this regard, the conventional probes may not select a nding sequence around a SNP involved in hybridization. The conventional technologies have serious limitations due to surrounding sequences to SNPs.

The VD-PCE—SH assay of the present invention will be described in more detail as follows: Since the VD-PCE-SH assay of the present invention is one of ations of the PCE-SH assay described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

Step (a): Hybridization of an upstream oligonucleotide and a PTO with a target nucleic acid sequence According to the present invention, a target nucleic acid sequence is first ized with an upstream oligonucleotide and a PTO.

The PTO used in the detection ofnucleotide variations comprises (i) a 3’- targeting portion serving as a probe, (ii) a 5’-tagging portion with a nucleotide sequence non-complementary to the target nucleic acid sequence, and (iii) a nucleotide variation discrimination site, sing a complementary sequence to the nucleotide variation on the target nucleic acid, positioned on a 5’-end part of the 3’— ing portion. The 5’-tagging portion is nucleolytically released from the PTO after hybridization with the target nucleic acid sequence. The ging portion and the 3’- targeting portion in the PTO have to be positioned in a 5’ to 3’ order. The PTO is tically illustrated in Fig. 9.

The PTO comprises the nucleotide variation discrimination site comprising a complementary ce to the nucleotide variation positioned on a 5’-end part of the 3’-targeting portion.

Where the PTO is hybridized with the target nucleic acid. sequence having the nucleotide variation complementary to the variation mination site, the 5’—end part of the 3’-targeting portion forms a double strand with the target nucleic acid sequence. Where the PTO is hybridized with a target nucleic acid sequence having a nucleotide variation non-complementary to the variation mination site, the 5’-end part of the 3’-targeting portion does not form a double strand with the target nucleic acid sequence. Such distinct hybridization ns on the nucleotide variation of interest are responsible for differences in cleavage sites of the PTO, thereby producing 2012/005281 two types of PTO fragments to give signal differentiation ing on the presence of~the nucleotide variation of interest. The 5’-end part of the 3’-targeting portion of the PTO may be also described as a single strand-forming 5’-end portion of the 3’— targeting portion of the PTO when hybridized with a target nucleic acid sequence having a tide variation non-complementary to the variation discrimination site.

The nucleotide variation discrimination site positioned on a 5’-end part of the 3’—targeting portion of the PTO comprises a complementary sequence to the nucleotide variation. For instance, where a nucleotide variation to be detected is a SNP, the nucleotide variation discrimination site comprises a complementary nucleotide to the SNP.

According to a preferred embodiment, the nucleotide variation discrimination site is d within 10 nucleotides, more preferably 8 nucleotides, still more preferably 6 nucleotides, still much more-preferably 4 nucleotides, 3 nucleotides, 2 tides or 1 nucleotide apart from the 5’-end of the 3’-targeting n of the PTO. Preferably, the nucleotide variation discrimination site is located at the 5’-end of the 3’—targeting portion of the PTO.

The term “site” with reference to either nucleotide variation discrimination site of probes or nucleotide ion site on target sequences is used herein to encompass not only a single nucleotide but also a plurality of nucleotides.

Preferably, the hybridization in step (a) is med under stringent conditions that the 3’-targeting portion is ized with the target nucleic acid sequence and the 5’-tagging n is not hybridized with the target nucleic acid sequence.

Step (b): Release of a fragment from the PTO Afterwards, the resultant of the step (a) is ted to an enzyme having a 5’ nuclease activity under conditions for cleavage of the PTO.

Where the PTO is hybridized with the target nucleic acid sequence (/16., match template) having the nucleotide variation complementary to the variation discrimination site, and the 5’—end part of the 3’-targeting portion forms a double strand with the target nucleic acid ce to induce cleavage from a first initial cleavage site, a first fragment is released (see Fig. 9).

Where the PTO is hybridized with a target nucleic acid sequence (Ila, mismatch template) having a nucleotide variation non-complementary to the variation discrimination site, and the 5’—end part of the 3’-targeting portion does not form a double strand with the target nucleic acid ce to induce cleavage from a second initial cleavage site located downstream of the first initial cleavage site, a“ second fragment is released; wherein the second fragment comprises an additional 3’—end portion allowing the second fragment different from the first nt (see Fig. 9).

Where the target, c acid sequence is not present in a sample, the cleavage of the PTO does not occur.

As such, differences in cleavage sites and types of PTO fragments generated result in ent extension patterns dependingion the ce and absence of the tide variation of interest on the target nucleic acid sequence, buting to differential detection of the nucleotide variation on the target c acid sequence.

A cleavage site by extension of upstream primers is generally positioned in a 5’ to 3’ direction at an initial nucleotide of a double strand (i.e., bifurcation site) in structures including a single strand and a double strand or at 1-2 nucleotides apart from the initial nucleotide. By the cleavage reaction, fragments comprising the 5’- 2O tagging portion and a part of the geting portion are produced. Where the present invention is performed by upstream oligonucleotide extension-independent cleavage induction, the cleavage site of the PTO may be adjusted by location of upstream oligonucleotides.

The term used herein “a first initial cleavage site” in conjunction with the PTO means to a cleavage site of the PTO being firstly cleaved when the PTO is hybridized with the target nucleic acid sequence having the tide variation complementary to the variation discrimination site. The term used herein “a second initial cleavage site” in ction with the PTO means to a cleavage site of the PTO being firstly cleaved when the PTO is hybridized with a target nucleic acid sequence having a nucleotide variation non-complementary to the variation discrimination site.

The term used herein “a first fragment” refers to a fragment produced upon cleavage at the first initial cleavage site. The term is used interchangeably with “a first segment” and “a PTO first nt”. The term herein “a second fragment” refers to a fragment produced upon cleavage at the second initial ge site. The term is used interchangeably with “a second segment” and “a PTO second nt”.

Preferably, the first fragment and the second nt each comprises the 5’- tagging n or a part of the 5’-tagging portion.

The cleavage may successively occur after the cleavage of the first initial ge site (or the second initial cleavage site) depending on cleavage methods used. For instance, where a 5’ nuclease cleavage reaction together with extension of upstream primers is used, the initial cleavage site and its successive sequence are d. Where an upstream probe is used and the cleavage reaction occurs at a site- apart from a location site of the probe, the cleavage reaction may occur only at the site and cleavage at successive sites may not occur.

According to a preferred embodiment, an initial cleavage site dependent on extension of upstream primers may be positioned in a 5’ to 3’ direction at an l nucleotide of a double strand (/Ze., bifurcation site).

As shown in Fig. 9 representing an example of the present invention, the nucleotide variation discrimination site is positioned at the 5’-end of the 5’-end part of the 3’-targeting portion. In such case, the first initial cleavage site is positioned immediately adjacent, in a 5’ to 3’ direction, to the 5’—end part of the 3’-targeting portion. In other words, the first initial cleavage site is positioned immediately nt, in a 3’ direction, to the nucleotide variation discrimination site. The second initial cleavage site is generally positioned at 1 nucleotide apart, in a 3’ direction, from the nucleotide variation discrimination site.

Where the nucleotide ion discrimination site is positioned at 1 nucleotide apart from the 5’-end of the 5’-end part of the 3’-targeting portion, the first l cleavage site is oned immediately adjacent, in a 5’ direction, to the nucleotide ion discrimination site. The second l cleavage site is generally oned at . 1 nucleotide apart, in a 3’ direction, from the nucleotide variation discrimination site.

According to a preferred embodiment, the 5’-end part may partially comprise a non—hybridizable sequence (or a non-base pairing sequence). The introduction of a non—hybridizable sequence into the 5’—end part is very advantageous over single strand ion of the 5’-end part when the PTO is hybridized with a target nucleic acid sequence having a nucleotide variation non—complementary to the nucleotide variation discrimination site. In addition, the introduction of a non-hybridizable sequence enables the second initial cleavage site to be adjusted. ing to a preferred embodiment, the 5’-end part of the geting n of the PTO comprises a non-base pairing moiety located within 1—10 nucleotides (more preferably 1-5 nucleotides) apart from the nucleotide variation discrimination site. The non-base g moiety prevents the 5’—end part of the 3’- targeting portion from formation of a double strand with the target tide sequence when the PTO is hybridized with the target nucleic acid sequence having the nucleotide variation non-complementary to the variation discrimination site.

The use of the non-base pairing moiety (e.g., mismatch tide) enhances discrimination potential of the PTO to nucleotide variations.

According to a preferred embodiment, the non—base pairing moiety does not inhibit the formation of a double strand between the 5’—end part and the target nucleic acid sequence when the PTO is hybridized with the target nucleic acid sequence having the nucleotide variation complementary to the nucleotide variation discrimination site. ' According to a preferred embodiment, the se pairing moiety widens the distance between the first initial cleavage site and the second initial cleavage site.

Preferably, the non-base pairing moiety is located downstream of the nucleotide ion discrimination site.

For example, where a mismatch nucleotide as a non-base pairing moiety is introduced into a position 2 nucleotides apart, in a 3’ direction, from the nucleotide variation discrimination site, the second initial cleavage site is adjusted to a position 2 nucleotides apart from the nucleotide variation discrimination site. In case of not using the mismatch nucleotide, the second initial ge site is positioned 1 nucleotide apart from the nucleotide variation discrimination site. That is to say, the non-base pairing moiety may widen the distance between the first initial cleavage site and the second l cleavage site.

The non-base pairing moiety includes any moieties not forming a base pair between target nucleic acid sequences. Preferably, the non-base pairing moiety is (i) a nucleotide comprising an artificial mismatch base, a non-base pairing base modified to be ble of base pairing or a universal base, (ii) a non-base pairing nucleotide modified to be incapable of base g, or (iii) a non-base pairing chemical compound.

For example, the non-base pairing moiety includes alkylene group, ribofuranosyl naphthalene, deoxy ribofuranosyl alene, metaphosphate, phosphorothioate linkage, alkyl phosphotriester linkage, aryl phosphotriester e, alkyl phosphonate linkage, aryl phosphonate linkage, hydrogen phosphonate linkage, alkyl phosphoroamidate linkage and aryl phosphoroamidate linkage. Conventional carbon spacers are also used as non—base pairing moieties. sal bases as non— base pairing moieties are useful in adjusting cleavage sites of the PTO.

As base pairs containing universal bases such as deoxyinosine, deoxy- beta—D-ribofuranosyl)—3-nitropyrrole and 5-nitroindole have a lower binding th than those between natural bases, universal bases may be employed as non-base pairing moieties under n hybridization conditions.

The se pairing moiety introduced into the 5’-end part has preferably 1- 10, more preferably 1-5, still more preferably 1-2 moieties."A plurality of non-base pairing moieties in the 5’—end part may be present in a consecutive or ittent manner. Preferably, the non—base g moiety has 2-5 consecutive moieties.

Preferably, the non—base pairing moiety is a non-base pairing chemical compound.

According to a preferred embodiment, the tide variation discrimination . site and the non—baSe pairing moiety of the PTO are located within 10 nucleotides (more preferably 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides or 1 nucleotide, still more preferably 1 tide) apart from the 5’-end of the 3’—targeting n.

Alternatively, the cleavage reaction may be executed only at the first initial cleavage site not at the second initial cleavage site. For instance, where an am probe is used and the cleavage reaction occurs at a site apart from a location site of the probe, the cleavage reaction may occur only at the first initial cleavage site when the PTO is hybridized with the match template. When the PTO is hybridized with the mismatch template, the bifurcation site (the second initial cleavage site) may not be cleaved because of a long distance from the am probe.

According to a preferred embodiment, where the PTO is ized with the mismatch template, the second initial cleavage site comprises an initial site of a double strand (/Ze., bifurcation site) in ures including a single strand and a double strand.

According to an embodiment, the PTO has a blocker portion'containing as a": . blocker at least one nucleotide resistant to cleavage by the enzyme having 5’ nuclease ty and the blocker portion is positioned at the second initial cleavage site. The ‘ blocker portion prevents cleavage at the second initial cleavage site and successive cleavages.

The number of rs contained in the blocker n may be not limited, preferably, 1-10, more preferably 2—10, still more preferably 3-8, most preferably 3-6 blockers. The blockers present in the probes may be in a uous or intermittent manner, preferably a continuous manner. The nucleotides as rs with a backbone resistant to the 5’ to 3’ exonuclease activity include any one known to one of skill in the art. For example, it includes various phosphorothioate linkages, phosphonate linkages, phosphoroamidate linkages and 2'-carbohydrates ations.

According to a more preferred embodiment, nucleotides having a backbone resistant WO 15442 to the 5’ to 3’ lease include phosphorothioate linkage, alkyl phosphotriester linkage, aryl phosphotriester linkage, alkyl phosphonate linkage, aryl phosphonate linkage, hydrogen phosphonate linkage, alkyl phosphoroamidate linkage, aryl phosphoroamidate linkage, phosphoroselenate linkage, 2'-O-aminopropyl modiﬁcation, 2'—O-a|kyl modiﬁcation, 2'—O-ally| modiﬁcation, 2'-O-butyl modiﬁcation, a-anomeric oligodeoxynucleotide and 1-(4'-thio-B-D-ribofuranosyl) ation.

Step (c): Hybridization of the fragment released from the PTO with CT0 The fragment released from the PTO is hybridized with a CT0 (Capturing and ting Oligonucleotide).

The ﬁrst fragment and the second fragment have commonly a hybridizable sequence with the capturing portion of the CT0 and thus one of them is hybridized with the CT0.

The second nt produced when hybridized with the mismatch template comprises an additional 3’-end portion being ent from the ﬁrst fragment produced when ized with the match template.

According to a preferred embodiment, the CT0 has a ce selected such that the CT0 is not hybridized with the additional 3’-end portion of the second fragment to prevent the second fragment from extension when the second fragment is hybridized with the capturing portion of the CT0. For example, the sequence of the CT0 may be selected such that the CT0 has a mismatch nucleotide(s) opposed to the additional 3’-end portion of the second fragment. Alternatively, universal bases may be used instead of the mismatch nucleotide.

The first l cleavage site (or the second initial cleavage site) may not be ' fixed but rather multiple in a condition. For e, initial cleavage sites may be positioned in a 5’ to 3’ direction at an initial nucleotide of a double strand (i.e., bifurcation site) in structures ing a single strand and a double strand and 1-2 nucleotides apart from the initial nucleotide. In such case, ably, the sequence of the CTO is selected such that the shortest fragment released by the first initial ge is selectively extended in the present invention to generate the extended strand indicative of the presence of the nucleotide variation.

Step (d): Extension of the Fragment When the first fragment is hybridized with the capturing portion of the CT0, it is extended to form an extended strand comprising an ed ce complementary to the ting portion of the CT0. When the second fragment is hybridized with the capturing portion of the CT0, it is not extended.

Generally, the extension of primers may be controlled by hybridization between a 3’-end part of primers and a template. By ing primer sequences and reaction conditions (6.9. annealing temperature), the extension of primers having at their 3’— end part 1-3 mismatch nucleotides is allowable. Alternatively, the extension of primers may be allowable only when they have perfectly complementary sequence to target sequences.

According to a preferred embodiment, the sequence of the LTD is selected that either the ﬁrst fragment or the second fragment is selectively extended.

According to a preferred embodiment, the ion of the fragment is carried out under conditions such that the extension does not occur even when a single mismatch is present at the 3’-end part of the nt.

Step (e): Signal Generation by hybridization between the extended strand and 50 Following the extension reaction, the extended strand is hybridized with a signaling oligonucleotide (SO). The signal indicative of the ce of the nucleotide variation complementary to the nucleotide discrimination site of the PTO is provided.

Details of hybridization between the extended strand and the SO, labeling systems and signal generation will be described with nce to descriptions indicated above.

Step (f): Detection of signal Finally, the detectable signal provided in the step (e) is ed,.whereby the detection of the signal indicates the presence of the extended strand and the presence of the nucleotide variation complementary to the tide discrimination site of the PTO.

Details of the detection of the signal will be described with reference to descriptions indicated above. ing to an embodiment, the present invention for nucleotide ion detection may be performed with no help of upstream oligonucleotides. Enzymes having upstream oligonucleotide—independent 5’ nuclease activity are used.

Considering ampliﬁcation of target c acid sequences, reaction conditions and 5’ nuclease activity, the t invention is preferably performed using upstream oligonucleotides, more preferably upstream s.

Kits for Target Detect/0n In a further aspect of this invention, there is provided a kit for detecting a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE—SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay, comprising: (a) a probing and targeting Oligonucleotide (PTO); wherein the PTO comprises (i) a 3’-targeting portiOn sing a izing 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 ce; 'wherein the 3’-targeting n of the PTO is hybridized with the target c acid sequence and the 5’-tagging portion is not hybridized with the target nucleic acid sequence; (b) an upstream Oligonucleotide comprising a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; wherein 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 capturing and templating oligonucleotide (CTO); wherein the CFO 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 S’-tagging n of the PTO and (ii) a templating portion comprising a nucleotide ce non- complementary to the 5’-tagging portion and the 3’—targeting portion of the PTO; wherein the nt released from the PTO is hybridized with the capturing portion of the CT0; n the fragment hybridized with the capturing portion of the CT0 is extended to form an extended strand comprising an extended ce mentary to the templating n of the CT0, thereby forming an extended duplex; and (d) a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand.

Since the kit of this invention is constructed to perform the ion method i of the present invention described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this speciﬁcation.

According to a preferred ment, at least a portion of the SO comprises a complementary sequence to the extended sequence.

According to a preferred embodiment, the kit comprises (i) the label linked to the SO, (ii) a combination of the label linked to the SO and a label linked to the fragment from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the ed” , or (iv) a combination of the label linked to the SO and an intercalating dye.

According to a preferred embodiment, the SO is labeled with an interactive dual label comprising a reporter molecule and a quencher molecule.

According to a preferred ment, the SC is labeled with a single label.

According to a preferred embodiment, the kit further comprises an additional $0 comprising a complementary sequence to the extended strand, the two 805 are hybridized with the extended strand in an adjacent manner, the two $05 each comprises one label among a reporter molecule and a quencher le of an interactive dual label. ing to a preferred embodiment, the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label and the fragment from the PTO comprises the other label among the reporter molecule and the quencher molecule.

According to a preferred embodiment, the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, and the templating portion of the CT0 comprises a nucleotide having a first non-natural base; wherein the kit further comprises a nucleotide having both a second non-natural base with a specific binding affinity to the first non-natural base and the other among the reporter molecule and the quencher molecule.

According to a preferred embodiment, the SO comprises one label among a reporter le and a er le of an interactive dual label, and the kit further comprises a nucleotide having the other among the er molecule and the quencher molecule.

According to a preferred embodiment, the SO comprises an acceptor of a FRET (ﬂuorescence resonance energy transfer) and the kit further comprises an intercalating dye.

According to a preferred embodiment, the PTO, CT0 and/or SO is blocked at its 3’-end to prohibit its extension.

According to a preferred embodiment, the upstream ucleotide is an upstream primer or an upstream probe.

According to a red embodiment, the kit further ses yme having a 5’ nuclease activity.

According to a preferred embodiment, 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 ses at least two types of the PTOs, the CT0 comprises at least two types of the CT05 and the SO comprises at least two types of the 505. ing to a preferred embodiment, the kit further comprises a downstream primer.

The features and advantages of this invention will be summarized as follows: (a) The present invention does not use probes to be hybridized with target nucleic acid sequences for providing target signals. Interestingly, the present ion uses probes (signaling oligonucleotides) to be hybridized with the extended strand formed in a target-dependent manner in which the ed strand is synthesized using the CT0 artiﬁcially selected as templates. The present invention employs ﬁrstly the PTO for probing target nucleic acid sequences and then ly the SD for providing signals by hybridization with the target—dependent extended strand, contributing to dramatic increase in speciﬁcity and much better convenience in determining reaction conditions by ing conditions for signal generation irrespective of target nucleic acid sequences. Such features permit conditions for signal generation to be more y established in simultaneous multiplex target detection in diverse clinical samples, and false ve s to be prevented. (b) In conventional technologies using probes to be hybridized with target nucleic acid sequences, probes are hybridized with target nucleic acid ces in ition with complementary sequences, of target nucleic acid sequences.

However, the t invention is able to amplify only the extended strand using a controlled amount of the CT0 as templates and therefore ensure efficient hybridization of probes, making it possible to efficiently give'signals indicative of the presence of target nucleic acid sequences. (c) The present invention may detect the presence of target nucleic acid sequences in a real-time manner or by a g analysis. (d) The Trn value of the hybridization resultant between the extended strand and the SO may be adjustable by a sequence and/or length of the SO and ore arbitrarily pre-determined. By using such feature, (i) the present ion may detect target c acid sequences with differentiating false ve signals because signals generated at temperatures other than pre-determined Tm values correspond to false positive signals. (ii) The arbitrary determination of Trn values of the hybridization resultant becomes more advantageous in multiplex detection for at least two target nucleic acid sequences. (e) Tm value of conventional melting curve analysis of the hybrid between a probe and a target nucleic acid sequence is affected by a sequence variation on the target nucleic acid ce. However, an extended strand in the present ion ‘- provides a constant Trn value regardless of a sequence variation on the target nucleic acid sequences, permitting to ensure ent accuracy in melting curve analysis. (f) It is noteworthy that the sequences of the 5’-tagging portion of the PTO, the CT0 and the 50 can be selected with no consideration of target nucleic acid sequences. This makes it possible to pre-design a pool of sequences for the 5’-tagging ,3 9 portion of the PTO, the CT0 and the SO. Although the 3’-targeting portion of the PTO has to be prepared with considering target nucleic acid sequences, the CT0 and the SO can be prepared in a ready-made fashion with no consideration or knowledge of target nucleic acid sequences. (9) A wide variety of the conventional labeled probes are applicable to the present invention for target detection. (h) Where the hybridization resultants between the extended strands and the 505 have ent Tm values from each other, at least two target nucleic acid sequences may be detected by melting curve is even using a labeling system providing signals with the same \ﬂuorescence characteristics. The advantage permits to be free from limitations associated with the number of detectable cence labels in multiplex real—time detection.

The present invention will now be described in. further detail by examples. It would be obvious to those skilled in the art that these es are intended to be more concretely illustrative and the scope of the present invention as set forth in the ed claims is not limited to or by the es.

EXAMPLES E 1: tion of PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization (PCE-SH) assay A New assay, PTO Cleavage and Extension-Dependent Signaling: Oligonucleotide Hybridization (PCE-SH) assay, was evaluated for the detection of a target nucleic acid sequence in (i) real-time detection at a pre-determined temperature or (ii) melting analysis manner (see Fig. 2). 723q DNA polymerase having a 5’ nuclease activity was used for the extension of upstream primer, the cleavage of PTO, and the extension of PTO fragment.

PTO and CT0 have no label. PTO and CT0 are blocked with a carbon spacer at their 3’-ends. The synthetic ucleotide for Ne/sser/a gonorrhoeae (NG) gene was used as a target template. Signaling Oligonucleotide (SO) has a ﬂuorescent reporter molecule (CAL Fluor Red 610) at its 5’-end and has a quencher molecule (BHQ-Z) at its 3’-end.

The sequences of synthetic template, upstream primer, PTO, CT0 and SO used in this e. are: NG-T ‘5'— AAATATGCGAAACACGCCAATGAGGGGCATGATGC'l‘lTCTTTlTGl‘I'CTTGCTCGGCAGAGCGAGTGATA CCGATCCATTGAAAAA—3’ (SEQ ID NO: 1) NG-R S’-CAATGGATCGGTATCACTCGC~3’ (SEQ ID NO: 2) NG—PTO 5’-ACGACGGCFTGGCTGCCCCTCATTGGCGTG'l'lTCG[C3 spacer]—3’ (SEQ ID NO: 3) NG-CTO 5’-GCGCTGGATACCCTGGACGATATGCAGCCAAGCCGTCGT[C3 spacer]-3’ (SEQ ID NO: 4) NG-SO-l 5’—[CAL Fluor Red 610]GCGCTGGATACCCTGGACGATATG[BHQ-2]-3’ (SEQ ID NO: 5) (Underlined s indicate the 5’-tagging portion of PTO) 1-1. Real—time ion at a pre—determined temperature The reaction was conducted in the final volume of 20 pl containing 2 pmole of tic template (SEQ ID NO: 1) for N6 gene, 10 pmole of upstream primer (SEQ ID NO: 2), 5 pmole of PTO (SEQ ID NO: 3), 0.5 pmole of CTO (SEQ ID NO: 4), 0.5 pmole of SO (SEQ ID NO: 5) and 10 pl of 2X Master Mix containing 2.5 mM MgCl2, 200 uM of dNTPs and 1.6 units of H-7aq DNA polymerase (Solgent, Korea); the tube containing the reaction mixture was placed in the real-time thermocycler (CFX96, Bio- Rad); the reaction mixture was denatured for 15 min at 95°C and subjected to 30 cycles of 30 sec at 95°C, 60 sec at 60°C, and 30 sec at 72°C. Detection of the ted Signal was performed at the hybridization step (60°C) of each cycle. The ion temperature was determined to the extent that the extended strand-SO hybrid maintains a double-stranded form.

As shown Figure 10A, the fluorescent signal was detected in the presence of the template. No signal was detected in the absence of the template, PTO, CTO or SO. 1—"2. Melting is After the on in Example 1—1, melting curve was obtained by cooling the reaction mixture to 55°C, holding at 55°C for 30 sec, and heating slowly at 55°C to 85°C. The fluorescence was measured continuously during the temperature rise to r dissociation of an extended strand-SO hybrid. Melting peak was derived from the melting curve data.

As shown Figure 108, a peak at 685°C corresponding to the expected Tm value of the extended strand—SO hybrid was detected in the presence of template. No peak was detected in the absence of the template, PTO, CT0 or SO.

EXAMPLE 2: ion of a target nucleic acid sequence using PCE-SH assay We further examined whether PCE-SH assay can detect a target nucleic acid sequence in (i) real—time PCR manner or (ii) post-PCR'melting analysis . 72%; DNA polymerase having a 5’ nuclease activity was used for the extension of upstream primer and downstream primer, the cleavage of PTO and the ion of PTO fragment.

PTO and CT0 have no label. PTO and CFO are blocked with a carbon spacer at their 3’-ends. The genomic DNA of NG gene was used as a target template. SO has a ﬂuorescent er molecule (CAL Fluor Redr610) at its 5’-end and has a quencher molecule ) at its 3’—end.

The sequences of upstream primer, downstream primer, PTO, CT0 and SO used in this Example are: NG-F 5'-TACGCCTGCTACTITCACGCT—3’ (SEQ ID NO: 6) NG-R 5’-CAATGGATCGGTATCACTCGC—3' (SEQ ID NO: 2) NG-PTO 5'-ACGACGGCTTGGCTGCCCCI'CA'ITGGCGTGTTFCG[C3 spacer]-3’ (SEQ ID NO: 3) NG-CTO CTGGATACCCTGGACGATATGCAGCCAAGCCGTCGT[C3 ]-3’ (SEQ ID NO: 4) NG-SO-l 5'-[CAL Fluor Red 610]GCGCTGGATACCCTGGACGATATG[BHQ-2]-3’ (SEQ ID NO: 5) (Underlined letters indicate the 5’—tagging portion'of PTO) 2-1. Real-time detection at a pre-determined temperature during PCR The reaction was conducted in the final volume of 20 pl containing 100 pg of genomic DNA of N6, .10 pmole of upstream primer (SEQ ID NO: 2), 10 pmole of downstream primer (SEQ ID NO: 6), 5 pmole of PTO (SEQ ID NO: 3), 0.5 pmole of cro (SEQ ID NO: ‘4), 0.5 pmole of so (SEQ ID NO: 5) and 10 pl of 2x Master Mix containing 2.5 mM MgCl2, 200 pM of dNTPs and 1.6 units of H— 734 DNA polymerase (Solgent, Korea); the tube containing the reaction mixture was placed in the real—time thermocycler (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95°C and subjected to 50 cycles of 30 sec at 95°C, 60 sec at 60°C, and 30 sec at 72°C. Detection of the signal was performed at the hybridization step (60°C) of each cycle. The detection temperature was determined to the extent that the extended strand-SO hybrid maintains a double-stranded form.

As shown Figure 11A, the ﬂuorescent signal (Ct: 30.34) was detected in the presence of the template. No signal was detected in the absence of the template. 2-2. Post-PCR melting analysis After the reaction in e 2-1, g curve was obtained by cooling the reaction mixture to 55°C, holding at 55°C for 30 sec, and heating slowly at 55°C to 85°C. The ﬂuorescence was measured continuously during the temperature rise to monitor dissociation of an extended strand-SO hybrid. Melting peak was derived from the melting curve data.

As shown Figure 118, a peak at 685°C corresponding to the expected Tm value of the extended strand-SO hybrid was ed in the presence of te. No peak was detected in the e of the template.

EXAMPLE 3: Discrimination of a single tide variation of a target nucleic acid sequence using PCE-SH We further examined whether PCE-SH assay can discriminate a single tide variation of a target c acid sequence. 7.'aq DNA polymerase having a 5’ nuclease activity was used for the extension of upstream primer and downstream primer, the cleavage of PTO and the extension of PTO nt. PTO and CT0 have no label. PTO and CT0 are blocked with a carbon spacer at their s. Wild-type (C), hetero—type (C/T) and mutant-type (T) of human genomic DNA for C677T mutation of MTHFR gene were used as target c acids. SO has a quencher molecule (BHQ-Z) at its 5'-end and has a ﬂuorescent er molecule (CAL Fluor Red 610) at its 3’-end. , PTO—1(SEQ ID N029) and CTO-l (SEQ ID NO:11) were used to detect the wild-type, and PTO-2(SEQ ID NO:10) and CTO-2(SEQ ID NO:12) used to detect the mutant-type. Where the wi|d~type gene was present, the extended strand (hereinafter referred to as “wild—type extended strand”) was formed using CT0-1 as a template. In the event that the mutant—type gene was present, the extended strand (hereinafter ed to as “mutant-type ed strand”) was formed using CT0-2 as a template.

In the detection of the hybridization products between the extended strands and the 505 by a melting analysis, the two types of the extended strands can be differentially detected even using one type of the 50. For instance, where the extended strands are designed'to have different sequences from each other on a portion to be ized with the 50s, the hybridization products have different Tm values enabling to differentially detect the formation of extended strands.

The sequences of upstream primer, downstream primer, PTO, CTO and SO: used in this e are: M677-F 5’—GCAGGGAGCITTGAGGCTGIIIIIAAGCACITGA—3’ (SEQ ID NO: 7) M677-R S’-CCTCACCTGGATGGGAAAGATIIIIIGGACGATGG-3’ (SEQ ID NO: 8) M677-PTO-1 5'— CCCAGGCAACCCTCCGATTTCATCATCACGCAGCTITI'CTITGAGGCT[Spacer c3]-3' (SEQ ID NO: 9) M677-PTO-2 5’-CTCCTGCTCGCGTACTCCCGCAGACACCTICTCCITCAAG[Spacer c3] -3' (SEQ ID NO: 10) M677-CTO-1 5’eTCCGCTGCTTCACCACGCCTTCGAGAGGGTTGCCTGGG[Spacer c3] -3' (SEQ ID NO: M677-CTO-2 5’-TCCGCl‘GCITGACGACGCC'ITCGiATACGCGAGCAGGAG[Spacer c3] -3' (SEQ ID NO: O 5’- [BHQ-2]TCCGCTGClTCACCACGCCTTCGA[CAL Red 610] -3’ (SEQ ID NO: 13) .

(I : Deoxyinosine) (Underlined letters indicate the 5’—tagging portion of PTO) (Bold letter indicates the sequence at C677T mutation site of MTHFR gene) The reaction was conducted in the final volume of 20 pl ning 30 ng of MTHFR (C677T) wild (C), hetero (C/T) or mutant (T) type human genomic DNA, 10 pmole of upstream primer (SEQ ID NO: 7), 10 pmole of downstream primer (SEQ ID NO: 8), each 5 pmole of PTOs (SEQ ID NO: 9 and 10), each 0.1 pmole of CTOs (SEQ ID NO: 11 and 12), 0.5 pmole SO (SEQ ID NO: 13) and 10 pl of 2X Master Mix containing 2.5 mM MgCl2, 200 pM of dNTPs and 1.6 units of H-7éq DNA polymerase (Solgent, Korea); the tube containing the reaction mixture was placed in the real-time thermocycler (CFX96, Bio-Rad); the reaction mixture was denatured for ’15 min at 95°C and subjected to 40 cycles of 30 sec at 95°C, 60 sec at 55°C, and 30 sec at 72°C. After the reaction, melting curve was obtained by g the reaction mixture ‘ to 45°C, holding at 45°C for 30 sec, and heating slowly at 45°C to 85°C. The .- ﬂuorescence was measured continuously during the temperature rise to monitor a - dissociation of an ed strand—SO hybrid. Melting peak was derived from the melting curve data.

As shown Figure 12, a peak at 71.0°C corresponding to the expected Tm value of the wild-type extended strand-SO hybrid was detected in the presence of the Wild-type template. A peak at 555°C corresponding to the expected Tm value of the mutant-type extended strand-SO hybrid was detected in the presence of the mutant- type template. A peak at 71.0°C (wild—type) and a peak at 555°C (mutant-type) were detected in the presence of the hetero~type template. No peak was ed in the absence of any type of templates.

EXAMPLE 4: Evaluation of PCE-SH assay using am oligonucleotide- independent cleavage of PTO PCE—S’H assay was further evaluated for the ion of a target nucleic acid sequence without using upstream oligonucieotide in (i) real—time detection at a pre- determined temperature or (ii) melting analysis manner.

Taq DNA polymerase having a 5’ nuclease activity was used for the cleavage of PTO, and the extension of PTO fragment.

PTO and CT0 have no label. PTO and CT0 are blocked with a carbon spacer at their 3’-ends. The synthetic oligonucieotide for Nasser/23 gonorrhoeae (NG) gene was used as a target template. SO has a ﬂuorescent reporter molecule (CAL Fluor Red 610) at its 5’-end and has a quencher molecule (BHQ-Z) at its .

The sequences of synthetic template, PTO, CT0 and SO used in this Example are: NG—T 5'- AAATATGCGAAACACGCCAATGAGGGGCATGATGCTITClTlTTG‘lTC'ITGCTCGGCAGAGCGAGTGATA CATI’GAAAAA-3’ (SEQ ID NO: 1) NG-PTO ’-ACGACGGCTTGGCTGCCCCTCATTGGCGTGTITCG[C3 spacer]-3’ (SEQ ID NO: 3) NG-CTO 5'-GCGCTGGATACCCTGGACGATATGCAGCCAAGCCGTCGT[C3 spacer]—3’ (SEQ ID NO: 4) NG-SO-l 5’-[CAL Fluor Red 610]GCGCTGGATACCCTGGACGATATG[BHQ-2]-3’ (SEQ ID NO: 5) (Underlined s indicate the 5’—tagging portion of PTO) 4—1. Real-time detection at a pre-determined temperature The reaction was ted in the final volume of 20 ul containing 2 pmole of synthetic template (SEQ ID NO: 1) for N6 gene, 5 pmole of PTO (SEQ ID NO: 3), 0.5 pmole of CTO (SEQ ID NO: 4), 0.5 pmole of SO (SEQ ID NO: 5) and 10 pl of 2X Master Mix containing 2.5 mM MgCl2, 200 pM of dNTPs and 1.6 units of H- 72m DNA polymerase (Solgent, ; the tube ning the reaction mixture was placed in the real—time thermocycler (CFX96, Bio-Rad); the reaction e was denatured for min at 95°C and subjected to 30 cycles of 30 sec at 95°C, 60 sec at 60°C, and 30 sec at 72°C. Detection of the generated Signal was performed at the hybridization step (60°C) of each cycle. The detection temperature was determined to the extent that the extended strand—SO hybrid maintains a double-stranded form.

As shown Figure 13A, the ﬂuorescent signal was detected in the presence of the te. No signal was detected in the absence of the template. 4-2. Melting analysis After the reaction in Example 4-1, melting curve was obtained by g the reaction mixture to 55°C, g at 55°C for 30 sec, and heating slowly at 55°C to 85°C. The ﬂuorescence was measured continuously during the temperature rise to monitor dissociation of an extended strand—SO hybrid. Melting peak was derived from the melting curve data.

As shown Figure 138, a peak at 685°C corresponding to the expected Tm value of the extended strand-SO hybrid was detected in the presence of template. No peak was detected in the absence of the template.

Having described a preferred embodiment of the present ion, it is to be understood that variants and cations thereof falling within the spirit of the invention may become apparent to those d in this art, andythe scope of this invention is to be determined by appended claims and their equivalents.

Claims

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-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay, comprising: 5 (a) hybridizing the target nucleic acid ce with an am primer and a probing and targeting oligonucleotide (PTO); wherein the upstream primer comprises a hybridizing nucleotide sequence mentary to the target nucleic acid sequence; the PTO comprises (i) a 3’-targeting portion comprising a izing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5’- 10 tagging portion comprising a nucleotide sequence mplementary to the target nucleic acid ce; wherein the 3’-targeting n 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; the upstream primer is located upstream of the PTO; (b) contacting the resultant of the step (a) to an enzyme having a 5’ 15 nuclease activity under conditions for ge of the PTO; wherein the extended strand of the upstream primer 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 a capturing and 20 templating oligonucleotide (CTO); wherein the CTO ses 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 ging portion and the 3’-targeting portion of the PTO; wherein the fragment released from the PTO is 25 hybridized with the ing portion of the CTO; (d) performing an extension reaction using the resultant of the step (c) and a te-dependent nucleic acid polymerase; wherein the fragment hybridized with the capturing n of the CTO is extended to form an extended strand comprising an extended sequence complementary to the templating portion of the CTO, thereby 30 g an extended duplex; (e) hybridizing the extended strand with a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand; and 35 (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

2. The method according to claim 1, wherein at least a portion of the SO comprises a complementary sequence to the extended sequence. 5

3. The method according to claim 1 or claim 2, wherein the detectable signal is provided by (i) the label linked to the SO, (ii) a combination of the label linked to the SO and a label linked to the fragment from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the extended strand during the extension on of the step (d), or (iv) a combination of the label linked to the SO 10 and an intercalating dye.

4. The method according to claim 1 or claim 2, wherein the SO is labeled with an interactive dual label comprising a reporter molecule and a quencher molecule and the hybridization between the SO and the extended strand in the step (e) induces change 15 in signal from the interactive dual label to provide the able signal.

5. The method according to claim 1 or claim 2, wherein the SO is labeled with a single label and the hybridization n the SO and the extended strand in the step (e) induces change in signal from the single label to provide the able signal.

6. The method ing to claim 5, wherein the single label is a single fluorescent label.

7. The method according to claim 1 or claim 2, n the method uses an 25 additional SO comprising a complementary sequence to the extended strand, the two SOs are hybridized with the extended strand in an adjacent manner, the two SOs each ses one label among a reporter molecule and a quencher molecule of an interactive dual label; and the ization between the two SOs and the extended strand induces change in signal from the interactive dual label to provide the 30 detectable signal.

8. The method according to claim 1 or claim 2, wherein the SO comprises one label among a reporter molecule and a quencher le of an interactive dual label, the fragment from the PTO comprises the other label among the reporter molecule 35 and the quencher molecule; the extended strand comprises the label originated from the fragment from the PTO, and wherein the hybridization between the SO and the ed strand induces change in signal from the interactive dual label to provide the detectable signal.

9. The method according to claim 1 or claim 2, n the SO comprises one 5 label among a reporter molecule and a quencher molecule of an interactive dual label, and the templating portion of the CTO comprises a nucleotide having a first nonnatural base; n the extension reaction in the step (d) is performed in the presence of a nucleotide having both a second non-natural base with a specific binding affinity to the first non-natural base and the other among the reporter

10 molecule and the quencher molecule, thereby incorporating the label into the extended strand; wherein the hybridization between the SO and the extended strand induces change in signal from the interactive dual label to provide the detectable signal. 15 10. The method according to claim 1 or claim 2, n the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, and the extension reaction in the step (d) is performed in the presence of a nucleotide having the other among the reporter molecule and the quencher molecule, thereby orating the label into the extended strand; wherein the hybridization between 20 the SO and the extended strand induces change in signal from the interactive dual label to provide the detectable signal.

11. The method according to claim 1 or claim 2, wherein the SO comprises an acceptor of a FRET escence resonance energy er) and the hybridization in 25 the step (e) is performed in the presence of an intercalating dye; wherein the hybridization between the SO and the extended strand induces change in signal from the acceptor of the SO to provide the detectable signal.

12. The method according to any one of claims 1-11, wherein the PTO, CTO 30 and/or SO is d at its 3’-end to prohibit its extension.

13. The method according to any one of claims 1 to 12, wherein the capturing n of the CTO comprises at its 5’-end part a nucleotide sequence complementary to a part of the 3’-targeting portion of the PTO.

14. The method according to claim 13, wherein the nucleotide sequence complementary to the part of the 3’-targeting portion of the PTO is 1-10 nucleotides in length.

15. The method according to any one of claims 1 to 14, wherein the method 5 further comprises the step of providing a able signal between the steps (e) and (f) by melting the ization resultant of the step (e) or by melting and hybridizing the ization resultant of the step (e); wherein the step (f) is performed by detecting the signal to determine the presence of the extended strand. 10

16. The method according to any one of claims 1 to 15, wherein the method further comprises the step of providing and detecting a able signal after the step (f) by melting the hybridization resultant of the step (e) or by g and hybridizing the ization resultant of the step (e), whereby the presence of the extended strand is determined one more time.

17. The method according to claim 15 or 16, wherein the presence of the ed strand is determined by a melting curve analysis or a hybridization curve analysis. 20

18. The method according to any one of claims 1 to 17, wherein the method further comprises the step of denaturation between the steps (d) and (e).

19. The method according to any one of claims 1 to 17, wherein the method further comprises repeating all or some of the steps (a)-(f) with denaturation between 25 repeating cycles.

20. The method ing to claim 19, wherein the method further comprises repeating the steps (a)-(e) with denaturation between repeating cycles, and melting the hybridization resultant of the step (e) or melting and hybridizing the hybridization 30 resultant of the step (e) to provide a detectable signal; wherein the step (f) is performed by detecting the signal to determine the presence of the extended .

21. The method according to any one of claims 1 to 20, wherein the steps (a)-(f) are med in a reaction vessel or some of the steps (a)-(f) are performed in 35 separate reaction vessels.

22. The method according to any one of claims 1 to 21, wherein the method is performed to detect at least two types of target nucleic acid ces; wherein the upstream primer comprises at least two types of upstream primers, the PTO comprises at least two types of the PTOs, the CTO comprises at least two types of the 5 CTOs and the SO comprises at least two types of the SOs.

23. The method according to any one of claims 1 to 22, wherein the step (b) uses a template-dependent nucleic acid polymerase for the extension of the upstream

24. The method according to any one of claims 1 to 23, n the target nucleic acid sequence comprises a nucleotide variation.

25. The method according to any one of claims 1-24, wherein the method is 15 med in the ce of a downstream primer.

26. A method for detecting a target c acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) assay, comprising: 20 (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); wherein each of the upstream primer and the ream primer comprise a hybridizing tide sequence complementary to the target nucleic acid sequences; the PTO comprises (i) a 3’-targeting portion comprising a 25 hybridizing nucleotide sequence complementary to the target nucleic acid sequence and (ii) a 5’-tagging portion comprising a nucleotide ce non-complementary to the target nucleic acid sequence; wherein the geting portion is hybridized with the target nucleic acid sequence and the 5’-tagging portion is not hybridized with the target c acid sequence; the PTO is located between the upstream primer and the 30 downstream primer; wherein the PTO is blocked at its 3’-end to prohibit its extension; (b) contacting the ant of the step (a) to a template-dependent nucleic acid polymerase having a 5’ nuclease activity under conditions for extension of the primers and for ge of the PTO; wherein when the PTO is hybridized with the target nucleic acid sequence, the upstream primer is extended and the extended 35 strand induces cleavage of the PTO by the template-dependent nucleic acid polymerase 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 a capturing and templating oligonucleotide (CTO); wherein the CTO comprises in a 3’ to 5’ direction (i) a capturing portion comprising a nucleotide sequence complementary to the 5’-tagging 5 n or a part of the 5’-tagging portion of the PTO and (ii) a templating n comprising a nucleotide sequence non-complementary to the 5’-tagging portion and the 3’-targeting n of the PTO; wherein the fragment ed 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 10 a template-dependent nucleic acid polymerase; wherein the fragment hybridized with the ing 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 a signaling oligonucleotide (SO); 15 wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand; and (f) detecting the signal; whereby the detection of the signal indicates the presence of the extended strand and the presence of the target nucleic acid sequence.

27. A kit for detecting a target c acid sequence from a DNA or a mixture of nucleic acids by a PCE-SH (PTO Cleavage and ion-Dependent Signaling Oligonucleotide Hybridization) assay when used in the performance of the method of any one of claims 1-26, comprising: 25 (a) a probing and ing oligonucleotide (PTO); n 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 mplementary to the target nucleic acid sequence; wherein the 3’-targeting portion of the PTO is hybridized with the target 30 nucleic acid sequence and the 5’-tagging n is not hybridized with the target nucleic acid sequence; (b) an upstream primer comprising a hybridizing nucleotide sequence complementary to the target c acid sequence; wherein the am primer is located upstream of the PTO; wherein the extended strand of the upstream primer 35 induces cleavage of the PTO by an enzyme having a 5’ nuclease ty such that the ge releases a fragment comprising the 5’-tagging portion or a part of the 5’- tagging portion of the PTO; (c) a capturing and templating oligonucleotide (CTO); wherein the CTO comprises in a 3’ to 5’ direction (i) a capturing portion comprising a nucleotide ce complementary to the 5’-tagging portion or a part of the 5’-tagging portion 5 of the PTO and (ii) a ting portion comprising a nucleotide sequence noncomplementary to the 5’-tagging portion and the 3’-targeting portion of the PTO; wherein the fragment released from the PTO is hybridized with the ing portion of the CTO; wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended strand comprising an ed sequence 10 complementary to the templating portion of the CTO, thereby forming an extended duplex; and (d) a signaling oligonucleotide (SO); wherein the SO comprises a complementary sequence to the extended strand and at least one label; the SO provides a detectable signal by hybridization with the extended strand.

28. The kit according to claim 27, wherein at least a portion of the SO comprises a complementary sequence to the extended ce.

29. The kit according to claim 27 or claim 28, wherein the kit comprises (i) the 20 label linked to the SO, (ii) a combination of the label linked to the SO and a label linked to the nt from the PTO, (iii) a combination of the label linked to the SO and a label to be incorporated into the extended strand, or (iv) a combination of the label linked to the SO and an intercalating dye. 25

30. The kit according to any one of claims 27 to 29, wherein the SO is labeled with an interactive dual label comprising a reporter molecule and a quencher molecule.

31. The kit ing to any one of claims 27 to 29, wherein the SO is labeled 30 with a single label.

32. The kit according to any one of claims 27 to 31, wherein the kit further comprises an onal SO comprising a mentary sequence to the extended strand, the two SOs are hybridized with the extended strand in an adjacent manner, 35 the two SOs each comprises one label among a reporter molecule and a quencher molecule of an interactive dual label.

33. The kit according to claim 27, n the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label and the fragment from the PTO ses the other label among the reporter molecule and 5 the quencher molecule.

34. The kit according to claim 27, wherein the SO comprises one label among a reporter molecule and a quencher molecule of an interactive dual label, and the ting portion of the CTO comprises a nucleotide having a first non-natural base; 10 wherein the kit further comprises a tide having both a second non-natural base with a specific binding affinity to the first non-natural base and the other among the reporter molecule and the quencher molecule.

35. The kit according to claim 27, wherein the SO comprises one label among a 15 reporter molecule and a er molecule of an interactive dual label, and the kit further ses a nucleotide having the other among the reporter molecule and the quencher molecule.

36. The method according to claim 27, wherein the SO comprises an acceptor of a 20 FRET (fluorescence resonance energy transfer) and the kit further comprises an intercalating dye.

37. The kit according to any one of claims 27 to 36, wherein the PTO, CTO and/or SO is blocked at its 3’-end to prohibit its extension.

38. The kit according to any one of claims 27 to 37, wherein the kit r comprises an enzyme having a 5’ nuclease activity.

39. The kit according to any one of claims 27 to 38, wherein the kit is for 30 detection of at least two types of target nucleic acid sequences; wherein the upstream primer ses at least two types of upstream primers, 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. 35

40. The kit according to any one of claims 27 to 39, wherein the kit further comprises a downstream primer.