RU2588457C2 - Determination of target nucleic acid sequences by splitting oligonucleotide probe and hybridisation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to detection of a target nucleic acid sequence by a POCH (PO cleavage and hybridisation) assay on a solid substrate. Invention detects target nucleic acid sequence by use of POCH assay, in which PO (probing oligonucleotide) hybridised with target nucleic acid sequence is cleaved and cleavage of PO is detected by hybridisation with CO (capturing oligonucleotide). Uncleaved PO is hybridised with CO immobilised onto solid substrate. Design of PO and CO is convenient and optimisation of reaction conditions is routinely easy. Where detection of signal on solid substrate is continuously performed along with repetition of cleavage of POs, number of POs cleaved is increased upon repetition number of cleavage reaction and signal is changed in parallel with number of POs cleaved. In this case target nucleic acid sequence can be detected in a real-time manner. In contrast, change of signal is not observed in absence of target nucleic acid sequence.

EFFECT: use of group of inventions allows simultaneous detection of multiple target nucleic acid sequences using a mark, even of one type.

42 cl, 24 dwg, 7 tbl, 7 ex

Description

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to the detection of a target nucleic acid sequence in an analysis with POSH (PO cleavage and hybridization) on a solid support.

RELATED INFORMATION

DNA hybridization-based technologies would be an extremely useful tool in determining a specific nucleic acid sequence and would undoubtedly be needed in the areas of clinical diagnosis, genetic research and laboratory forensics. In addition to hybridization methods using probes, various approaches have been proposed using additional enzymatic reactions, for example, a method using TaqMan ™ probes.

In the TaqMan ™ probe method, a labeled probe hybridized to the target nucleic acid sequences is cleaved by the 5′-nuclease activity of the DNA polymerase depending on the upstream primer (US Patent Nos. 5210015, 5538848 and 6326145) . The release of the labeled fragment indicates the cleavage of the probe, ultimately indicating the presence of target sequences. This labeled fragment can be detected using size analysis such as gel electrophoresis, gradient sedimentation, gel permeation chromatography, and homochromatography. The splitting of the probes can be carried out in real time using a system of two interacting labels.

According to the method using TaqMan ™ probes, two approaches for signal generation are proposed: polymerization-dependent splitting and polymerization-independent splitting. With polymerization-dependent cleavage, elongation of the upstream primer must occur before nucleic acid polymerase meets the 5′-end of the labeled probe. As the extension reaction continues, the polymerase gradually cleaves the 5′-end of the labeled probe. When polymerization is independent of the cleavage, the upstream primer and the labeled probe hybridize to the target nucleic acid in close proximity, so that the binding of the nucleic acid polymerase to the 3 ′ end of the upstream primer brings it into contact with 5 ′ - The end of the labeled probe, resulting in the release of the label. In addition, the TaqMan ™ probe method describes that a labeled probe having a 5′-tail region at its 5′-end that does not hybridize with target sequences also cleaves to form a fragment containing this 5′-tail region .

Some methods have been reported in which probes having a 5′-tail region non-complementary to target sequences are cleaved by 5′-nuclease to release a fragment containing a given 5′-tail region and target detection is carried out using a fragment containing this 5′-tail section.

For example, US Pat. No. 5,691,142 describes a cleavable structure that must be digested by the 5′-nuclease activity of DNA polymerase. An example of this cleavable structure is given, in which an oligonucleotide containing a 5′-terminal region, non-complementary to the matrix, and a 3′-terminal region, complementary to the matrix, hybridizes with this matrix, and in the immediate vicinity, the upstream hybridizes with this matrix oligonucleotide. Such a cleavable structure is cleaved by a DNA polymerase having 5′-nuclease activity, or a modified DNA polymerase with a reduced ability to synthesize, releasing a 5′-terminal region incomplete to this matrix. The released 5′-terminal region then hybridizes with an oligonucleotide having a hairpin structure to form a cleavable structure, thereby inducing progressive cleavage reactions necessary for the detection of target sequences.

US Pat. No. 7,381,532 describes a method in which a cleavable structure comprising an upstream oligonucleotide with a 3 ′ end blocked is cleaved by DNA polymerase having 5′ nuclease activity or FEN nuclease (flap endonuclease) to be released non-complementary 5′-terminal single-chain “hanging” section (flap), and the detection of this released 5′-terminal “hanging” section is carried out using size analysis or a system of two interacting labels.

U.S. Patent Application Publication No. 2008/0241838 describes a method for detecting a target using the cleavage of single-labeled probes having a 5′-terminal portion non-complementary to the target and capture probes immobilized on a solid substrate. A single label is located on the non-complementary 5′-terminal portion of the labeled probe. Labeled probes hybridized to the target are cleaved to release fragments, after which these fragments then hybridize to capture probes to detect the presence of the target sequence. In this method, it is necessary that the undigested / intact probe does not hybridize with the capture probe. In order to do this in this way, it is necessary to prevent the hybridization of an undigested target probe with an immobilized capture probe by controlling the orientation of the immobilized oligonucleotide during its immobilization and the distance from it to the surface of the solid substrate. However, this limitation leads to a decrease in the efficiency of hybridization on a solid substrate and to difficulties in optimizing the reaction conditions.

US Patent Application Publication 2008/0193940 also describes a method for detecting a target using probes that contain a non-complementary target sequence (a sequence containing a tag (or marking; tag) or a “hanging” portion (flap)), as well as capture probes immobilized on solid substrates. The label is also located on the non-complementary portion of the probes. Unsplit probes form a hairpin structure and do not hybridize to capture probes. In contrast, when probes are cleaved, label-containing fragments are then hybridized to capture probes, thereby detecting the presence of target nucleic acid sequences. However, there are serious problems associated with the need for careful monitoring of the reaction conditions when considering the value of T _m for hybridization of target sequences with probes, as well as when considering the value of T _m for the hairpin structure of unsplit probes, as well as the value of T _m for hybridization of split fragments with capture probes.

Thus, in this area there remains a long-overdue need to develop new approaches to detecting a target sequence on a solid phase, in particular, devoid of the disadvantages of traditional technologies, by using probes carrying a tag-containing sequence and capture probes immobilized on solid substrates.

Throughout this application, references are made to various patents and publications, and references to them are given in parentheses. Thus, the description of these patents and publications in their entirety is incorporated into this application by reference in order to more fully describe the present invention and the state of the field to which this invention relates.

SUMMARY OF THE INVENTION

The authors of the present invention have carried out intensive studies to develop new approaches for the detection of target sequences, characterized by higher accuracy and convenience, among other things, in the mode of multiple detection. As a result, the inventors have developed new protocols for detecting target sequences using a probe oligonucleotide (PO) and an capture oligonucleotide (CO), in which the target is detected by a probe cleavage reaction and additional probe hybridization (i.e., a 5′-nucleolytic reaction involving PO and hybridization reactions between split / non-split PO and CO). The protocols of the present invention with sharply increased specificity for the target are well adapted to reactions in the solid phase, and this makes it possible to carry out multiple detection of target sequences with higher accuracy and in a more convenient manner.

Accordingly, this invention solves the problem of developing a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSN (PO cleavage and hybridization) on a solid substrate.

Another objective of the present invention is to develop a kit for the detection of a target nucleic acid sequence from DNA or a mixture of nucleic acids in the analysis with ROSN (cleavage and hybridization of PO) on a solid substrate.

Other objects and advantages of the present invention will become apparent from the following detailed description in conjunction with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. Figure 1 schematically shows the structures of PO (probing oligonucleotide) and CO (capturing oligonucleotide) used in the analysis with ROSH (cleavage and hybridization of PO). A RO includes an untagged (i.e., unmarked; non-tagged) RO and a tagged RO, which is further subdivided into 3′-tagged RO and 5′-tagged RO. Preferably, the 3 ′ end of the PO is blocked to prevent elongation (FIG. 1A). CO contains a nucleotide sequence capable of hybridization with PO. CO are immobilized on a solid support through its 3′-end or 5′-end. Preferably, the 3′-end of CO immobilized through the 5′-end is blocked to prevent elongation (FIG. 1B).

In FIG. Figure 2 shows schematically a ROSH analysis using an untagged PO having a single fluorescent label at the 5 ′ end of its targeting portion.

In FIG. Figure 3 shows schematically a ROSH analysis using an untagged PO having a single fluorescent label at the 3 ′ end of its target-recognizing region.

In FIG. Figure 4 is a schematic representation of ROSN analysis using a 3′-tagged PO having a single fluorescent label at the 5′-end of its target-recognizing region.

In FIG. Figure 5 shows schematically a ROSH analysis using a 5′-tagged PO having a single fluorescent label at the 3′-end of its target-recognizing region.

In FIG. Figure 6 is a schematic representation of ROSN analysis using a 5′-tagged PO having a single fluorescent label at the 3′-end of its target-recognizing region, and CO additionally containing a templating portion serving as a matrix for extending the tagging region portion) hybridized with CO.

In FIG. Figure 7 shows schematically a ROSH analysis using an untagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the acceptor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. Figure 8 shows schematically a ROSH analysis using a 3′-tagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the acceptor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. Figure 9 shows schematically a ROSH analysis using a 5′-tagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the acceptor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. Figure 10 is a schematic representation of a POSH analysis using an untagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the donor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is not suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. Figure 11 shows schematically a POSH analysis using a 3′-tagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the donor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. Figure 12 is a schematic representation of an ROSH analysis using a 5′-tagged PO having a donor molecule and an acceptor molecule in a system of two interacting labels to measure the signal from the donor molecule. The donor molecule and the acceptor molecule are localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex.

In FIG. 13 is a schematic representation of POCH analysis using an intercalating agent.

In FIG. Figure 14 shows the results of target detection by ROSH analysis using a single-tagged untagged PO.

In FIG. Figure 15 shows the results of target detection by ROSH analysis using a 3'-tagged single-label PO.

In FIG. 16 shows the results of target detection by ROSH analysis using a 5′-tagged single-label PO.

In FIG. 17 shows the results of target detection by ROSH analysis using a 3′-tagged PO with a double label.

In FIG. Figure 18 shows the results of target detection by ROSH analysis using PCR amplification. A PO is a single-tagged untagged PO.

In FIG. 19 shows the results of target detection by ROSH analysis using PCR amplification. A PO is a 3′-tagged PO with a single label.

In FIG. Figure 20 shows the results of real-time detection of target nucleic acid sequences by ROSH analysis using a 3'-tagged PO with a single label. In FIG. 20A shows fluorescence images depending on the number of cycles in the POCH analysis, and FIG. 20B shows the change in fluorescence intensity depending on the number of cycles in the ROSN analysis.

In FIG. 21 is a schematic representation of ROSH analysis using a 5′-tagged PO to detect a single nucleotide variation.

In FIG. Figure 22 is a schematic representation of ROSN analysis using a 5′-tagged PO having an artificial mismatch nucleotide as a non-base pairing moiety to detect a single nucleotide variation.

DETAILED DESCRIPTION OF THIS INVENTION

The authors of the present invention have carried out intensive studies to develop new approaches for the detection of target sequences, characterized by higher accuracy and convenience, among other things, in the mode of multiple detection. As a result, the inventors have developed new protocols for detecting target sequences using a probe oligonucleotide (PO) and an capture oligonucleotide (CO), in which the target is detected by a probe cleavage reaction and additional probe hybridization (i.e., a 5′-nucleolytic reaction involving PO and hybridization reactions between split / non-split PO and CO). The protocols of the present invention with sharply increased specificity for the target are well adapted to reactions in the solid phase, and this makes it possible to carry out multiple detection of target sequences with higher accuracy and in a more convenient manner.

The present invention relates to a new protocol for the detection of target sequences on a solid substrate using a combination of PO and CO.

A fundamental principle of the present invention is to detect the presence of PO cleavage by using CO immobilized on a solid support. In addition, it should be noted that when the target sequence is absent in the sample, undigested PO hybridizes with CO immobilized on a solid substrate. According to the present invention, the final signal to be measured will be different depending on whether or not a duplex — unsplit PO / CO — is generated, which may indicate the presence or absence of a target sequence.

In the present invention, the target sequence is detected in accordance with the principle of implementation described above, and the invention is divided into three embodiments depending on the selected label systems.

The present invention will be described below in more detail.

I. Method for target detection by ROSN using a single label

In one aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; RO has a single label; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;

(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by an enzyme having 5′-nuclease activity to obtain a single label-containing fragment;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex; and

(d) detecting the presence of PO cleavage by measuring the signal from a single label on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

It is proposed to determine the presence of the target sequence by hybridization of the cleaved fragment of the target probe with an oligonucleotide immobilized on a solid support (see US Patent Application Publication Nos. 2008/0241838 and 2008/0193940). According to a conventional method, the presence or absence of a target sequence is determined only by hybridization of the cleaved probe with an oligonucleotide immobilized on a solid support. The unsplit probe is not involved in hybridization with the immobilized oligonucleotide. To do this in the traditional way, it is necessary to prevent the hybridization of the undigested target probe with the immobilized oligonucleotide by constructing the target probe with the tagging region so that it has a hairpin structure, or by adjusting the orientation of the immobilized oligonucleotide when it is immobilized and the distance from it to the surface of the solid substrate.

The traditional method, requiring the presence of a target probe having a hairpin structure, has serious drawbacks in that the design of the target probe and reaction conditions must be determined taking into account both the hybridization conditions between the target probe having a hairpin structure and the target sequence, and conditions under which hybridization does not occur between the target probe and the immobilized oligonucleotide. Thus, traditional methods are very inconvenient and impractical from the point of view of designing the target probe and the immobilized oligonucleotide and determining the reaction conditions.

In contrast to the traditional methods of the present invention, hybridization between undigested PO and immobilized CO is used without the disadvantages characteristic of traditional methods.

According to the present invention, the final signal to be measured, indicating the presence or absence of the target sequence, will be different depending on whether the cleaved PO fragment is hybridized with CO immobilized on a solid support, or the uncleaved PO hybridizes with immobilized CO.

Therefore, the method of the present invention is called "Analysis with cleavage and hybridization of PO (ROSN)."

The present invention will be described in more detail below.

Stage (a). Hybridization of the upstream oligonucleotide and PO with the target nucleic acid sequence

According to the present invention, the target nucleic acid sequence first hybridizes to the upstream oligonucleotide and PO (probe oligonucleotide).

As used herein, the term “target nucleic acid”, “target nucleic acid sequence”, or “target sequence” refers to a nucleic acid sequence of interest to detect on which a probe or primer is annealed, or which is hybridized with a probe or primer under hybridization conditions annealing or amplification.

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

The PO used in the present invention is preferably a probe.

As used herein, the term “probe” refers to a single-stranded nucleic acid molecule containing a region or regions that are substantially complementary to the target nucleic acid sequence.

The term “primer,” as used herein, refers to an oligonucleotide that is capable of acting as a point of synthesis initiation when placed under conditions under which the synthesis of a primer extension product that is complementary to the nucleic acid chain (template) is induced, i.e. . in the presence of nucleotides and a polymerization agent, such as DNA polymerase, and at suitable temperatures and pH. Preferably, the primer is a single chain deoxyribonucleotide molecule.

The probes or primers used in this invention may contain natural dNMP (deoxynucleoside monophosphate) (i.e., dAMP, dGMP, dCMP and dTMP), a modified nucleotide or a non-natural nucleotide. Probes or primers may also include ribonucleotides.

The primer must be of sufficient length to prime the synthesis of elongation products in the presence of a polymerization agent. The exact length of the primers will depend on many factors, including the temperature of the method of application and the source of the primer. The term “annealing” or “priming,” as used herein, refers to the attachment of an oligodeoxynucleotide or nucleic acid to a nucleic acid matrix, which addition allows the polymerase to polymerize nucleotides to form a nucleic acid molecule that is complementary to a nucleic acid that is matrix, or parts thereof.

The term “hybridization” as used herein refers to the formation of a double stranded nucleic acid from complementary single stranded nucleic acids. Hybridization can occur between two nucleic acid chains that are completely comparable or substantially comparable with a number of mismatches. The complementarity required for hybridization may depend on hybridization conditions, in particular temperature.

Hybridization of target nucleic acid sequences with an upstream oligonucleotide and PO can be carried out under suitable hybridization conditions determined in the established manner by optimizing the procedures. Conditions such as temperature, concentration of components, duration of hybridization and washing, buffer components, their pH and ionic strength, can be changed depending on various factors, including the length and GC composition of the oligonucleotide (located upstream oligonucleotide and PO) and nucleotide target sequences. For example, when a relatively short oligonucleotide is used, it is preferred that low stringency conditions are adopted. Detailed hybridization conditions can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc., N.Y. (1999).

He does not anticipate differences between the terms "annealing" and "hybridization", and these terms will be used interchangeably.

The upstream oligonucleotide and PO contain hybridizing nucleotide sequences complementary to the target nucleic acid sequence. The term “complementary” is used herein to indicate that primers or probes are sufficiently complementary to selectively hybridize to a target nucleic acid sequence under intended annealing conditions or under stringent conditions, encompassing the terms “substantially complementary” and “fully complementary ", preferably" fully complementary ".

As used herein, the term “probing oligonucleotide (PO)” means an oligonucleotide containing a target-recognizing region serving as a probe,

According to a preferred embodiment, the RO includes an untagged PO without a tagging region having a nucleotide sequence incomplete with a target nucleic acid sequence and a tagged PO with a tagging region having a nucleotide sequence non-complementary to a target nucleic acid sequence (Fig. 1A).

According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising in its 5′-terminal portion a tagging region having a nucleotide sequence non-complementary to the target nucleic acid sequence (Fig. 1A).

The PO tagging region is preferably a tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence. The term “non-complementary” is used herein to indicate that primers or probes are sufficiently non-complementary so as not to selectively hybridize to the target nucleic acid sequence under the intended annealing conditions or under stringent conditions, encompassing the terms “substantially non-complementary” and “ completely non-complementary ", preferably" completely non-complementary.

Preferably, tagged PO is used for selective hybridization with CO due to the presence of the tagging region.

No specific length is provided for RO. A non-tagged PO can be of any length sufficient for specific hybridization with the target nucleic acid sequence, for example, its length can be 10-60 nucleotides, 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 15-60 nucleotides, 15- 50 nucleotides, 15-40 nucleotides, 15-30 nucleotides, 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides or 20-30 nucleotides. Tagged PO can have a length of 15-80 nucleotides, 15-60 nucleotides, 15-40 nucleotides, 20-80 nucleotides, 20-60 nucleotides, 20-40 nucleotides, 30-80 nucleotides, 30-60 nucleotides or 30-40 nucleotides. The PO region recognizing the target can be of any length, provided that it specifically hybridizes with the target nucleic acid sequences. For example, the length of a target-recognizing region of PO can be 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 15-50 nucleotides, 15-40 nucleotides, 15-30 nucleotides, 20-50 nucleotides, 20-40 nucleotides or 20 -30 nucleotides. The tagging region of the tagged PO can be of any length, provided that it specifically hybridizes with CO. For example, the length of the tagging region of a PO can be 5-50 nucleotides, 5-40 nucleotides, 5-30 nucleotides, 5-20 nucleotides, 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.

At the 3′-end of the PO, there may be a 3′-OH group. Preferably, the 3′-end of the PO is “blocked” to prevent elongation.

Blocking can be achieved by traditional methods. For example, blocking can be accomplished by adding to the 3′-hydroxyl group of the last nucleotide a chemical moiety such as biotin, label, phosphate group, alkyl group, non-nucleotide linker, phosphorothionate or alkanediol. Alternatively, blocking can be accomplished by removing the 3′-hydroxyl group of the last nucleotide or by using a nucleotide lacking a 3′-hydroxyl group, such as a dideoxynucleotide.

Alternatively, the PO can be designed to adopt a secondary structure such as a hairpin structure. Since in the present invention, unsplit PO is hybridized with oligonucleotides immobilized on a solid support, the hairpin structure should be designed so as not to impede the hybridization of the unsplit PO with immobilized CO. Preferably, the hairpin structure of the PO has a value of T _m less than that of the duplex formed between the unsplit PO and the immobilized CO.

Meanwhile, in accordance with the conventional method, in order to detect a target sequence by using the cleavage reaction of a target probe having a tagging region and hybridization with an oligonucleotide immobilized on a solid support, it is necessary that the target probe has a hairpin structure that prevents hybridization of the unsplit target probe with immobilized oligonucleotide.

When a tagged PO is used, hybridization with the target nucleic acid sequence is carried out under stringent conditions under which its target recognition region hybridizes with the target nucleic acid sequence and its tagging region does not hybridize with the target nucleic acid sequence. The phrase “the tagging region of the PO does not hybridize to the target nucleic acid sequence” means that it does not form a stable duplex with the target nucleic acid sequence under conditions of a specific stiffness. Preferably, the tagged region of the tagged PO does not hybridize to the target nucleic acid sequence, remaining as a single chain.

The upstream oligonucleotide is located upstream relative to the PO when it is hybridized to the target nucleic acid. The upstream oligonucleotide or its extended chain hybridized to the target nucleic acid sequence induces the cleavage of PO hybridized to the target nucleic acid sequence, an enzyme having 5′-nuclease activity.

Induction of PO cleavage via an upstream oligonucleotide can occur in two ways: (1) as an induction of cleavage independent of the elongation (ie, polymerization) of an upstream oligonucleotide; and (2) as the induction of cleavage, dependent on the elongation of the upstream oligonucleotide.

In the case where the upstream oligonucleotide is localized close enough to PO to induce the cleavage of PO with an enzyme having 5′-nuclease activity, then the enzyme that binds to the upstream oligonucleotide cleaves the PO without an extension reaction. In contrast, when an upstream oligonucleotide is localized away from PO, then an enzyme having polymerase activity (eg, matrix polymerase) catalyzes the elongation of an upstream oligonucleotide (eg, upstream) primer), and an enzyme with 5′-nuclease activity, bound to an elongated product, cleaves PO.

Therefore, the upstream oligonucleotide can be localized relative to PO in two ways. The upstream oligonucleotide can be localized close enough to the PO to induce the cleavage of the PO independent of elongation (ie, polymerization). Alternatively, an upstream oligonucleotide can be localized far enough from PO to induce PO cleavage in an elongation-dependent manner.

Used in this description, the term "located in close proximity" in relation to locations or localizations means that located "upstream" of the oligonucleotide is localized near the target-recognizing region of PO with the formation of a nick (single-stranded gap). In addition, this term means that an upstream oligonucleotide is located at a distance of 1-30 nucleotides, 1-20 nucleotides, or 1-15 nucleotides from the target region of the PO.

According to a preferred embodiment, the upstream oligonucleotide is located far enough from the PO to induce the cleavage of the PO in an elongation-dependent manner.

As used herein, the term “distant” with respect to locations or locations is not limited, as is the term “located in close proximity,” including any locations or locations sufficient to permit extension reactions to occur. For example, the term "remote" may include localization with the formation of a nickname.

According to a preferred embodiment, the upstream oligonucleotide is an upstream primer or an upstream probe. The upstream primer is suitable for inducing extension-independent cleavage or for inducing extension-dependent cleavage, and the upstream probe is suitable for inducing extension-independent cleavage.

Alternatively, the upstream oligonucleotide has a sequence partially overlapping with the target recognition region PO. Preferably, the length of the overlapping sequence is 1-10 nucleotides, more preferably 1-5 nucleotides, even more preferably 1-3 nucleotides. In the case where a 5′-tagged PO having a partially overlapping sequence is used, the 3′-terminal target-recognizing region can be partially cleaved together with the cleavage of the tagging region in the cleavage reaction in step (b). In addition, the presence of an overlapping sequence allows the cleavage of the desired site of the target recognition site.

According to a preferred embodiment, the upstream primer induces, through its extended chain, PO cleavage with an enzyme having 5′-nuclease activity.

Conventional techniques for cleavage reactions using upstream oligonucleotides may be applied in the present invention, provided that the upstream oligonucleotide induces cleavage of the PO hybridized with the target nucleic acid sequence. For example, the methods described in US Pat. Nos. 5,221,015, 5,489,972, 5,691,142, 5,994,069, and 7381,532 and US Patent Application Publication No. 2008/0241838 can be applied in the present invention.

According to a preferred embodiment, the method of the present invention is carried out in the presence of a downstream primer. The downstream primer further forms a target nucleic acid sequence that will hybridize with PO, increasing the sensitivity of target detection.

In those cases where an upstream primer is additionally used, in addition a second PO is used, localized downstream relative to the downstream primer.

According to a preferred embodiment, when an upstream primer and a downstream primer are used, matrix nucleic acid polymerase is used to extend these primers. Matrix nucleic acid polymerase can serve as an enzyme with 5′-nuclease activity to cleave PO.

According to a preferred embodiment, the upstream oligonucleotide (upstream primer or upstream probe) and / or downstream primer have a double priming oligonucleotide (DPO) structure developed by the present inventors inventions. Oligonucleotides having a DPO structure exhibit significantly improved target specificity compared to traditional 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)).

According to a preferred embodiment, the target-recognizing region of PO has the structure of a modified double specificity oligonucleotide (mDSO) developed by the present inventors. The structure of the modified double specificity oligonucleotide (mDSO) demonstrates significantly improved target specificity compared to traditional probes (see WO 2011/028041).

The term "traditional oligonucleotide" refers to oligonucleotides (primer or probe) that do not have a DSO or mDSO structure.

The PO used in this invention has a single label. A single label provides a signal indicating the presence or absence of a target nucleic acid sequence. This label will be described in more detail in step (d).

Stage (b). RO cleavage

Further, the product from step (a) is brought into contact with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO.

PO hybridized with the target nucleic acid sequence is digested with an enzyme having 5′-nuclease activity, releasing a single label fragment (see FIGS. 2-6). If the target nucleic acid sequence is absent, the PO is not digested by the enzyme having 5′-nuclease activity.

Used in this description, the term "conditions for the cleavage of PO" means conditions sufficient for the digestion of PO hybridized with the nucleic acid sequence of the target, an enzyme having 5′-nuclease activity, such as temperature, pH, ionic strength, buffer, length and sequence of oligonucleotides and enzymes. For example, when Taq DNA polymerase is used as an enzyme having 5′-nuclease activity, conditions for PO cleavage include Tris-HCl buffer, KCl, MgCl ₂ and temperature.

A large number of conventional technologies can be used to carry out the PO cleavage reaction. PO cleavage sites vary depending on the type of upstream oligonucleotides (upstream probe or upstream primer), hybridization sites upstream oligonucleotides and cleavage conditions (see US Patents No. 5210015, 5487972, 5691142, 5994069 and 7381532 and publication of patent application US No. 2008/0241838).

The length and sequence of a single label containing fragment may vary depending on the cleavage technology selected. In particular, when a tagged PO is used, a fragment thereof containing a partial or full sequence of the tagging portion can be obtained. Due to the adjustment of the localization of a single label on a tagged PO, this single label may not be in a fragment containing a partial or complete sequence of the tagging region.

As a rule, the initial cleavage site of PO after extension of the upstream primer is the starting point of the double chain between the PO and the target nucleic acid sequence or a site 1-3 nucleotides from this starting point. The length of the cleaved PO becomes shorter; therefore, it dissociates from the complex with the target nucleic acid sequence. When a 3′-tagged PO or 5′-tagged PO is used, a fragment can be obtained containing the tagging region and part of the target-recognizing region. When a cleavage reaction is used that is independent of the elongation of the upstream oligonucleotides, the PO cleavage site is determined depending on the location of the upstream oligonucleotides. When a 3′-tagged PO or 5′-tagged PO is used, the resulting PO fragment may contain (1) a portion of the tagging portion, (2) a tagging portion or (3) a tagging portion and a portion of the target-recognizing portion.

As used herein, the term “fragment containing a tagging region or part of a tagging region” with respect to cleavage of a tagged PO with an enzyme having 5′-nuclease activity is used to denote (1) the tagging region, (2) the tagging region and the nearby incomplete target recognition sequence plot and (3) parts of the tagging plot.

The term "part" used in relation to PO, such as a portion of the 5′-terminal tagging region of the PO and the 5′-terminal portion of the 3′-terminal target recognition region of the PO, refers to a nucleotide sequence containing 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 5′-nuclease activity is a DNA polymerase having 5′-nuclease activity, or FEN nuclease, more preferably thermostable DNA polymerase having 5′-nuclease activity, or FEN nuclease.

A suitable DNA polymerase having 5′-nuclease activity in the present invention is a thermostable DNA polymerase obtained from a number of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antranikianii, thermus caldophilus, thermus chliarophilus, thermus igniterrae, thermus lacteus, thermus oshimai, thermus ruber, thermus rubens, thermus scotoductus, thermus silvanus, thermus species z05, thermus species sps 17, thermus thermophilus, thermotoga maritima Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyss /, Pyrodictium occultum, Aquifex pyr ophilus and Aquifex aeolieus. Most preferably, the thermostable DNA polymerase is Taq polymerase

Alternatively, DNA polymerases having 5′-nuclease activity, modified to reduce polymerase activity, can be used in the present invention.

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

Suitable for the present invention, FEN nuclease includes FEN nucleases obtained from a number of bacterial species, including Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis, Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens, Desulfurococococcoccus amcus, Desulfurococococcus amcus Thermococcus zilligii, Methanopyrus kandleri, Methanococcus igneus, Pyrococcus horikoshii, Aeropyrum pernix and Archaeaglobus veneficus.

When the upstream primer is used in step (a), it is preferred that the conditions for PO cleavage include an extension reaction of the upstream primer.

According to a preferred embodiment, an upstream primer is used in step (a), matrix polymerase is used to extend the upstream primer, and this matrix polymerase is identical to an enzyme having 5′-nuclease activity.

It is possible that upstream primer is used in step (a), matrix polymerase is used to extend the upstream primer, and this matrix polymerase is different from an enzyme having 5′-nuclease activity.

Stage (s). Solid phase hybridization

The product from step (b) is contacted with CO (an capture oligonucleotide) immobilized on a solid support to carry out a hybridization reaction.

In the present invention, the detection of PO cleavage is carried out using CO immobilized on a solid support. When the PO is not cleaved in step (b), the single-label non-cleaved PO hybridizes with CO immobilized on a solid support, resulting in a signal from a single mark on the solid support. When the PO is cleaved in step (b), the single label containing the cleaved fragment does not hybridize with CO, resulting in no signal from the single label on the solid support.

Since the possibility of PO cleavage depends on the presence of the target nucleic acid sequence, the presence of this target nucleic acid sequence can be detected by measuring the blanking or attenuation of the signal from a single label on a solid substrate.

CO contains a nucleotide sequence capable of hybridization with PO. The term, as used herein, or “hybridizable sequence” in combination with CO, refers to a sequence capable of forming a stable duplex with uncleaved PO in step (c). For example, a CO sequence capable of hybridization with PO may comprise a sequence complementary to the entire PO region or part of this region recognizing the target; the whole tagging section of the RO or part of this section; the entire site recognizing the target and the part of the tagging site of the RO; parts of the site recognizing the target and the entire tagging site of the RO; or parts of the target-recognizing site and parts of the tagging site of the RO.

No distinction is made between the terms “nucleotide sequence capable of hybridization” and “nucleotide sequence complementary”, and these terms will be used interchangeably.

The hybridization reaction in step (c) is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex.

In one embodiment of the invention, the CO contains a nucleotide sequence capable of hybridization with the target recognition region O. The nucleotide sequence capable of hybridization with the target recognition region PO contains a sequence that is complementary to the whole region of the target recognition region or part of this region, and is complementary and long enough to form a stable duplex between the unsplit PO and CO in the hybridization reaction in step (c).

As illustrated in FIG. 2 and 3, CO contains a nucleotide sequence capable of hybridization with a target recognition region of PO. When there is a target nucleic acid sequence containing a single label, the fragment formed as a result of the cleavage reaction has a shorter length than the unsplit PO, and therefore does not have the ability to hybridize with CO under conditions of a certain stringency (in particular, at a certain temperature). As a result, a single label will not be present on a solid substrate.

Alternatively, CO can be designed so that it does not have a sequence capable of hybridizing with a single label fragment to prevent hybridization of a single label fragment with CO.

Therefore, in the presence of a target nucleic acid sequence, duplex formation between undigested PO and CO does not occur and no signal from a single label is received.

In the absence of a target nucleic acid sequence, uncleaved PO forms a duplex with CO, and a single label will be present on the solid support.

When the processes illustrated in FIG. 2 and 3, carried out using a polymerase having 5′-nuclease activity, untagged PO hybridized with the target nucleic acid sequence is not cleaved. Since non-tagged PO hybridizes with the target nucleic acid sequence, its hybridization with CO can be prevented under carefully verified conditions ensuring the absence of any signal from a single label. However, in the case of using enzymes having 5′-nuclease activity, as in the present invention, the PO hybridized with the target nucleic acid sequence is cleaved and the presence of cleavage is determined under convenient generally accepted conditions to accurately detect the presence of the target nucleic acid sequence.

As illustrated in FIG. 4 and 5, CO contains a nucleotide sequence capable of hybridization with the tagging region of PO. The single label on the tagged PO is positioned so that this single label does not remain on the fragment containing the tagging region, which is released as a result of cleavage of the tagged PO and hybridizes with CO. Even when a fragment containing a tagging region hybridizes with CO, no signal from a single label is received, since a fragment containing a tagging region does not carry a single label. The signal comes from the hybridization of an unsplit tagged PO with CO.

In the case of the presence of a target nucleic acid sequence, the formation of a duplex between unsplit PO and CO does not occur due to PO cleavage leading to damping (or reduction) of the signal from a single label on a solid substrate, which indicates the presence of a target nucleic acid sequence.

According to a preferred embodiment using tagged PO, the nucleotide sequence of CO capable of hybridization with the tagging portion of PO contains a sequence complementary to the entire tagging portion of PO or a portion of this portion, and has complementarity and length sufficient to form a stable duplex between non-cleaved tagged PO and CO in the hybridization reaction in step (c).

Alternatively, when a tagged PO is used, the CO may contain a nucleotide sequence capable of hybridization with part (or all) of the tagging region (s) and part (or all) of the target region (ohm) of the tagged PO . The location of a single label on a tagged PO is determined taking into account the cleavage method, the cleavage site, and the CO sequence, and the tagged PO is cleaved under such conditions that a fragment containing a single tag does not hybridize with CO. Preferably, the single label is positioned so that it remains on the fragment that is released as a result of cleavage of the tagged PO and does not hybridize with CO.

According to a preferred embodiment, using a 5′-tagged PO, the CO may further comprise a matrix region serving as a matrix for lengthening the tagging region hybridized with CO (see FIG. 6). The extension product allows for more stable hybridization with CO. An extension may require additional DNA polymerase.

CO is immobilized on a solid support through its 5′-end or 3′-end.

According to a preferred embodiment, the PO is a 3′-tagged PO, and the CO CO is immobilized on a solid support through its 5′-end (FIG. 4). Preferably, the PO is a 5′-tagged PO, and the CO is immobilized on a solid support through its 3′-end (FIG. 5).

According to a preferred embodiment, the solid support on which CO is immobilized is a microchip. CO is immobilized directly or indirectly (preferably indirectly) through its 5′-end or 3′-end on the surface of a solid substrate. In addition, CO can be immobilized on the surface of a solid substrate in a covalent or non-covalent manner. In cases where the immobilized COs are COs immobilized indirectly on the surface of the solid support, suitable linkers are used. Linkers useful in this invention may include any linkers used to immobilize probes on the surface of a solid substrate. For example, alkyl or aryl compounds with an amine functional group or alkyl or aryl compounds with a thiol functional group serve as linkers for immobilizing CO. In addition, a poly (T) tail or a poly (A) tail can serve as linkers. Preference is given to a poly (T) tail or a poly (A) tail for the reason that it is able to reduce space hindrance under the action of an enzyme (for example, in an enzymatic cleavage reaction) and increase the efficiency of hybridization. A poly (T) tail or a poly (A) tail is not considered a sequence of probes.

A microchip ensuring compliance with the reaction conditions in this invention may include any of the microchips that are known to a person skilled in the art. All processes in the present invention, that is, hybridization with target nucleic acid sequences, cleavage and detection are carried out on a microchip. The CO immobilized on a microchip serve as hybridisable chip elements. A solid substrate for the manufacture of a microchip includes metals (e.g., gold, an alloy of gold and copper, aluminum), metal oxide, glass, ceramics, quartz, silicon, a semiconductor, a Si / SiO ₂ plate, germanium, galium arsenide, carbon, a carbon nanotube, polymers (e.g., polystyrene, polyethylene, polypropylene and polyacrylamide), sepharose agarose and colloids, but are not limited to this. Most of the immobilized COs used in this invention can be immobilized on an accessible site or two or more accessible sites on a solid substrate, which may contain 2-1000000 available sites. In order to obtain a microchip or microchips for a given application, immobilized CO can be manufactured using traditional manufacturing techniques such as photolithography, inkjet technology, mechanical spot spotting, and variants thereof.

According to the present invention, carried out on the solid phase, it is possible to carry out the simultaneous detection of multiple nucleic acid target sequences using labels of even one type, since the labels on immobilized CO are physically separated from each other. In this regard, the number of target nucleic acid sequences to be detected on the solid phase of the present invention is unlimited.

Using confocal devices for detecting on a solid substrate, it is possible to detect only a signal from a solid substrate without any influence of the signal from the labels present in the reaction solution.

The length of CO can vary within wide limits. CO can be of any length, provided that it is sufficient for the formation of a duplex with unsplit PO. For example, the length of CO is 5-80 nucleotides, 5-60 nucleotides, 5-40 nucleotides, 5-30 nucleotides, 10-80 nucleotides, 10-60 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 15-80 nucleotides, 15-60 nucleotides, 15-40 nucleotides, 15-30 nucleotides or 15-20 nucleotides.

According to a preferred embodiment, using a 5′-tagged PO, when the CO further comprises a template region for lengthening the tagging region hybridized with the CO, the CO can also further comprise 5-100 nucleotides in the matrix region.

According to a preferred embodiment, the 3′-end of the CO is blocked to prevent elongation. Elongation-free blocking of CO can be achieved by conventional methods. For example, blocking can be accomplished by adding a chemical moiety, such as biotin, labels, phosphate group, alkyl group, non-nucleotide linker, phosphorothionate or alkanediol, to the 3′-hydroxyl group of the last CO nucleotide. Alternatively, blocking can be accomplished by removing the 3′-hydroxyl group of the last nucleotide or by using a nucleotide lacking a 3′-hydroxyl group, such as a dideoxynucleotide.

Hybridization in step (c) can be described in more detail with reference to the descriptions given in step (a).

The hybridization in step (c) is carried out under such conditions that a single-label fragment does not hybridize with CO, and uncleaved PO hybridizes with CO to form an uncleaved PO / CO duplex. Hybridization conditions can be determined in the prescribed manner by traditional methods known to a person skilled in the art. For example, hybridization conditions can be controlled by changing the temperature, concentration of components, duration of hybridization, components of the buffer and its pH and ionic strength.

According to a preferred embodiment, the hybridization conditions in step (c) are controlled by changing the temperature during the hybridization. Alternatively, the hybridization conditions in step (c), in particular the conditions that allow a single-label fragment to not hybridize with CO, can be achieved by excluding from the CO a sequence capable of stably hybridizing to the single-label fragment.

PO and CO may be composed of natural dNMPs. Alternatively, PO and CO may be composed of modified nucleotides or non-natural nucleotides, such as PNA (peptide-nucleic acid), see PCT publication No. WO 92/20702) and LNA ("closed" nucleic acid), see PCT publication No. WO 98/22489, WO 98/39352 and WO 99/14226). PO and CO may contain universal bases such as deoxyinosine, inosine, 1- (2′-deoxy-beta-O-ribofuranosyl) -3-nitropyrrole and 5-nitroindole. The term "universal base" refers to a base capable of forming base pairs with each of the natural DNA / RNA bases with a slight difference in their ratio.

Stage (d). PO cleavage detection indicating the presence of a target sequence

At the end of the hybridization reaction, the presence of PO cleavage is detected by measuring the signal from a single label on a solid substrate, thereby the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

As discussed above, the patterns of hybridization of PO with CO clearly differ depending on whether or not the cleavage of PO occurs. This difference in the hybridization pattern causes a difference in the signal on the solid substrate. Therefore, the presence or absence of a target nucleic acid sequence can be determined by detecting a signal from a single label on a solid substrate.

Stage (d) is carried out by measuring the signal from a single label associated with PO on a solid substrate.

A single label on a PO can be described as a reporter molecule.

The single label used includes, but is not limited to, chemical labels (e.g., biotin), enzymatic labels (e.g., alkaline phosphatase, β-galactosidase and β-glucosidase peroxidase), fluorescent labels, luminescent labels, chemiluminescent labels, electrochemical labels and metal . Preferably, a single label includes a fluorescent label.

The single label is positioned in such a way that it does not remain on the fragment that is released as a result of PO cleavage and hybridizes with CO.

When an untagged PO is used, a single tag can be attached anywhere. Preferably, a single label is attached to the 5′-terminal or 3′-terminal portion of the untagged PO. More preferably, it is localized at the 5′-end or 3′-end or at a distance of 1-20 nucleotides (even more preferably 1-10 nucleotides) from the 5′-end or 3′-end of untagged PO, even more preferably 5′- end or 3′-end.

According to a preferred embodiment using tagged PO, a single label is positioned so that it does not remain on the fragment containing the tagging region that is released as a result of cleavage of the tagged PO. More preferably, the single label is positioned so that it does not remain on the fragment containing the tagging region, which is released as a result of cleavage of the tagged PO and hybridizes with CO.

The location of a single label can be determined taking into account the methods of cleavage, cleavage sites and the release of cleaved PO from the target nucleic acid sequence.

More preferably, a single label is localized at the 5′-terminal region or 1-20 nucleotides (even more preferably 1-10 nucleotides) from the 5′-end of the 3′-tagged PO, and is located at the 3′-terminal region or at a distance of 1 -20 nucleotides (even more preferably 1-10 nucleotides) from the 3′-end of the 5′-tagged PO. Even more preferably, a single label is localized at the 5′-end of the 3′-tagged PO and localized at the 3′-end of the 5′-tagged PO.

According to a preferred embodiment, the tagged PO has a single label; a single label is located on the target-recognized region of the tagged PO; a single label is positioned so that it does not remain on the fragment containing the tagging region, which is released as a result of PO cleavage in step (b); and the duplex between the fragment containing the tagging region and the CO does not have a single label, and the duplex non-cleaved PO / CO has a single label (see Figs. 4 and 5). Duplex - (a fragment containing a tagging site) / CO immobilized on a solid substrate is not able to provide a signal due to the absence of a single label; however, duplex - unsplit PO / CO provides a signal due to the presence of a single label on the unsplit PO.

As described above, the presence of a target nucleic acid sequence is detected by measuring the signal from a single label on a solid substrate.

According to a preferred embodiment, using a single-labeled PO, the final measured signal is compared to a signal from a negative control lacking a target nucleic acid sequence. Further, to detect the nucleic acid sequence of the target, damping (or reduction) of the signal is determined.

According to a preferred embodiment using a single-label PO, when detection on a solid substrate is carried out continuously along with repetition of the PO cleavage, the number of cleaved POs increases as the number of cleavage reactions repeats, and the signal attenuates simultaneously with an increase in the number of cleaved POs. In this case, the detection of the target nucleic acid sequence can be carried out in real time. In contrast, in the absence of a target nucleic acid sequence, no signal change is observed.

Single fluorescence labels useful in the present invention may include any molecule known in the art. Examples of these are: Su2 ™ (506), YO-PRO ™ -1 (509), YOYO ™ -1 (509), calcein (517), FITC (518), FluorX ™ (519), Alexa ™ (520), Rhodamine 110 (520), Oregon Green ™ 500 (522), Oregon Green ™ 488 (524), RiboGreen ™ (525), Rhodamine Green ™ (527), Rhodamine 123 (529), Magnesium Green ™ (531), Calcium Green ™ (533), TO-PRO ™ -1 (533), TOT01 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568), BODIPY564 / 570 (570), Cy3 ™ (570), Alexa ™ 546 (570), TRITC (572), Magnesium Orange ™ (575), phycoerythrin R&B (575), rhodamine-phalloidin (575), Calcium Orange ™ (576 ), pyronine Y (580), rhodamine B (580), TAMRA (582), Rhodamine Red ™ (590), Cy3.5 ™ (596), ROX (608), Calcium Crimson ™ (615), Alexa ™ 594 ( 615), Texas Red (615), Nile Red (628), YO-PRO ™ -3 (631), YOYO ™ -3 (631), R-Phycocyanin (642), C-Phycocyan in (648), TO-PRO ™ -3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5 ™ (670), thiadicarbocyanine (671) and Cy5.5 (694), HEX (556) ), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 ( 637), FAM (520), fluorescein (520), fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The numbers in brackets represent the wavelength corresponding to the maximum radiation, in nanometers. Preferred single fluorescent labels include JOE, FAM, TAMRA, ROX, and a fluorescein-based label.

A single label can be attached to the PO by a number of methods known to a person skilled in the art. Preferably, a single label can be attached to the PO through a spacer containing at least three carbon atoms (for example, a 3-carbon spacer, a 6-carbon spacer and a 12-carbon spacer).

II. A method for detecting a target using ROSN using a double label

The present invention demonstrates excellent performance when using a system of two interacting labels for the detection of nucleic acid sequences of the target.

In another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO has a system of two interacting labels containing a donor molecule and an acceptor molecule; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;

(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by the enzyme with 5′-nuclease activity, with the separation of the system of two interacting labels, resulting in the formation of a fragment containing a donor molecule and a fragment containing an acceptor molecule;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that at least one of the fragments containing the donor molecule fragment and the fragment containing the acceptor molecule does not hybridize with CO, and unsplit PO is hybridized with CO with the formation of an unsplit PO / CO duplex; wherein the signal from the duplex unsplit PO / CO differs from the signal arriving when at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO; and

(d) detecting the presence of PO cleavage by measuring the signal from a system of two interacting labels on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

Since the second embodiment of the present invention is the same as the first embodiment using a single label, with the exception of the use of a label system, a general description is omitted to avoid unnecessary duplication, which complicates this description.

A system of two interacting labels - donor / acceptor attached to the PO.

The system of two interacting labels is a signal-generating system in which energy is transferred between a donor molecule and an acceptor molecule without the participation of radioactivity. As a representative system of interacting labels, the label system for FRET (resonance fluorescence energy transfer) includes a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). With FRET, the energy donor is fluorescent, and the energy acceptor may be fluorescent or not fluorescent. For another form of interacting label systems, the energy donor is not fluorescent, for example, a chromophore, and the energy acceptor is fluorescent. For another form of interacting label systems, the energy donor is luminescent, for example bioluminescent, chemiluminescent, electrochemiluminescent, and the acceptor is fluorescent.

According to a preferred embodiment, the PO has a system of two interacting tags, more preferably a FRET tag, even more preferably a double tag containing a donor molecule and an acceptor molecule (see Figs. 7-13).

A donor molecule and an acceptor molecule useful in the present invention include any molecules known to one skilled in the art, and examples thereof may be described with reference to the fluorescence labels mentioned above.

Suitable donor-acceptor pairs are described in a number of publications, which are listed below: Pesce et al., Editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2 ^nd 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 RP, Handbook of Fluorescent Probes and Research Chemicals, 6 ^{th Edition (Molecular Probes, Eugene,} Oreg, 1996.); U.S. Patent Nos. 3,996,345 and 4,351,760.

In a signal transmission system comprising a reporter and a damper adapted to the PO, the reporter includes a FRET donor, and the damper includes another FRET donor (acceptor). For example, a fluorescein-based colorant is used as a reporter, and a rhodamine-based colorant is used as a quencher.

The donor (reporter) molecule and the acceptor molecule (quencher) attached to the PO can be fluorescent or not fluorescent. For example, a non-fluorescent dark quencher capable of damping fluorescence over a wider range of wavelengths or at a specific wavelength can be used in the present invention. When the acceptor molecule (quencher) is fluorescent, the signal from the fluorescent acceptor molecule (quencher) can be used to detect the target nucleic acid sequence.

The present invention will be described in more detail below.

Stage (a). Hybridization of the upstream oligonucleotide and PO with the target nucleic acid sequence

Stage (a) of the second embodiment of the present invention can be understood with reference to the description of stage (a) of the first embodiment.

PO has a system of two interacting labels containing a donor molecule and an acceptor molecule, which provides a signal indicating the presence of a target nucleic acid sequence.

Stage (b). RO cleavage

Step (b) of the second embodiment of the present invention can be understood with reference to the description of step (b) of the first embodiment.

The product from step (a) is brought into contact with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO.

PO hybridized with the target nucleic acid sequence is digested with an enzyme having 5′-nuclease activity, with the separation of the system of two interacting labels, resulting in the formation of a fragment containing a donor molecule and a fragment containing an acceptor molecule (see Fig. 7-13).

According to a preferred embodiment, the donor molecule and the acceptor molecule at the PO are separated by a cleavage site for an enzyme having 5′-nuclease activity.

In the absence of the target nucleic acid sequence, the PO is not digested by the enzyme possessing 5′-nuclease activity, and therefore there is no separation of the system of two interacting labels.

Stage (s). Solid phase hybridization

The product from step (b) is contacted with CO (an capture oligonucleotide) immobilized on a solid support to carry out a hybridization reaction.

Step (c) of the second embodiment of the present invention can be understood with reference to the description of step (c) of the first embodiment. The hybridization reaction is carried out under such conditions that at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO, and unsplit PO hybridizes with CO to form an unsplit PO / CO duplex. Hybridization conditions can be determined in the prescribed manner by traditional methods known to a person skilled in the art. For example, hybridization conditions can be controlled by changing the temperature, concentration of components, duration of hybridization, components of the buffer and its pH and ionic strength.

According to a preferred embodiment, such hybridization conditions that at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO are controlled by changing the temperature during hybridization. Alternatively, the hybridization conditions in step (c) can be achieved by excluding from the CO a sequence capable of stably hybridizing with the label containing fragment.

When a target nucleic acid sequence is present, at least one of the fragments containing the donor molecule fragment and the fragment containing the acceptor molecule does not hybridize to CO. In the absence of a target nucleic acid sequence, an unsplit PO having a system of two interacting labels hybridizes with CO.

The signal arriving from an uncleaved PO / CO duplex (that is, in the absence of a target nucleic acid sequence) differs markedly from a signal arriving when at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO (i.e., in the presence of a target nucleic acid sequence). Therefore, the signal from the system of two interacting labels is generated differently depending on the presence or absence of the target nucleic acid sequence.

Stage (d). Detection of PO cleavage indicating the presence of a target sequence

At the end of the hybridization reaction, the presence of PO cleavage is detected by measuring the signal from the system of two interacting labels on a solid substrate, thereby the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

As discussed above, the patterns of hybridization of PO with CO clearly differ depending on whether or not the cleavage of PO occurs. This difference in the hybridization pattern causes a difference in the signal on the solid substrate. Therefore, the presence or absence of a target nucleic acid sequence can be determined by detecting a signal from a system of two interacting labels on a solid substrate.

In the case of using a system of two interacting labels, the signal is measured in two ways. The first method consists in measuring the signal generated by the acceptor molecule, and the second method consists in measuring the signal generated by the donor molecule.

The first measurement method is illustrated in FIG. 7-9.

In FIG. 7 depicts an embodiment using untagged PO to measure a signal from an acceptor molecule. According to a preferred embodiment, the nucleotide sequence in CO capable of hybridization with PO comprises a nucleotide sequence capable of hybridization with a target recognition region of PO. In this case, the system of two interacting labels is preferably localized in such a way that the signal from the donor molecule is quenched by the acceptor molecule during the formation of the unsplit PO / CO duplex, and during stage (d), the signal from the acceptor molecule is detected.

As shown in FIG. 7, when the donor molecule and the acceptor molecule are located in close proximity to each other on an untagged PO so that between the donor molecule and the acceptor molecule (for example, located in close proximity, which contributes to the occurrence of the FRET phenomenon), energy transfer occurs, untagged PO, hybridized with the target nucleic acid sequence, cleaves to form a fragment containing a donor molecule, and a fragment containing an acceptor molecule. Therefore, there is no interaction between the donor molecule and the acceptor molecule until both fragments, both the fragment containing the donor molecule and the fragment containing the acceptor molecule are hybridized with CO in stage (c), as a result, no signal from the solid substrate comes .

Used in this description, the expression "donor molecule and the acceptor molecule are located in close proximity" means that the donor molecule and the acceptor molecule are separated by a certain number of nucleotides in the probe so that energy transfer occurs between the donor molecule and the acceptor molecule. Preferably, the donor molecule and the acceptor molecule are separated by 1-20, 1-15 and 1-10 nucleotides.

In the case when the target nucleic acid sequence is absent, the unsplit untagged PO hybridizes with CO, and the interaction between the donor molecule and the acceptor molecule is carried out, thereby providing a signal from the acceptor molecule. Irradiation with a wavelength of the excitation wave for the donor molecule causes the fluorescence of the donor molecule, which is then quenched by the acceptor molecule, which ultimately provides a fluorescent signal from the acceptor molecule.

In the presence of a target nucleic acid sequence, the formation of a duplex of undigested PO / CO does not occur due to the cleavage of PO, and therefore, no signal is received from the acceptor molecule. Measurement of the damping (or decrease) of the signal from the acceptor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

In FIG. 8 and 9 show embodiments using tagged POs to measure the signal from an acceptor molecule. According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising in its 5′-terminal portion a tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence, and the nucleotide sequence in CO capable of hybridization with PO, soda rzhit nucleotide sequence capable of hybridizing to the tagged portion RO. The system of two interacting labels is preferably localized in such a way that the signal from the donor molecule is quenched by the acceptor molecule during the formation of the unsplit PO / CO duplex, while during stage (d) the signal from the acceptor molecule is detected.

As illustrated in FIG. 8 using a 3′-tagged PO, when the donor molecule and the acceptor molecule are conformationally located in close proximity to each other so that energy transfer occurs between the donor molecule and the acceptor molecule, 3′-tagged PO hybridized with the target nucleic acid sequence, cleaved to form a fragment containing a donor molecule, and a fragment containing an acceptor molecule. The fragment containing the acceptor molecule containing the tagging region is hybridized with CO containing the nucleotide sequence capable of hybridization with the tagging region of the 3′-tagged PO, but the fragment containing the donor molecule is not involved in hybridization with CO, thus, the signal from the acceptor molecule not coming.

Used in this description, the expression "donor molecule and the acceptor molecule are conformationally located in close proximity" means that the donor molecule and the acceptor molecule are spatially close to each other due to the presence of a conformational structure in part of the PO or PO, such as a random helix or hairpin structure.

In the case when the target nucleic acid sequence is absent, the unsplit 3′-tagged PO hybridizes with CO and the interaction between the donor molecule and the acceptor molecule is carried out, thereby obtaining a signal from the acceptor molecule.

In the presence of a target nucleic acid sequence, the formation of a duplex, undigested PO / CO does not occur due to the cleavage of the 3′-tagged PO, and therefore no signal is received from the acceptor molecule. Measurement of the damping (or decrease) of the signal from the acceptor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

According to a preferred embodiment, the donor molecule and the acceptor molecule on a 3′-tagged PO are located in close proximity to each other so that energy transfer occurs between them. The donor molecule and the acceptor molecule are localized in a 3′-tagged PO in the direction 5 ′ → 3 ′ or in the direction 3 ′ → 5 ′.

According to a preferred embodiment, both the donor molecule and the acceptor molecule are located on the target-recognizing region of the 3′-tagged PO, or one of them is located on the target-recognizing region, and the other is located on the tagging region of the 3′-tagged PO.

As illustrated in FIG. 9 using a 5′-tagged PO, when the donor molecule and the acceptor molecule are conformationally located in close proximity to each other so that energy transfer occurs between the donor molecule and the acceptor molecule, 5′-tagged PO hybridized with the target nucleic acid sequence, cleaved to form a fragment containing a donor molecule, and a fragment containing an acceptor molecule. A fragment containing a donor molecule containing a tagging region is hybridized with CO containing a nucleotide sequence capable of hybridizing with a tagging region of a 5′-tagged PO, but a fragment containing an acceptor molecule is not involved in hybridization with CO, thus, a signal from an acceptor molecule not coming.

In the case when the target nucleic acid sequence is absent, the unsplit 5′-tagged PO hybridizes with CO and the interaction between the donor molecule and the acceptor molecule is carried out, whereby a signal is received from the acceptor molecule.

In the presence of a target nucleic acid sequence, the formation of a duplex, undigested PO / CO does not occur due to the cleavage of the 5′-tagged PO, and therefore no signal is received from the acceptor molecule. Measurement of the damping (or decrease) of the signal from the acceptor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

According to a preferred embodiment, the donor molecule and the acceptor molecule on the 5′-tagged PO are located in close proximity to each other so that energy transfer occurs between them. The donor molecule and the acceptor molecule are localized in a 5′-tagged PO in the direction 5 ′ → 3 ′ or in the direction 3 ′ → 5 ′.

According to a preferred embodiment, both the donor molecule and the acceptor molecule are located on the target recognition region of the 5′-tagged PO, or one of them is located on the target-recognizing region, and the other is located on the tagging region of the 5′-tagged PO.

A second measurement method is illustrated in FIG. 10-12.

In FIG. 10 shows an embodiment using untagged PO to measure the signal from a donor molecule. According to a preferred embodiment, the nucleotide sequence in CO capable of hybridization with PO comprises a nucleotide sequence capable of hybridization with a target recognition region of PO. The hybridization reaction in step (c) is carried out under such conditions that the fragment containing the donor molecule does not hybridize with CO; in this case, the system of two interacting labels is localized in such a way that the signal from the donor molecule is not suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex, and during stage (d), the signal from the acceptor molecule is detected.

As shown in FIG. 10, when the donor molecule and the acceptor molecule are conformationally located in close proximity to each other on the untagged PO so that energy transfer occurs between the donor molecule and the acceptor molecule, the untagged PO hybridized with the target nucleic acid sequence is split to form a fragment containing the donor a molecule and a fragment containing an acceptor molecule. Hybridization is carried out in step (c) under conditions such that a fragment containing a donor th molecule does not hybridize with CO. Therefore, there is no signal from the donor molecule on the solid support.

In the case where the target nucleic acid sequence is absent, the unsplit untagged PO hybridizes with CO, separating the donor molecule and the acceptor molecule, which prevents the interaction between the donor molecule and the acceptor molecule. Irradiation with a wavelength of excitation for a donor molecule (e.g., a reporter molecule) causes the fluorescence of the donor molecule, which is not quenched by the acceptor molecule, which ultimately provides a fluorescence signal from the donor molecule.

In the presence of a target nucleic acid sequence of the formation of a duplex, undigested PO / CO does not occur due to PO cleavage and because the fragment containing the donor molecule does not hybridize with CO, therefore, there is no signal from the donor molecule. Measurement of the damping (or decrease) of the signal from the donor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

According to a preferred embodiment, the donor molecule and the acceptor molecule are located in the untagged PO in the direction 5 ′ → 3 ′ or in the direction 3 ′ → 5 ′, more preferably in the direction 5 ′ → 3 ′.

In FIG. 11 and 12, using tagged POs, embodiments are shown for measuring a signal from a donor molecule. According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising in its 5′-terminal portion a tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence, and the nucleotide sequence in CO capable of hybridization with PO, soda rzhit nucleotide sequence capable of hybridizing to the tagged portion RO. The fragment containing the donor molecule contains a tagging region capable of hybridization with CO, and in the hybridization reaction in step (c), the fragment containing the donor molecule hybridizes with CO. The system of two interacting labels is preferably localized in such a way that the signal from the donor molecule is quenched by the acceptor molecule during the formation of an unsplit PO / CO duplex, while during stage (a) the signal from the donor molecule is detected.

As illustrated in FIG. 11 using a 3′-tagged PO, when the donor molecule and the acceptor molecule are conformationally located in close proximity to each other so that energy transfer occurs between the donor molecule and the acceptor molecule, 3′-tagged PO hybridized with the target nucleic acid sequence, cleaves to form a fragment containing a donor molecule and a fragment containing an acceptor molecule A fragment containing a donor molecule containing a tagging site, hybridization is a C SB containing a nucleotide sequence capable of hybridizing to the 3'-tagged site tagged PO, but a fragment containing the acceptor molecule not involved in hybridization with CO that thus provides a signal from the donor molecule.

In the case where the target nucleic acid sequence is absent, the unsplit 3′-tagged PO hybridizes with CO and the interaction between the donor molecule and the acceptor molecule occurs, which leads to the transfer of energy from the donor molecule to the acceptor molecule, thereby not causing any fluorescence from donor molecule.

In the presence of the target nucleic acid sequence, the 3′-tagged PO is cleaved and the fragment containing the donor molecule containing the tagging region is hybridized with CO, providing a signal from the donor molecule. Measurement of the generation (or increase) of the signal from the donor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

According to a preferred embodiment, the donor molecule and the acceptor molecule on a 3′-tagged PO are located in close proximity to each other so that energy transfer occurs between them. The acceptor molecule and the donor molecule are localized in a 3′-tagged PO in the direction 5 ′ → 3 ′.

According to a preferred embodiment, both the acceptor molecule and the donor molecule are localized in the target recognition region of the 3′-tagged PO, or the acceptor molecule is localized in the target-recognition region, and the donor molecule is localized in the tagging region of the 3′-tagged PO.

As illustrated in FIG. 12 using a 5′-tagged PO, when the donor molecule and the acceptor molecule are conformationally located in close proximity to each other so that energy transfer occurs between the donor molecule and the acceptor molecule, 5′-tagged PO hybridized with the target nucleic acid sequence, cleaves to form an acceptor molecule-containing fragment and a donor-containing fragment containing a tagging site. A fragment containing a donor molecule containing a tagging region is hybridized with CO containing a nucleotide sequence capable of hybridizing with a tagging region of a 5′-tagged PO, but a fragment containing an acceptor molecule is not involved in hybridization with CO, which thus provides a signal from the donor a molecule indicating the presence of a target nucleic acid sequence. In the case when the target nucleic acid sequence is absent, the unsplit 5′-tagged PO hybridizes with CO and the interaction between the donor molecule is an acceptor molecule, which leads to the transfer of energy of the donor molecule to the acceptor molecule, thereby not causing any fluorescence from the donor molecule .

In the presence of the target nucleic acid sequence, 5′-tagged PO is cleaved, and the fragment containing the donor molecule containing the tagging region hybridizes with CO, providing a signal from the donor molecule. Measurement of the generation (or increase) of the signal from the donor molecule on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence.

According to a preferred embodiment, the donor molecule and the acceptor molecule on the 5′-tagged PO are located in close proximity to each other so that energy transfer occurs between them. The donor molecule and the acceptor molecule are localized in a 5′-tagged PO in the direction 5 ′ → 3 ′.

According to a preferred embodiment, both the donor molecule and the acceptor molecule are localized in the target recognition region of the 5′-tagged PO, or the acceptor molecule is localized in the target recognition region, and the donor molecule is localized in the tagging region of the 5-tagged PO.

In an alternative use of tagged PO, CO contains a nucleotide sequence capable of hybridization with part (or all) of the tagging (s) region (ohm) and part (or all) of the target region (ohms) of the tagged PO that recognizes (them). The location of the system of two interacting labels on the PO is determined taking into account the cleavage method and the cleavage site, as well as the sequence of CO.

III. Method for target detection by ROSN using intercalating agent

The present invention demonstrates excellent performance when using an intercalating agent to detect a target nucleic acid sequence.

In another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;

(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by an enzyme having 5′-nuclease activity to produce a cleaved fragment;

(c) carrying out a hybridization reaction in the presence of an intercalating agent by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the cleaved fragment does not hybridize with CO, and the unsplit PO hybridizes with CO with the formation of an unsplit PO / CO duplex; and (a) detecting the presence of PO cleavage by measuring the signal from the intercalating agent on a solid support; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

Since the third embodiment of the present invention is the same as the first embodiment using a single label, except for the use of a label system, a general description is omitted to avoid unnecessary duplication, which complicates the description.

According to a preferred embodiment, the hybridization reaction is carried out under such conditions that the cleaved fragment resulting from the PO cleavage does not hybridize with CO.

Examples of intercalating dyes useful in this invention include SYBR ™ Green I, PO-PRO ™ -1, BO-PRO ™ -1, SYTO ™ 43, SYTO ™ 44, SYTO ™ 45, SYTOX ™ Blue, POPO ™ -1, POPO ™ -3, BOBO ™ -1, BOBO ™ -3, LO-PRO ™ -1, JO-PRO ™ -1, YO-PRO ™ 1, TO-PRO ™ 1, SYTO ™ 11, SYTO ™ 13, SYTO ™ 15, SYTO ™ 16, SYTO ™ 20, SYTO ™ 23, TOTO ™ -3, YOYO ™ 3, GelStar ™ and triazole orange. These intercalating dyes specifically intercalate into double-stranded nucleic acid molecules, generating signals.

When the target nucleic acid sequence is present, duplexing of unsplit PO / CO does not occur due to PO cleavage, and therefore no signal from the intercalating agent is received. Measurement of the damping (or decrease) of the signal from the intercalating agent on a solid substrate makes it possible to determine the presence of the target nucleic acid sequence (see Fig. 13).

According to a preferred embodiment, the method further comprises repeating steps (a) - (b), (a) - (c) or (a) - (d) with denaturation between repeating cycles. This repetition allows amplification of the target nucleic acid sequence and / or signal from the target.

According to a preferred embodiment, steps (a) to (d) are carried out in a reaction vessel or in separate reaction vessels. More preferably, steps (a) - (b) and steps (c) - (d) are carried out in a reaction vessel or in separate reaction vessels.

Preferably, steps (a) - (b) and steps (c) - (d) can be carried out separately even in one reaction vessel. For example, when hybridization between the target region of the PO and the nucleic acid sequence of the target is carried out under conditions of higher stringency than the conditions for hybridization between the fragment of PO and CO, it is possible to repeat steps (a) - (b) without proceeding to stages ( c) - (d). Upon completion of the repetition of steps (a) to (b), steps (c) to (d) can be performed sequentially.

In the event that steps (a) - (b) and steps (c) - (d) can be carried out separately, steps (a) - (b) are performed repeatedly with denaturation between repeated cycles.

When the present invention using the upstream primer is carried out by repeating the steps with denaturation between repeating cycles, it is preferable to apply this method in the presence of an upstream primer. Most preferably, the method of the present invention is carried out in accordance with PCR (polymerase chain reaction).

When the present invention using the upstream primer is carried out by repeating the steps with denaturation between repeating cycles, it is preferable to apply this method in the presence of an upstream primer.

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

If mRNA is used as the starting material, a reverse transcription step is necessary before the annealing step, details of which can be 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., 16: 10366 (1988)). For reverse transcription, a random hexamer or dT oligonucleotide can be used as a primer capable of hybridizing with mRNA.

Target nucleic acid sequences that can be detected and / or amplified include any prokaryotic, eukaryotic (e.g., from protozoa and parasites, fungi, yeast, higher plants, lower and higher animals, including mammals and humans), viral (e.g. , from herpes virus, HIV, influenza virus, Epstein-Barr virus, hepatitis virus, poliovirus, etc.) or naturally occurring viroid nucleic acid.

The target nucleic acid sequence to be detected according to the present invention includes a wide variety of nucleic acid sequences, for example, sequences in the genome, artificially isolated or fragmented sequences, and synthesized sequences (for example, cDNA sequences and barcode sequences). For example, the target nucleic acid sequence includes nucleic acid marker sequences for immuno-PCR (iPCR). B and PCR use conjugates formed between nucleic acid marker sequences and antibodies, together with PCR, which is widely used to detect various types of targets, including proteins (see Sano et al., Science, 258, pp. 120-122 (1992), U.S. Patent No. 5,665,539, Niemeyer et al., Trends in Biotechnology, 23. pp. 208-216 (2005), U.S. Patent Application Publication No. 2005/0239108 and Ye et al., Journal of Environmental Science, 22, pp. 796 -800 (2010)).

The term "nucleotide variation", as used herein, refers to any single or multiple nucleotide substitutions, deletions or insertions in a DNA sequence at a specific position among continuous segments of DNA that are otherwise identical in sequence.

Such continuous DNA segments include a gene or any other part of the chromosome. Such nucleotide variations may be the result of a mutation or represent variations of polymorphic alleles. For example, the nucleotide variation detected in the present invention includes SNP (single nucleotide polymorphism), mutation, deletion, insertion substitution, and translocation. Nucleotide variations are exemplified by numerous variations in the human genome (e.g., variations in the MTHFR (methylenetetrahydrofolate reductase) gene), variations involved in the emergence of drug resistance in pathogenic microorganisms, and leading to oncogenesis of the variation. The term “nucleotide variation” as used herein includes any variation at a particular position in a DNA molecule. In other words, the term “nucleotide variation” includes the wild type and any mutant type thereof at a particular position in the DNA molecule.

In the present invention, for detecting a nucleotide variation in a target nucleic acid sequence when the primers or probes used have a sequence complementary to that nucleotide variation in a given target nucleic acid sequence, a target nucleic acid sequence containing such a nucleotide variation is described in this invention as comparable matrix (matching or match template). In the case where the primers or probes used have a sequence incompatible with this nucleotide variation in a given target nucleic acid sequence, a target nucleic acid sequence containing such a nucleotide variation is described in this invention as an incompatible matrix (mismatching or mismatch template).

To detect nucleotide variations, the 3′-end of the upstream primer can be designed to be located opposite the site of the nucleotide variation in the target nucleic acid sequence. According to a preferred embodiment, the 3′-end of the upstream primer has a sequence complementary to the nucleotide variation in the target nucleic acid sequence. The 3 ′ end of the upstream primer having a sequence complementary to a given nucleotide variation in this target nucleic acid sequence is annealed with a comparable matrix and extended to induce PO cleavage. In contrast, when the 3′-end of the upstream primer does not correspond to the nucleotide variation in the disparate matrix, it does not lengthen under conditions necessary for annealing the 3′-end of the primers and are essential for elongation, even when upstream downstream, the primer hybridizes with an incomparable matrix; therefore, no PO cleavage occurs.

Alternatively, PO cleavage can be used depending on the hybridization of the PO having a sequence complementary to the nucleotide variation in the target nucleic acid sequence. For example, under controlled conditions, a PO having a sequence complementary to this nucleotide variation in a given target nucleic acid sequence will hybridize to a comparable template and then cleave. At the same time, under these controlled conditions, PO does not hybridize with an incompatible matrix having an incomplete sequence at the location of the nucleotide variation, and does not split. In this case, it is preferable that the sequence complementary to the sequence of a given nucleotide variation in the PO is located in the middle of its 3′-terminal target recognition region PO.

Alternatively, the present invention uses a PO having a nucleotide variation recognition site located at the 5′-terminal portion of the 3′-terminal target recognition region for selectively recognizing the PO of a particular nucleotide variation. The 5′-terminal portion of the 3′-terminal target-recognizing region of the PO is oriented with respect to the nucleotide variation in the target nucleic acid sequence to detect this nucleotide variation, and the 5′-terminal portion of the 3′-terminal target-recognizing region of PO has a sequence complementary to the sequence of this nucleotide variation in the target nucleic acid sequence.

As used herein, the term “nucleotide variation recognition site” with respect to PO means a site (1) containing a sequence complementary to the sequence of the nucleotide variation in the target nucleic acid, and (2) located at the 5′-terminal portion of the 3′-terminal recognition plot target.

When a PO hybridizes with a target nucleic acid sequence having a nucleotide variation, the sequence of which is complementary to the recognition site sequence of a given nucleotide variation (i.e., with a comparable matrix), the 5′-terminal portion of the 3′-terminal target recognition region forms a double strand with a comparable matrix ; however, when a PO hybridizes with a target nucleic acid sequence having a nucleotide variation, the sequence of which is not complementary to the recognition site sequence of a given nucleotide variation (i.e., with a disparate matrix), the 5′-terminal portion of the 3′-terminal target recognition region does not form a double disparate matrix chains.

It should be noted that such different hybridization patterns in the case of the nucleotide variation of interest are responsible for differences in the initial PO cleavage sites.

The 5′-nuclease used in the present invention is an enzyme capable of cleaving a single strand of a double-stranded nucleic acid molecule in the direction of 5 ′ → 3 ′ exonucleolytically or endonucleolytically. As a rule, the PO cleavage site under the action of 5′-nuclease may vary depending on the hybridization of the 5′-terminal part of the PO.

When a PO hybridizes with a target nucleic acid sequence having a nucleotide variation, the sequence of which is complementary to the sequence of the recognition site of this variation (i.e., with a comparable matrix), the 5′-terminal portion of the 3′-terminal target recognition site forms a double chain with a nucleic acid sequence - target, inducing cleavage at the first initial cleavage site.

When a PO hybridizes with a target nucleic acid sequence having a nucleotide variation, the sequence of which is not complementary to the sequence of the recognition site of this variation (i.e., with a disparate matrix), the 5′-terminal portion of the 3′-terminal target recognition region does not form a double chain with a nucleic acid the target sequence, inducing cleavage in the second initial cleavage site, localized "downstream" relative to the first initial cleavage site.

It should be noted that the second initial cleavage site is located "downstream" relative to the first initial cleavage site.

As used herein, the term “first initial cleavage site” with respect to PO means a PO cleavage site that is cleaved first when the PO is hybridized to a target nucleic acid sequence having a nucleotide variation, the sequence of which is complementary to the recognition site sequence of this variation. As used herein, the term “second initial cleavage site” with respect to PO means a PO cleavage site that is cleaved first when the PO is hybridized to a target nucleic acid sequence having a nucleotide variation, the sequence of which is not complementary to the recognition site sequence of this variation.

According to a preferred embodiment, the nucleotide variation recognition site is located at a distance of 10 nucleotides, more preferably 8 nucleotides, even more preferably 6 nucleotides, even more preferably 4 nucleotides, 3 nucleotides, 2 nucleotides or 1 nucleotide from the 5′-end of the 3′-terminal target recognition site RO. Preferably, the nucleotide variation recognition site is located at the 5 ′ end of the 3 ′ terminal target recognition region PO.

The term "site" with reference either to the site of recognition of nucleotide variation in probes, or to the site of nucleotide variation in target sequences, is used in this description, covering not only one nucleotide, but also a certain number of nucleotides.

A PO having a nucleotide variation recognition site located in the 5′-terminal portion of the 3′-terminal target-recognizing region can be used to detect nucleotide variation together with a blocker resistant to cleavage by an enzyme having 5′-nuclease activity.

For example, a PO has a blocking region containing at least one nucleotide as a blocker that is resistant to digestion with an enzyme having 5′-nuclease activity, and this blocking region is located at the site to be initially cleaved after hybridization of the PO with an incompatible matrix. When a PO having a blocking region hybridizes to a comparable matrix, this affects the cleavage starting from the first initial cleavage site. However, when a PO having a blocking region hybridizes with a disparate matrix, the blocking region prevents cleavage starting from the second starting cleavage site.

The number of blockers contained in the blocking portion may be unlimited, preferably 1-10, more preferably 2-10, even more preferably 3-8, most preferably 3-6 blockers. Blockers present in the probes may be located continuously or at intervals, preferably continuously. Nucleotides serving as blockers having a backbone resistant to 5 ′ → 3′-exonuclease activity include any nucleotide known to one skilled in the art. For example, they contain various phosphorothioate bonds, phosphonate bonds, phosphoamidate bonds and modifications at the 2′-position of carbohydrates. According to a more preferred embodiment, nucleotides having a 5 ′ → 3′-exonuclease-resistant backbone contain a phosphorothioate bond, an alkylphosphotriether bond, an arylphosphotriether linkage, an alkylphosphonate linkage, a hydrophosphonate linkage, an alkylphosphoric sulfate-phosphorodamide -O-aminopropyl, 2′-O-alkyl modification, 2′-O-allyl modification, 2′-O-butyl modification, α-anomeric oligodeoxynucleotide and 1- (4′-thio-β-O-ribofuranosyl) modification.

It is possible that the detection of nucleotide variations can be carried out by appropriate selection of the label localization, taking into account the localization of the first initial cleavage site and the second initial cleavage site without any use of blockers.

When a single label is used, suitable locations for the single label make it possible for the following: a single label containing fragment resulting from the cleavage of PO hybridized with a comparable matrix does not hybridize with CO, and a single label containing fragment generated from the PO cleavage hybridized with disparate matrix, hybridizes with CO. Hybridization between CO and a single-label fragment formed as a result of the cleavage of PO hybridized with an incompatible matrix provides a signal identical to the signal obtained as a result of hybridization between unsplit PO and CO, thereby allowing detection of nucleotide variations.

According to a more preferred embodiment, a single label attached to the PO is located on the nucleotide between the first starting cleavage site and the second starting cleavage site.

In the case where a system of two interacting tags is used, suitable locations for such a double tag make it possible for the following: (1) after splitting the PO hybridized with a comparable matrix, the tags in this double tag are separated from each other, and at least one of fragments containing a donor molecule fragment and containing an acceptor molecule fragment does not hybridize with CO; (2) after cleavage of the PO hybridized with a disparate matrix, the labels in this double label do not separate from each other, and the fragment containing the double label hybridizes with CO. Hybridization between CO and the double-labeled fragment generated by the cleavage of the PO hybridized with an incompatible matrix provides a signal identical to the signal obtained by hybridization between the uncleaved PO and CO, thereby allowing the detection of nucleotide variations.

According to a more preferred embodiment, when a double-labeled PO is used, one of the labels in this double-label is attached to the nucleotide located upstream of the first initial cleavage site, and the other to the nucleotide located between the first initial cleavage site and the second initial cleavage site.

For example, when using a 5′-tagged PO having a nucleotide variation recognition site located in the 5′-terminal portion of the 3′-terminal target recognition region, together with the CO to be hybridized with the 5′-tagging region, suitable label localization allows detection nucleotide variations without any use of blockers.

When using a 5′-tagged PO with a single label, this single tag attached to the 5′-tagged PO is preferably located on the nucleotide between the first starting cleavage site and the second starting cleavage site.

In FIG. 21 shows embodiments for detecting nucleotide variation using a 5'-tagged single-label PO. The nucleotide variation recognition site is located at a distance of 1 nucleotide from the 5′-end of the 5′-terminal portion of the 3′-terminal target-recognizing region. A single label is attached to the nucleotide located between the first starting cleavage site and the second starting cleavage site.

When a 5′-tagged PO containing a single label hybridizes with a comparable matrix, it is cleaved at the first initial cleavage site, and a fragment containing a 5′-tagged region without a single label is formed. Since CO contains a nucleotide sequence capable of hybridization with the tagging region of PO, a single-label fragment generated by cleavage of PO hybridized with a comparable matrix does not hybridize with CO. When a 5′-tagged PO containing a single label hybridizes with a disparate matrix, it splits at the second initial cleavage site, and a single-label fragment containing a 5′-tagged region is formed. Since CO contains a nucleotide sequence capable of hybridization with the tagging region of PO, a fragment containing a single label formed by cleavage of PO hybridized with an incompatible matrix hybridizes with CO. Hybridization between CO and a single-label fragment formed by the cleavage of PO hybridized with an incompatible matrix provides a signal identical to the signal obtained by hybridization between uncleaved PO and CO.

Therefore, depending on the presence of nucleotide variations, various signals can be obtained.

When a 5-tagged double-labeled PO is used, it is preferable that one of the tags in this double-tag be attached to a nucleotide located upstream of the first starting cleavage site (for example, at the 5'-end of the 5'-tagging region PO), and the other to the nucleotide located between the first initial cleavage site and the second initial cleavage site.

When a 5′-tagged PO containing a double label hybridizes with a comparable matrix, it cleaves at the first initial cleavage site, and the labels in this double label are separated from each other to form a fragment containing a donor molecule and a fragment containing an acceptor molecule. One of these fragments contains a 5′-tagging region, and it hybridizes with CO containing a nucleotide sequence capable of hybridization with this tagging region of a 5′-tagged PO. When a 5′-tagged PO containing a double label hybridizes with a disparate matrix, it cleaves at the second initial cleavage site to form a fragment containing a 5′-tagged region with a double label. The double-labeled fragment containing the 5′-tagged region hybridizes with CO, and the hybridization product provides a signal identical to the signal obtained as a result of hybridization between the uncleaved 5′-tagged PO and CO.

Therefore, depending on the presence of nucleotide variations, various signals can be obtained.

According to a preferred embodiment, it is desirable that the 5′-terminal portion of the 3′-terminal target recognition region PO contains a moiety not suitable for base pairing located at a distance of 1-10 nucleotides (more preferably 1-5 nucleotides) from the nucleotide variation recognition site.

In FIG. 22 shows embodiments for detecting nucleotide variation using a moiety not suitable for base pairing.

A group not suitable for base pairing does not allow the 5′-terminal portion of the 3′-terminal target recognition site to double chain with the target nucleotide sequence when the PO hybridizes with the target nucleic acid sequence having a nucleotide variation whose sequence is not complementary to the site sequence recognition of this variation.

The use of a group that is not suitable for base pairing (for example, a non-complementary nucleotide) enhances the recognition of PO nucleotide variations.

According to a preferred embodiment, a moiety not suitable for base pairing does not inhibit the formation of a double chain between the 5′-terminal portion and the target nucleic acid sequence when the PO hybridizes with the target nucleic acid sequence having a nucleotide variation, the sequence of which is complementary to the recognition site sequence of this nucleotide variation .

According to a preferred embodiment, a moiety unsuitable for base pairing increases the distance between the first starting cleavage site on the hybrid structure formed by the PO and the comparable matrix, and the second starting cleavage site on the hybrid structure formed by the PO and the incompatible matrix.

A group that is not suitable for base pairing includes any groupings that do not form base pairs between nucleic acid target sequences. Preferably, the moiety not suitable for base pairing is (1) a nucleotide containing an artificial non-complementary base, a base not suitable for base pairing (non-base pairing base), modified so that it is not capable of forming a base pair, or universal a base, (2) a nucleotide that is not suitable for base pairing (non-base pairing nucleotide), modified so that it is not able to form a base pair, or (3) a chemical compound that is not suitable for pairing dreams.

For example, a moiety unsuitable for base pairing contains an alkylene group, ribofuranosyl-naphthalene, deoxyribofuranosyl-naphthalene, metaphosphate, phosphorothioate bond, alkylphosphotriether bond, arylphosphotriether bond, arylphosphonate, phosphorylphosphate, arylphosphate-phosphate, arylphosphate-phosphate. Conventional carbon spacers are also often used as moieties not suitable for base pairing. Universal bases as moieties not suitable for base pairing are useful for regulating PO cleavage sites.

A group not suitable for base pairing introduced in the 5'-terminal part preferably contains 1-10, more preferably 1-5, even more preferably 1-2 groups. A number of moieties not suitable for base pairing can be located continuously or at intervals in the 5′-terminal portion. Preferably, the moiety not suitable for base pairing contains 2-5 successive moieties.

Preferably, the moiety not suitable for base pairing is a chemical compound not suitable for base pairing.

According to a preferred embodiment, the nucleotide variation recognition site and the moiety not suitable for base pairing are located in the PO at a distance of 10 nucleotides (more preferably 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides or 1 nucleotide, even more preferably 1 nucleotide) from the 5′-end of the 3′-terminal target recognition region.

According to a preferred embodiment, the moiety not suitable for base pairing is localized “downstream” with respect to the first initial cleavage site.

According to a preferred embodiment, the target nucleic acid sequence used in the present invention is a nucleic acid sequence previously amplified using an amplification primer.

The advantages of the present invention can be particularly noted in the simultaneous (multiple) detection of at least two target nucleic acid sequences. According to a preferred embodiment, the method is carried out to detect at least two types (more preferably at least three types, even more preferably at least five types) of target nucleic acid sequences. The upstream oligonucleotide contains at least two types (more preferably at least three types, even more preferably at least five types) of oligonucleotides, PO contains at least two types (more preferably at least three types, still more preferably at least five types) of PO and CO contains at least two types (more preferably at least three types, even more preferably at least five types) of CO.

In an embodiment using tagged POs, the tagged regions of at least two POs may have an identical sequence or a different sequence. For example, if the present invention is carried out for screening target nucleic acid sequences (for example, detecting a nucleic acid sequence among a plurality of target nucleic acid sequences), POs having tagging regions with an identical sequence can be used.

In addition, one type of CO can be used to detect multiple target nucleic acid sequences. For example, when POs having an identical sequence in their tagging regions are used for screening target nucleic acid sequences, one type of CO can be used.

According to a preferred embodiment, the method of the present invention can be carried out using at least two downstream primers.

Preferred Embodiments

In a preferred embodiment of the present invention, there is provided a method for detecting target nucleic acid sequences from DNA or a mixture of nucleic acids in an analysis with ROSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequences with a pair of primers containing an upstream primer and an upstream primer and a PO (probe oligonucleotide); wherein each of the primers located upstream of the primer and located downstream of the primer contains a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; RO has a single label; PO is blocked at its 3′-end to prevent its extension; the RO site recognizing the target is localized between the upstream primer and the downstream primer; the elongated chain of the upstream primer induces PO cleavage under the action of matrix nucleic acid polymerase having 5′-nuclease activity;

(b) contacting the product from step (a) with a nucleic acid template polymerase having 5′-nuclease activity under conditions suitable for elongation of primers and for cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequences, the PO is cleaved by the action of a matrix nucleic acid polymerase having 5′-nuclease activity to obtain a single label-containing fragment;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex;

(d) denaturing the product from step (c);

(e) repeating steps (a) to (d) at least twice; and

(f) detecting the presence of PO cleavage by measuring the signal from a single label on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

Alternatively, after step (b), step (b-1) is performed by denaturing the product from step (b) and step (b-2) by repeating steps (a) to (b-1) at least twice. In this case, it is possible to carry out stages (d) and (e).

The measurement of the signal from a single label can be carried out for each of the repeating cycles after repeating stage (d) or at predetermined time intervals in the process of repeating stage (d).

In another preferred embodiment of the present invention, there is provided a method for detecting target nucleic acid sequences from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequences with a pair of primers consisting of an upstream primer and a downstream primer and a PO (probe oligonucleotide); wherein each of the primers located upstream of the primer and located downstream of the primer contains a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO has a system of two interacting labels containing a donor molecule and an acceptor molecule; PO is blocked at its 3′-end to prevent its extension; the RO site recognizing the target is localized between the upstream primer and the downstream primer; the elongated chain of the upstream primer induces PO cleavage under the action of matrix nucleic acid polymerase having 5′-nuclease activity;

(b) contacting the product from step (a) with a nucleic acid template polymerase having 5′-nuclease activity under conditions suitable for elongation of primers and for cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequences, the PO is cleaved by the enzyme with 5′-nuclease activity, with the separation of the system of two interacting labels, resulting in a fragment containing a donor molecule and a fragment containing an acceptor molecule;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; moreover, the hybridization reaction is carried out under such conditions that at least one of the fragments containing the donor molecule fragment and the fragment containing the acceptor molecule does not hybridize with CO, and unsplit PO is hybridized with CO with the formation of an unsplit PO / CO duplex; wherein the signal from the duplex unsplit PO / CO differs from the signal received when at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO; and

(d) denaturing the product from step (c);

(e) repeating steps (a) to (d) at least twice; and

(f) detecting the presence of PO cleavage by measuring a signal from a system of two interacting labels on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

Alternatively, after step (b), step (b-1) is performed by denaturing the product from step (b) and step (b-2), repeating steps (a) to (b-1) at least twice. In this case, it is possible to carry out stages (d) and (e).

In yet another preferred embodiment of the present invention, there is provided a method for detecting target nucleic acid sequences from DNA or a mixture of nucleic acids in an analysis with ROSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequences with a pair of primers containing an upstream primer and an upstream primer and a PO (probe oligonucleotide); wherein each of the primers located upstream of the primer and located downstream of the primer contains a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO is blocked at its 3′-end to prevent its extension; the RO site recognizing the target is localized between the upstream primer and the downstream primer; the elongated chain of the upstream primer induces PO cleavage under the action of matrix nucleic acid polymerase having 5′-nuclease activity;

b) contacting the product from step (a) with a nucleic acid template polymerase having 5′-nuclease activity under conditions suitable for primer extension and PO cleavage; in this case, when the PO hybridizes with the target nucleic acid sequences, the PO is cleaved by the action of a matrix nucleic acid polymerase having 5′-nuclease activity to obtain a single label-containing fragment;

(c) carrying out a hybridization reaction in the presence of an intercalating agent by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex;

(d) denaturing the product from step (c);

(e) repeating steps (a) to (d) at least twice; and

(f) detecting the presence of PO cleavage by measuring the signal from the intercalating agent on a solid support; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

Alternatively, after step (b), step (b-1) is performed by denaturing the product from step (b) and step (b-2) by repeating steps (a) to (b-1) at least twice. In this case, it is possible to carry out stages (d) and (e).

IV. Method for target detection in ROSN analysis based on the use of 5′-nuclease activity independent of the upstream oligonucleotide

In a further aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POCH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with the probe oligonucleotide (PO); wherein the PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; RO has a single label;

b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by an enzyme having 5′-nuclease activity to obtain a single label-containing fragment;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex;

(d) detecting the presence of PO cleavage by measuring the signal from a single label on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

In yet another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with the probe oligonucleotide (PO); wherein the PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO has a system of two interacting labels containing a donor molecule and an acceptor molecule;

b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by the enzyme with 5′-nuclease activity, with the separation of the system of two interacting labels, resulting in a fragment containing a donor molecule and a fragment containing an acceptor molecule;

(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that at least one of the fragments containing the donor molecule fragment and the fragment containing the acceptor molecule does not hybridize with CO, and unsplit PO is hybridized with CO with the formation of an unsplit PO / CO duplex; wherein the signal from the duplex unsplit PO / CO differs from the signal received when at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO; and

(d) detecting the presence of PO cleavage by measuring the signal from a system of two interacting labels on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

In yet another aspect of the present invention, there is provided a method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) hybridization of the target nucleic acid sequence with the probe oligonucleotide (PO); wherein the PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence;

b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by the enzyme with 5′-nuclease activity to produce a cleaved fragment;

(c) carrying out a hybridization reaction in the presence of an intercalating agent by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the cleaved fragment does not hybridize with CO, and the unsplit PO hybridizes with CO with the formation of an unsplit PO / CO duplex;

(d) detecting the presence of PO cleavage by measuring the signal from the intercalating agent on a solid support; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence.

The present invention may be practiced without using an upstream oligonucleotide. The cleavage of PO can be carried out using 5′-nuclease activity, independent of the upstream oligonucleotide. In this case, you can use traditional enzymes with 5′-nuclease activity, independent of the upstream oligonucleotide.

For example, you can use 5′-FEN nuclease with 5′-exonuclease activity and / or 5′-endonuclease activity independent of the upstream oligonucleotide.

Among matrix polymerases, there are several enzymes with 5′-nuclease activity (5′-exonuclease activity and / or 5′-endonuclease activity) independent of the upstream oligonucleotide, for example, Taq DNA polymerase (see lawyer et al., Genome Res., 2, pp. 275-287 (1993), WO 2008/011004 and Lyamichev et. al., Science, 260, pp. 778-783 (1993)).

According to a preferred embodiment, the cleavage of PO under the action of a matrix polymerase having independent of the upstream oligonucleotide 5′-nuclease activity depends on the location of the labels or the type of association with the labels present in the PO. Preferably, when the label is attached to the 5′-end of untagged PO, cleavage of the untagged PO by matrix polymerase having 5′-nuclease activity can be more effective if this tag is attached to the phosphate group of the 5′-end of untagged PO, in particular through the carbon spacer. When a tag is attached to the base at the 5′-end of an untagged PO, or when no carbon spacer is used, splitting of the untagged PO is unlikely.

The cleavage efficiency in the case of 5′-nuclease activity dependent on the upstream oligonucleotide may be higher than in the case of 5′-nuclease activity independent of the upstream oligonucleotide.

5′-nuclease activity, independent of the upstream oligonucleotide, is more sensitive to reaction conditions (eg, types of enzymes, buffer compositions, and PO sequences).

Given the amplification of the target nucleic acid sequences and the PO cleavage efficiency, the POCH analysis of the present invention is preferably performed using upstream oligonucleotides.

Target Detection Kits

In another aspect of the present invention, there is provided a kit for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) a probe oligonucleotide (PO) comprising a target recognition region comprising a hybridizable nucleotide sequence complementary to a target nucleic acid sequence;

(b) an upstream oligonucleotide comprising a hybridizable nucleotide sequence complementary to a target nucleic acid sequence; wherein the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity, and

(c) an capture oligonucleotide (CO) immobilized on a solid support; in this case, the CO contains a nucleotide sequence capable of hybridization with PO, as a result of which the PO, which is not cleaved by the enzyme with 5′-nuclease activity, hybridizes with the CO with the formation of an uncleaved PO / CO duplex.

Since the kit of this invention is made to implement the above detection method of the present invention, a general description is omitted to avoid unnecessary duplication, which complicates the description.

According to a preferred embodiment, the PO contains a single label or a system of two interacting labels.

According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising in its 5′-terminal portion a tagging region having a nucleotide sequence non-complementary to the target nucleic acid sequence, and a nucleotide sequence in CO capable of hybridization with PO, soda rzhit nucleotide sequence capable of hybridizing to the tagged portion RO.

According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising in its 5′-terminal portion a tagging region having a nucleotide sequence non-complementary to the target nucleic acid sequence, and a nucleotide sequence in CO capable of hybridization with PO, soda rzhit nucleotide sequence capable of hybridizing with a portion of tagged portion and the recognizing part RO target portion.

According to a preferred embodiment, the PO is a 3′-tagged PO, further comprising in its 3′-terminal portion a tag region having a nucleotide sequence incomplete to the target nucleic acid sequence, and the CO is immobilized on a solid support through its 5′-end.

According to a preferred embodiment, the PO is a 5′-tagged PO, further comprising in its 5′-terminal portion a tag region having a nucleotide sequence non-complementary to the target nucleic acid sequence and the CO immobilized on a solid support through its 3′-end.

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

According to a preferred embodiment, the kit further comprises an enzyme having 5′-nuclease activity, more preferably a thermostable DNA polymerase having 5′-nuclease activity, or FEN nuclease.

According to a preferred embodiment, the upstream oligonucleotide is located in the immediate vicinity of the PO so that this upstream oligonucleotide induces the cleavage of the PO with an enzyme having 5′-nuclease activity.

According to a preferred embodiment, the upstream oligonucleotide has a partially overlapping sequence with a target-recognizing region of PO.

According to a preferred embodiment, the upstream primer induces, through its extended chain, PO cleavage with an enzyme having 5′-nuclease activity.

According to a preferred embodiment, the upstream oligonucleotide is an upstream primer, and the enzyme having 5′-nuclease activity is a nucleic acid matrix polymerase having 5′-nuclease activity.

According to a preferred embodiment, the kit further comprises an intercalating agent.

According to a preferred embodiment, the kit is used to detect at least two types of target nucleic acid sequences. The upstream oligonucleotide contains at least two types of upstream oligonucleotides, the PO contains at least two types of PO, and the CO contains at least two types of CO.

According to a preferred embodiment, the kit further comprises a downstream primer.

According to another additional aspect of the present invention, there is provided a kit for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with POSH (PO cleavage and hybridization) on a solid support, comprising:

(a) a probe oligonucleotide (PO) comprising a target recognition region comprising a hybridizable nucleotide sequence complementary to a target nucleic acid sequence; and

(b) a capture oligonucleotide (CO) immobilized on a solid support; in this case, the CO contains a nucleotide sequence capable of hybridization with PO, as a result of which the PO, which is not cleaved by the enzyme with 5′-nuclease activity, hybridizes with the CO with the formation of an uncleaved PO / CO duplex.

The features and advantages of this invention will be summarized below.

(a) In the present invention, a target nucleic acid sequence is detected using a POSH analysis according to which a PO (probe oligonucleotide) hybridized with a target nucleic acid sequence is cleaved and a PO cleavage is detected by hybridization with CO (capture oligonucleotide). In the present invention, the unsplit probe is hybridized to an oligonucleotide immobilized on a solid support.

According to traditional technologies using a target probe and an immobilized capture probe, it is necessary to prevent the hybridization of unsplit target probes with immobilized oligonucleotides by (1) constructing a target probe having a hairpin structure and controlling both hybridization conditions with target sequences and hybridization conditions with immobilized oligonucleotides; or (2) the construction of immobilized oligonucleotides, taking into account the orientation of the immobilized oligonucleotides when they are immobilized and the distance from them to the surface of the solid substrate. In view of this, traditional methods are very inconvenient in terms of constructing target probes and immobilized oligonucleotides and developing reaction conditions.

In contrast, the present invention is devoid of such disadvantages and limitations. In the present invention, the design of PO and CO is a convenient procedure, and optimization of the reaction conditions is usually easy.

(b) When, in the present invention, the detection of a signal on a solid substrate is carried out continuously along with the repetition of PO cleavage cycles, the number of cleaved POs increases as the number of cleavage reactions is repeated, and the signal changes in parallel with the change in the number of cleaved POs. In this case, the detection of the target nucleic acid sequence can be carried out in real time.

(c) The present invention enables the simultaneous detection of multiple target nucleic acid sequences using a label of even one type. Since the detection of PO cleavage is carried out by hybridization with CO immobilized on a solid substrate, the detection of multiple target nucleic acid sequences does not require the presence of different labels for each target nucleic acid.

(d) In general, it will be apparent to one skilled in the art that using target detection methods that use direct hybridization between target nucleic acid sequences and probes immobilized on solid substrates, it is impossible to obtain effective and reliable hybridization results due to reaction-limiting external conditions inherent reactions in the solid phase. In contrast, the present invention uses hybridization between PO and CO without the participation of target nucleic acid sequences on a solid support, which provides more effective and reliable results in typical environments and conditions for a reaction on a solid support.

(e) Where a tagged PO and CO having a nucleotide sequence capable of hybridizing with the tagging portion of PO are used in the present invention, the sequence of the tagging portion of PO and the sequence of CO can be selected without any consideration of the target nucleic acid sequence. This makes it possible to pre-construct a set of sequences useful in the present invention. In particular, CO can be obtained in a standard way without any consideration or any knowledge of the target nucleic acid sequences. Such features provide significant advantages in microarray analysis using CO immobilized on a solid support.

Now the present invention will be described in more detail in the examples. It will be apparent to those skilled in the art that these examples are intended to illustrate more specifically, and the scope of the present invention as set forth in the appended claims is not limited to these examples.

EXAMPLES

EXAMPLE 1. Evaluation of the analysis with cleavage and hybridization of the probing oligonucleotide (ROSN) using a non-tagged probing oligonucleotide (PO) with a single label

The applicability of the new assay was evaluated — an assay with cleavage and hybridization of the probing oligonucleotide (POSH) for the detection of the target nucleic acid sequence using an untagged single-label PO (see Fig. 2). PO cleavage was carried out in vitro and an aliquot of the obtained product was transferred to a microchip on which an exciting oligonucleotide (CO) was immobilized.

To lengthen the upstream primer and PO cleavage, Taq DNA polymerase having 5′-nuclease activity was used. An untagged PO contains a target recognition region complementary to the target nucleic acid sequence and Quasar570 as a fluorescent reporter molecule at its 5′-end. CO contains a nucleotide sequence capable of hybridization with a target recognition region of PO. PO and CO are blocked by a carbon spacer at their 3′-ends. CO has poly (T) ₁₀ as a linker leg, and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end, a marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of a glass plate using an amino group at its 3′-end. A synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used as a matrix.

The sequences of the synthetic matrix located upstream of the primer, PO, CO and marker used in this example are:

For the manufacture of CO and marker (SEQ ID NOs: 4 and 5), NSB9 NHS plates (NSBPOSTECH Korea) were used. CO and a marker dissolved in a spot-coated NSB buffer at a final concentration of 50 μm were printed on an NSB9 NHS plate using a microarray spotter PersonalArrayer ™ 16 (CapitalBio, China). CO and the marker were applied as spots in a single line in 2 × 1 format (spots in two repetitions) and the resulting microchip was incubated in a chamber while maintaining ~ 85% humidity overnight. Then the plates were washed in a buffer solution containing 2 × SSPE (0.3 M sodium chloride; 0.02 M sodium hydrogen phosphate and 2.0 mm EDTA), pH 7.4, and 7.0 mm SDS, at 37 ° C for 30 minutes to remove non-specifically bound CO and marker and washed with distilled water. Then, DNA-functionalized plates were dried using a plate centrifuge and stored in the dark at 4 ° C until use.

The cleavage reaction was carried out in a final volume of 50 μl containing 2 pmol of the synthetic matrix (SEQ ID NO: 1) for the NG gene, 10 pmol of the upstream primer (SEQ ID NO: 2), 1 pmol of PO (SEQ ID NO: 3) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 30 cycles of 30 s at 95 ° C and 60 s at 60 ° C.

30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 4). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Images were obtained using the Ahop GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution of 5 μm pixel size. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 14 and in Table 1, in the presence of a target nucleic acid sequence, a reduced fluorescence signal (RFU: 9006 ± 20.5) was detected compared to a fluorescent signal (RFU: 65484 ± 0.7) in the absence of a target nucleic acid sequence.

These results indicate that the POSH analysis is applicable for the detection of the target nucleic acid sequence.

EXAMPLE 2. Evaluation of ROSN analysis using a 3′-tagged PO with a single label

Further, the inventors evaluated the applicability of the POSH analysis for the detection of the target nucleic acid sequence using a 3'-tagged PO with a single label (see Fig. 4). PO cleavage was carried out in vitro and an aliquot of the obtained product was transferred to a microchip on which CO was immobilized.

To lengthen the upstream primer and PO cleavage, Taq DNA polymerase having 5′-nuclease activity was used. A 3′-tagged PO contains a target recognition region complementary to the target nucleic acid sequence and a 3′-tagged region non-complementary to the target nucleic acid sequence. The 3′-tagged PO has Quasar570 as a fluorescent reporter molecule at its 5′-end. CO contains a nucleotide sequence capable of hybridization with the 3′-tagged region of PO. 3′-Tagged PO and CO are blocked by a carbon spacer at their 3′-ends. CO has poly (T) 5 as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end. A marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of the glass plate using an amino group at its 3′-end. A synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used as a matrix.

The sequences of the synthetic matrix located upstream of the primer, 3′-tagged PO, CO and marker used in this example are:

(Underlined letters mark the 3′-tagging region of the RO).

The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 1, except that they used CO with SEQ ID NO: 7 instead of CO with SEQ ID NO: 4.

The cleavage reaction was carried out in a final volume of 50 μl containing 2 pmol of the synthetic matrix (SEQ ID NO: 1) for the NG gene, 10 pmol of the upstream primer (SEQ ID NO: 2), 1 pmol of PO (SEQ ID NO: 6) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 30 cycles of 30 s at 95 ° C and 60 s at 60 ° C.

30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 7). The plate was placed on a block in a thermal cycler (GenePro B41, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was performed using an AchoN GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 15 and in Table 2, in the presence of the target nucleic acid sequence, a reduced fluorescent signal (RFU: 10217 ± 73.5) was detected compared to the fluorescent signal (RFU: 65464 ± 6.4) in the absence of the target nucleic acid sequence.

These results indicate that the POSH analysis using a 3'-tagged single-label PO is applicable for the detection of a target nucleic acid sequence.

EXAMPLE 3. Evaluation of ROSN analysis using a 5′-tagged PO with a single label

Further, the inventors evaluated the applicability of the POSH analysis for the detection of a target nucleic acid sequence using a 5'-tagged single-label PO (see Fig. 5). PO cleavage was carried out in vitro and an aliquot of the obtained product was transferred to a microchip on which CO was immobilized.

To lengthen the upstream primer and PO cleavage, Taq DNA polymerase having 5′-nuclease activity was used. A 5′-tagged PO contains a target-recognizing region complementary to the target nucleic acid sequence and a 5′-tagged region non-complementary to the target nucleic acid sequence. A 5′-tagged PO has Quasar570 as a fluorescent reporter molecule at its 3′-end. CO contains a nucleotide sequence capable of hybridization with the 5′-tagged region of PO. CO has poly (T) ₁₀ as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC7) through its 3′-end. A marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of the glass plate using an amino group at its 3′-end. A synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used as a matrix.

The sequences of the synthetic matrix located upstream of the primer, 5′-tagged PO, CO and marker used in this example are:

(Underlined letters mark the 5′-tagging site of the RO). The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 1, except that CO with SEQ ID NO: 9 was used instead of CO with SEQ ID NO: 4.

The cleavage reaction was carried out in a final volume of 50 μl containing 2 pmol of the synthetic matrix (SEQ ID NO: 1) for the NG gene, 10 pmol of the upstream primer (SEQ ID NO: 2), 1 pmol of PO (SEQ ID NO: 8) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 30 cycles of 30 s at 95 ° C and 60 s at 60 ° C.

30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 9). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was carried out using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 16 and in Table 3, in the presence of a target nucleic acid sequence, a reduced fluorescent signal (RFU: 17586 ± 152.0) was detected compared to a fluorescent signal (RFU: 65455 ± 0.7) in the absence of a target nucleic acid sequence.

These results indicate that the POSH analysis using a 5'-tagged single-label PO is applicable for the detection of a target nucleic acid sequence.

EXAMPLE 4. Evaluation of ROSN analysis using 3′-tagged PO with double label

Further, the inventors evaluated the applicability of the POSH assay for detecting a target nucleic acid sequence using a 3-tagged double-labeled PO (see FIG. 11). PO cleavage was carried out in vitro and an aliquot of the obtained product was transferred to a microchip on which CO was immobilized.

To lengthen the upstream primer and PO cleavage, Taq DNA polymerase having 5′-nuclease activity was used. A 3′-tagged PO contains a target recognition region complementary to the target nucleic acid sequence and a 3′-tagged region non-complementary to the target nucleic acid sequence. The 3′-tagged PO contains BHQ-2 as an acceptor molecule at its 5′-end and Quasar570 as a donor molecule at its 3′-end of the target recognition site. CO contains a nucleotide sequence capable of hybridization with the 3′-tagged region of PO. To detect the target nucleic acid sequence, the signal from the donor molecule was measured. 3′-tagged PO and CO are blocked by a carbon spacer at their 3′-ends. CO has poly (T) ₅ as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end. A marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of the glass plate using an amino group at its 3′-end. A synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used as a matrix.

The sequences of the synthetic matrix located upstream of the primer, 3′-tagged PO, CO and marker used in this example are:

(Underlined letters mark the 3′-tagging region of the RO).

The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 1, except that they used CO with SEQ ID NO: 7 instead of CO with SEQ ID NO: 4.

The cleavage reaction was carried out in a final volume of 50 μl containing 2 pmol of the synthetic matrix (SEQ ID NO: 1) for the NG gene, 10 pmol of the upstream primer (SEQ ID NO: 2), 1 pmol of PO (SEQ ID NO: 10) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 30 cycles of 30 s at 95 ° C and 60 s at 60 ° C.

30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 7). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was carried out using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

Since the acceptor molecule dampens the fluorescent signal from the donor molecule, there is no signal from the donor molecule in the duplex (unsplit 3′-tagged PO) / CO. Moreover, a fragment of a cleaved 3′-tagged PO hybridized with CO containing a donor molecule is able to provide a fluorescent signal.

As shown in FIG. 17 and in Table 4, in the presence of the target nucleic acid sequence, an increased fluorescent signal (RFU: 65469 ± 0.7) was detected compared to the fluorescent signal (RFU: 13349 ± 441.2) in the absence of the target nucleic acid sequence.

These results indicate that POSH analysis using a 3′-tagged double-labeled PO is useful for detecting a target nucleic acid sequence.

EXAMPLE 5. Detection of the target nucleic acid sequence in ROSN analysis

The inventors used ROSH analysis to detect the target nucleic acid sequence, complemented by amplification of the target sequence. To study the possibility of such an application, untagged PO with a single label and 3′-tagged PO with a single label were used, respectively. The cleavage of PO during the amplification of the target by PCR was carried out in vitro and an aliquot of the obtained product was transferred to a microchip on which CO was immobilized.

The upstream primer is involved in the cleavage of PO under the action of an enzyme with 5′-nuclease activity, and is also involved in the amplification of the target nucleic acid sequence along with the downstream primer during PCR. Taq DNA polymerase having 5′-nuclease activity was used to extend the upstream primer and downstream primer and PO cleavage.

An untagged PO contains a target recognition region complementary to the target nucleic acid sequence. CO for non-tagged PO contains a nucleotide sequence capable of hybridization with the target-recognizing region of PO. The 3′-tagged PO contains a target-recognizing region complementary to the target nucleic acid sequence and an S′-tagged region non-complementary to the target nucleic acid sequence. CO for a 3′-tagged PO contains a nucleotide sequence capable of hybridization with the 3′-tagged region of PO.

Untagged PO and 3′-Tagged PO contain Quasar570 as a fluorescent reporter molecule at their 5′-ends. PO and CO are blocked by a carbon spacer at their 3′-ends. CO for untagged PO has poly (T) ₁₀ as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end. CO for a 3-tagged PO has poly (T) ₅ as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end. A marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of the glass plate using an amino group at its 3′-end. Genomic DNA from Neisseria gonorrhoeae (NG) was used as the target matrix.

5-1. ROSN analysis using untagged PO with a single label

The sequences of the upstream primer, the downstream primer, PO, CO, and marker used in this example are:

The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 1.

The cleavage reaction was carried out in a final volume of 50 μl containing 100 pg of genomic DNA from NG, 10 pmol of the upstream primer (SEQ ID NO: 11), 10 pmol of the upstream primer (SEQ ID NO: 12) , 1 pmol of PO (SEQ ID NO: 3) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Tag DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 60 cycles of 30 s at 95 ° C and 60 s at 60 ° C. 30 μl of the resulting mixture was placed in a chamber collected on the surface of an NSB glass plate to which was sewn CO (SEQ ID NO: 4). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was carried out after each washing using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 18 and in Table 5, in the presence of the target nucleic acid sequence, a reduced fluorescent signal (RFU: 1650 ± 97.6) was detected compared to the fluorescent signal (RFU: 64474 ± 0.0) in the absence of the target nucleic acid sequence.

5-2. ROSN analysis using a 3′-tagged PO with a single label

The sequences of the upstream primer, the downstream primer, 3′-tagged PO, CO, and marker used in this example are:

(Underlined letters mark the 3′-tagging region of the RO). The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 2.

The cleavage reaction was carried out in a final volume of 50 μl containing 100 pg of genomic DNA from NG, 10 pmol of the upstream primer (SEQ ID NO: 11), 10 pmol of the upstream primer (SEQ ID NO: 2) , 1 pmol of PO (SEQ ID NO: 6) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 60 cycles of 30 s at 95 ° C and 60 s at 60 ° C. 30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 7). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was carried out after each washing using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 19 and in Table 6, in the presence of the target nucleic acid sequence, a reduced fluorescent signal (RFU: 9969 ± 217.1) was detected compared to the fluorescent signal (RFU: 65470 ± 1.4) in the absence of the target nucleic acid sequence.

These results indicate that the detection of the target nucleic acid sequence can be carried out in ROSH analysis, supplemented by amplification of the target using the PCR process.

EXAMPLE 6. Detection of the target nucleic acid sequence in ROSN analysis in real time

The inventors used ROSN analysis to detect the target nucleic acid sequence in real time. To study the possibility of such an application, a 3′-tagged PO was used. The PO cleavage and the hybridization of PO with CO were performed together with the amplification of the target during PCR on a microchip with immobilized CO. The change in the fluorescent signal was measured as a function of the number of cycles. The genomic DNA of Neisseria gonorrhoeae (NG) was used as the target matrix.

In this Example, the same oligonucleotides were used (upstream primer, downstream primer, 3′-tagged PO, CO and marker) as in Example 5-2. The preparation of the plates was carried out in accordance with a protocol similar to that used in Example 2.

A mixture for ROSH analysis was prepared in a final volume of 30 μl containing 100 pg of genomic DNA from NG, 10 pmol of an upstream primer (SEQ ID NO: 11), 10 pmol of an upstream primer (SEQ ID NO : 2), 1 pmol of PO (SEQ ID NO: 6) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); the whole mixture was placed in a chamber assembled on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 7). The plate was placed on a block in a thermal cycler (GenePro B4I, China). For the analysis in cyclic mode, five identical plates were prepared. The reaction with ROSN was carried out as follows: denaturation for 15 min at 95 ° C and then 0, 5, 30, 40 or 60 cycles of 30 s at 95 ° C, 60 s at 60 ° and 30 min at 55 ° C. After passing the appropriate number of cycles, the images were obtained using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution of 5 μm pixel size. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

As shown in FIG. 20A and 20B and in Table 7, the fluorescent signal decreased after 30 cycles (0 cycles_RFU: 65438 ± 0.0; 20 cycles_RFU: 65445 ± 2.1; 30 cycles_RFU: 65480 ± 0.0; 40 cycles_RFU: 8844 ± 1485.6 and 60 cycles_RFU: 4878 ± 169.7) in the presence of a matrix. No change in the fluorescent signal was observed depending on the number of cycles in the absence of the matrix.

These results indicate that the target nucleic acid sequence can be detected in real time in the ROSH analysis.

EXAMPLE 7. Evaluation of ROSN analysis using the cleavage of PO, independent of the upstream oligonucleotide

Further, the inventors evaluated the applicability of the POSH assay for detecting a target nucleic acid sequence without using an upstream oligonucleotide. The PO cleavage, independent of the upstream oligonucleotide, was carried out in a test tube without an upstream oligonucleotide and an aliquot of the obtained product was transferred to a microchip on which CO was immobilized.

For the cleavage of PO, independent of the upstream oligonucleotide, Taq DNA polymerase having 5′-nuclease activity was used. An untagged PO contains a target-recognizing region complementary to the target nucleic acid sequence and FAM as a fluorescent reporter molecule at its 5 ′ end. CO contains a nucleotide sequence capable of hybridization with a target recognition region of PO. PO and CO are blocked by a carbon spacer at their 3′-ends. CO has poly (T) ₁₀ as a linker leg and is immobilized on the surface of a glass plate using an amino group (aminoC6) through its 5′-end. A marker probe having a fluorescent reporter molecule (Quasar570) at its 5′-end was immobilized on the surface of the glass plate using an amino group at its 3′-end. A synthetic oligonucleotide for Neisseria gonorrhoeae (NG) was used as a matrix.

The sequences of the synthetic matrix, PO, CO, and marker used in this example are:

For the manufacture of CO and marker (SEQ ID NOs: 4 and 5), NSB9 NHS plates (NSBPOSTECH Korea) were used. CO and a marker dissolved in a spot-coated NSB busreter at a final concentration of 50 μm were printed on NSB9 NHS plates using a PersonalArrayer ™ 16 microchip spotter (CapitalBio, China). CO and the marker were applied as spots in a single line in 2 × 1 format (spots in two repetitions) and the resulting microchip was incubated in a chamber while maintaining ~ 85% humidity overnight. Then the plates were washed in a buffer solution containing 2 × SSPE (0.3 M sodium chloride; 0.02 M sodium hydrogen phosphate and 2.0 mm EDTA), pH 7.4 and 7.0 mm SDS, at 37 ° C for 30 min to remove non-specifically bound CO and marker and washed with distilled water. Then, DNA-functionalized plates were dried using a plate centrifuge and stored in the dark at 4 ° C until use.

The cleavage reaction was carried out in a final volume of 50 μl containing 2 pmol of the synthetic matrix (SEQ ID NO: 1) for the NG gene, 1 pmol of PO (SEQ ID NO: 12) and 25 μl of a 2 × Master Mix containing 2.5 mM MgCl ₂ , 200 μM dNTP and 4 units of H-Taq DNA polymerase (Solgent, Korea); a tube containing the reaction mixture was placed in a thermal cycler for real-time PCR (CFX96, Bio-Rad); the reaction mixture was denatured for 15 min at 95 ° C and subjected to 30 cycles of 30 s at 95 ° C and 60 s at 60 ° C.

30 μl of the resulting mixture was placed in a chamber collected on the surface of a NSB glass plate to which CO was sewn (SEQ ID NO: 4). The plate was placed on a block in a thermal cycler (GenePro B4I, China). The hybridization reaction was carried out for 30 min at 55 ° C. Image acquisition was carried out using an Axon GenePix4100A confocal laser scanning device (Molecular Device, US) with scanning at a resolution with a pixel size of 5 μm. Fluorescence intensity was analyzed using GenePix pro7.0 microarray quantitation software (Molecular Device, US). The fluorescence intensity was expressed as mean values for the spots after subtracting the local background. To check reproducibility, each spot was analyzed in duplicate. Fluorescence intensity indicates the average for duplicate spots.

In the presence of the target nucleic acid sequence, a reduced fluorescence signal was detected compared to the fluorescent signal in the absence of the target nucleic acid sequence.

These results indicate that the POSH analysis using the PO cleavage independent of the upstream oligonucleotide is applicable for the detection of the target nucleic acid sequence.

Based on the description of a preferred embodiment of the present invention, it should be understood that its variations and modifications corresponding to the essence of the invention may become apparent to experts in this field, and the scope of this invention should be determined by the attached claims and their equivalents.

Claims (42)

1. A method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSN (PO cleavage and hybridization) on a solid support, including:
(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; RO has a single label; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;
(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by an enzyme having 5′-nuclease activity to obtain a single label-containing fragment;
(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the single-label containing fragment does not hybridize with CO, and the uncleaved PO hybridizes with the CO to form an uncleaved PO / CO duplex; and
(d) detecting the presence of PO cleavage by measuring the signal from a single label on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence. 2. The method according to claim 1, wherein the nucleotide sequence in CO capable of hybridization with PO contains a nucleotide sequence capable of hybridization with a target recognition region of PO. 3. The method of claim 1, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO, further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide sequence capable of hybridizing to a localized target site PO. 4. The method according to p. 3, where a single label is positioned so that it does not remain on the fragment containing the tagging region, which is released as a result of PO cleavage. 5. The method of claim 1, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO, further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide a sequence capable of hybridizing with part tagged portion and a portion of the recognizing target area PO. 6. The method according to p. 1, where the RO and / or CO is blocked at its 3′-end, to prevent its extension. 7. The method of claim 1, wherein the upstream oligonucleotide is a upstream primer or upstream probe. 8. The method of claim 1, wherein the upstream oligonucleotide has a partially overlapping sequence with a target recognition region of the PO. 9. The method of claim 1, wherein the enzyme having 5′-nuclease activity is a thermostable DNA polymerase having 5′-nuclease activity, or FEN nuclease. 10. The method of claim 1, wherein the upstream oligonucleotide is an upstream primer and the enzyme having 5′-nuclease activity is a matrix nucleic acid polymerase having 5′-nuclease activity. 11. The method according to p. 1, further comprising repeating steps (a) - (b), (a) - (c) or (a) - (d) with denaturation between repeating cycles. 12. The method of claim 1, wherein steps (a) to (d) are carried out in a reaction vessel or in separate reaction vessels. 13. The method of claim 1, wherein the target nucleic acid sequence comprises at least two types of target nucleic acid sequences. 14. The method of claim 1, wherein the target nucleic acid sequence comprises a nucleotide variation. 15. The method according to any one of paragraphs. 1-14, which is carried out in the presence of a downstream primer. 16. A method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSN (PO cleavage and hybridization) on a solid support, including:
(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; PO has a system of two interacting labels containing a donor molecule and an acceptor molecule; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;
(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by the enzyme with 5′-nuclease activity, with the separation of the system of two interacting labels, resulting in the formation of a fragment containing a donor molecule and a fragment containing an acceptor molecule;
(c) carrying out a hybridization reaction by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that at least one of the fragments containing the donor molecule fragment and the fragment containing the acceptor molecule does not hybridize with CO, and unsplit PO is hybridized with CO with the formation of an unsplit PO / CO duplex; wherein the signal from the duplex unsplit PO / CO differs from the signal arriving when at least one of the fragments containing the donor molecule and the fragment containing the acceptor molecule does not hybridize with CO; and
(d) detecting the presence of PO cleavage by measuring the signal from a system of two interacting labels on a solid substrate; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence. 17. The method according to p. 16, where the nucleotide sequence in CO, capable of hybridization with PO, contains a nucleotide sequence capable of hybridization with the target-recognizing region of PO, and the system of two interacting labels is localized so that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of a duplex, unsplit PO / CO, and during stage (d), a signal from an acceptor molecule is detected. 18. The method of claim 16, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO, further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide The sequence capable of hybridizing to the tagged portion RC and a system of two interacting labels is localized so that the signal from the donor molecule is quenched acceptor molecule in the formation of duplex uncleaved PO / CO, wherein during step (d) is carried out signal detection from the acceptor molecule. 19. The method according to p. 16, where the nucleotide sequence in CO, capable of hybridization with PO, contains a nucleotide sequence capable of hybridization with the target-recognizing region of PO, and the hybridization reaction in step (c) is carried out under such conditions that a fragment containing donor molecule does not hybridize with CO; in this case, the system of two interacting labels is localized in such a way that the signal from the donor molecule is not suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex, and during stage (d), the signal from the donor molecule is detected. 20. The method of claim 16, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising at its 5′-terminal portion, a tagging region having a nucleotide sequence non-complementary to the target nucleic acid sequence; a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide sequence capable of hybridization with a tagging region of PO; the fragment containing the donor molecule contains a tagging region capable of hybridization with CO, and in the hybridization reaction in step (c), the fragment containing the donor molecule hybridizes with CO; in this case, the system of two interacting labels is localized in such a way that the signal from the donor molecule is suppressed by the acceptor molecule during the formation of the unsplit PO / CO duplex, and during stage (d), the signal from the donor molecule is detected. 21. The method of claim 16, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO, further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide The sequence capable of hybridizing to a portion or part of the tagged part of recognizer target area RA.            22. The method of claim 16, wherein the upstream oligonucleotide is an upstream primer and template nucleic acid polymerase is used to extend the upstream primer in step (b). 23. The method according to p. 16, further comprising repeating steps (a) - (b), (a) - (c) or (a) - (d) with denaturation between repeating cycles. 24. The method of claim 16, wherein steps (a) to (d) are carried out in a reaction vessel or in separate reaction vessels. 25. The method according to any one of paragraphs. 16-24, which is carried out in the presence of a downstream primer. 26. A method for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSN (PO cleavage and hybridization) on a solid support, including:
(a) hybridization of the target nucleic acid sequence with an upstream oligonucleotide and a probe oligonucleotide (PO); wherein the upstream oligonucleotide comprises a hybridizable nucleotide sequence complementary to the target nucleic acid sequence; PO contains a target-recognizing region containing a hybridizing nucleotide sequence complementary to the target nucleic acid sequence; the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity;
(b) contacting the product from step (a) with an enzyme having 5′-nuclease activity under conditions suitable for the cleavage of PO; in this case, when the PO hybridizes with the target nucleic acid sequence, the PO is cleaved by an enzyme having 5′-nuclease activity to produce a cleaved fragment;
(c) carrying out a hybridization reaction in the presence of an intercalating agent by contacting the product from step (b) with a capture oligonucleotide (CO) immobilized on a solid support; wherein CO contains a nucleotide sequence capable of hybridization with PO; moreover, the hybridization reaction is carried out under such conditions that the cleaved fragment does not hybridize with CO, and the unsplit PO hybridizes with CO with the formation of an unsplit PO / CO duplex; and (d) detecting the presence of PO cleavage by measuring the signal from the intercalating agent on a solid support; thus, the presence of PO cleavage indicates the presence of a target nucleic acid sequence. 27. The method according to p. 26, where the nucleotide sequence in WITH, capable of hybridization with PO, contains a nucleotide sequence capable of hybridization with a target-recognizing region of PO. 28. The method of claim 26, wherein the upstream oligonucleotide is an upstream primer and template nucleic acid polymerase is used to extend the upstream primer in step (b). 29. The method according to p. 26, further comprising repeating steps (a) - (b), (a) - (c) or (a) - (d) with denaturation between repeating cycles. 30. The method according to p. 26, where stage (a) to (d) is carried out in a reaction vessel or in separate reaction vessels. 31. The method according to any one of paragraphs. 26-30, which is carried out in the presence of a downstream primer. 32. A kit for detecting a target nucleic acid sequence from DNA or a mixture of nucleic acids in an analysis with ROSH (PO cleavage and hybridization) on a solid support for use in the method according to any one of claims. 1, 16 and 26, containing:
(a) a probe oligonucleotide (PO) comprising a target recognition region comprising a hybridizable nucleotide sequence complementary to a target nucleic acid sequence;
(b) an upstream oligonucleotide comprising a hybridizable nucleotide sequence complementary to a target nucleic acid sequence; wherein the upstream oligonucleotide is located upstream relative to the PO; an upstream oligonucleotide or an extended chain thereof induces PO cleavage with an enzyme having 5′-nuclease activity; and
(c) a capture oligonucleotide (CO) immobilized on a solid support; in this case, the CO contains a nucleotide sequence capable of hybridization with PO, as a result of which the PO, which is not cleaved by the enzyme with 5′-nuclease activity, hybridizes with the CO with the formation of an uncleaved PO / CO duplex. 33. The set of claim 32, wherein the RO has a single label or a system of two interacting labels. 34. The kit of claim 32, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide a sequence capable of hybridizing to the tagged portion RO. 35. The kit of claim 32, wherein the PO is a 3′-tagged PO, further comprising a tag region in its 3′-terminal portion having a nucleotide sequence non-complementary to the target nucleic acid sequence, or a 5′-tagged PO further comprising of its 5′-terminal part, the tagging region having a nucleotide sequence incomplete with the target nucleic acid sequence and a nucleotide sequence in CO capable of hybridization with PO contains a nucleotide a sequence capable of hybridizing with a portion of tagged portion and the recognizing part RO target portion. 36. The kit of claim 32, wherein the upstream oligonucleotide is a upstream primer or upstream probe. 37. The kit of claim 32, further comprising an enzyme having 5′-nuclease activity. 38. The kit of claim 32, wherein the upstream oligonucleotide has a partially overlapping sequence with a target recognition region of the PO. 39. The kit of claim 37, wherein the enzyme having 5′-nuclease activity is a thermostable DNA polymerase having 5′-nuclease activity, or FEN nuclease. 40. The kit of claim 32, further comprising an intercalating agent. 41. The kit of claim 32, wherein said kit is used to detect at least two types of target nucleic acid sequences, wherein the upstream oligonucleotide contains at least two types of upstream oligonucleotides, the PO contains at least at least two types of PO, and CO contains at least two types of CO. 42. The kit of claim 32, wherein said kit further comprises a primer located downstream.

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