JPH08238100A - Method for specifically detecting nucleic acid having prescribed sequence, and probe and kit used in the same method - Google Patents

Abstract

PURPOSE: To provide a method of specifically detecting a prescribed sequence of a nucleic acid in high sensitivity, especially a method increasing the detecting sensitivity by utilizing a signal amplifying reaction, and a probe and a kit used in the methods. CONSTITUTION: In the objective method, a probe capable of hybridizing with a specific nucleic acid in a specimen, having AP-site bonded to DNA at 5'-side and 3'-side, the site is able to be cut by AP-endonuclease only when the probe is hybridized with the nucleic acid and only the cut probe is able to be dissociated from the nucleic acid is used. The probe and the AP-endonuclease are reacted with a target nucleic acid and a reaction in which the nucleic acid in a specimen hybridizes with the probe, a reaction in which an AP-site of the hybridized probe is cut by a specific AP-endonuclease of a double stranded DNA and a reaction in which the cut probe is dissociated from the nucleic acid, are repeated in the same turn.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for specifically detecting a sequence of a predetermined nucleic acid with high sensitivity, particularly a method for increasing detection sensitivity by utilizing a signal amplification reaction, and a probe and a kit used therefor. Regarding

[0002]

2. Description of the Related Art The probe method based on the complementarity of nucleic acid sequences is a powerful means for identifying genetic diseases, cancers, microorganisms, etc., because the genetic characteristics can be directly analyzed. However, in conventional analysis techniques that simply utilize complementarity, the DNA in a sample and the probe bind one-to-one, so there is a limit to detection sensitivity, and detection is performed when the target gene amount in the sample is small. It was not easy and it was often necessary to amplify the target gene itself or the detection signal.

As a method for amplifying a target gene, PC
The R (Polymerase Chain Reaction) method is generally performed. The PCR method is the most general method for amplifying nucleic acids in vitro, but there are some problems. For example, a special temperature control device is required, amplification efficiency varies depending on the primer used, and the amplification proceeds logarithmically, which causes a problem in quantification, and contamination by the amplified product is likely to occur. Contamination becomes a serious problem especially when a large number of specimens are handled at the same time.

A signal amplification method has been developed which requires no special equipment and is not easily affected by contamination in a simple reaction system. For example, a chimeric probe having a DNA-RNA-DNA sequence is hybridized with a target nucleic acid, RNA sequence in the probe is decomposed by RNaseH, and as a result, a series of reactions in which the decomposed probe dissociates from the target nucleic acid is repeated. "Cycling probe reaction method" described above (see Japanese Patent Publication No. 2-504110). Since this method has good detection sensitivity and can solve various drawbacks of the PCR method, it has recently attracted attention. Also,
As a method for detecting a target nucleic acid by utilizing hybridization of a chimeric probe such as DNA-RNA-DNA, there is a method described in JP-A-62-190086.

However, these methods include DNA-RN
The method for synthesizing the A-DNA chimera probe is difficult and expensive. To synthesize DNA-RNA,
This is because it is necessary to first stop the reaction and then newly synthesize RNA after the DNA synthesis. In addition, RNA has a chemically unstable structure, and at the same time, nonspecific degradation is likely to occur due to contamination with various RNA degrading enzymes.
It is expected that the handling of the probe will be difficult due to the inclusion of a part of RNA degrading enzymes are also routinely present on the surface of laboratory equipment and technicians, and it is difficult to completely remove their effects.

In addition, since one molecule of RNA (ribonucleoside) remains at the end of the degradation product of RNase H of a chimeric probe having a DNA-RNA-DNA structure, it is unlikely to be a substrate for DNA ligase like ordinary oligo DNA. Hard to become. Moreover, the decomposition reaction of RNase H is
There is also a problem that depending on the base sequence of the RNA portion in the A-RNA chimera structure, the easiness of decomposition differs depending on the RNA sequence. For example, compared to cytosine, baredenine is less likely to be decomposed. Due to such problems, DNA-RNA-
At present, the method using the DNA chimera probe is excellent in principle but not yet put into practical use. Furthermore, the method disclosed in Japanese Patent Laid-Open No. 62-190086 contains the problem of the conventional probe method without amplification. In this method, only the hybridization between the chimeric probe and the target nucleic acid is used, and the specific cleavage of the probe does not lead to the cycle reaction, and the signal amplification cannot be expected.

[0007]

As described above, there is a problem in that it is difficult to obtain sensitivity in the conventional analysis technique utilizing the complementarity of gene sequences, and in known signal amplification techniques such as PCR and LCR. Has a problem that it is not quantitative, it is weak against noise, and temperature control is complicated.

Against this background, the present inventors have conducted a study on a signal amplification method utilizing the 3 ′ → 5 ′ exonuclearase activity of Escherichia coli exonureaase III (hereinafter abbreviated as “Exo III”), and have already patented it. An application was filed (JP-A-6-327499). Japanese Patent Laid-Open No. 6-327499 discloses that a hybridized probe is decomposed from the end by Exo III and becomes a length that cannot maintain a double strand, and is disengaged. This was decomposed from the end by Exo III, and when it became a length that could not maintain the double strand, it was disengaged, and this cycle was repeated. It describes a method of detecting a target nucleic acid by detecting a probe that becomes an unusable length and deviates from the target nucleic acid.

This method is a useful nucleic acid detection method that can quantitatively realize repeated reactions by a simple reaction system at a constant temperature, but the length of the degradation product of the probe produced is not always constant. Therefore, when detecting degradation products by a physical method such as electrophoresis, it may be necessary to measure the total amount of multiple bands, and there are still points that need to be improved in terms of operability. It was

The present invention has been made in view of the above problems, and improves stability which is a problem with a probe having a structure of DNA-RNA-DNA. This method (probe cleavage-single strand separation) Cycle) to improve the practicality of a nucleic acid detection method, and to provide a new probe capable of constructing various detection systems in the nucleic acid detection method using a probe cleavage to single-strand separation cycle And Moreover, it aims at providing not only a probe but the detection method and reagent composition using a new probe.

[0011]

In order to solve the above problems, the present inventors have devised a signal amplification method using AP endonuclease such as Exo III.

The present inventors have found that various AP endonucleases have an enzymatic activity of hydrolyzing various types of phosphodiester bonds of double-stranded DNA, specifically, an apurinic acid or apyrimidic acid site ( Below "A
The present invention has been completed by focusing on the fact that 5'-AP endonuclease activity that hydrolyzes a phosphodiester bond is present in the "P site".

The probe according to the present invention essentially has a structure in which DNA is bound to the 5'side and 3'side of the AP site (DNA-
AP-DNA), which is capable of hybridizing with a specific nucleic acid in a sample, and has at least one AP site to which DNA is bound on the 5'side and 3'side, The AP site is characterized in that it can be cleaved by an AP endonuclease only when the probe hybridizes with the specific nucleic acid, and only the cleaved probe can be dissociated from the specific nucleic acid.

The probe according to the present invention as described above is a probe used for analysis of a specific nucleic acid in a sample, and is characterized in that the following reactions occur repeatedly in sequence.

(1) a reaction in which the specific nucleic acid in the sample hybridizes with the probe, (2) double-stranded DNA-specific A
Reaction in which the AP site of the probe hybridized with P endonuclease is cleaved, (3) Reaction in which the cleaved probe is dissociated from the specific nucleic acid.

The method for analyzing a specific nucleic acid in a sample using the probe according to the present invention detects a cleaved probe (hereinafter referred to as a fragmented probe) generated by the above-mentioned reactions repeatedly occurring in sequence. Is characterized by detecting a specific nucleic acid. The above reaction can be stopped in one cycle by appropriately setting the conditions.

The scope of the present invention also includes a kit containing the probe according to the present invention. This kit is a kit for analyzing a nucleic acid, and a nucleic acid can be analyzed by detecting a fragmented probe generated by the above-described reaction (that is, a cycle reaction).

[Reaction Filling] Next, the reaction filling of the present invention will be specifically described with reference to FIG.

A reaction solution comprising a target nucleic acid (single strand), an AP site-introduced probe DNA in excess of the target nucleic acid, and an AP endonuclease is prepared. Since the target nucleic acid must be single-stranded, if it is double-stranded, it is heated or treated with an alkali as necessary. In this reaction solution, first, probe DN
A hybridizes to the target sequence and forms a double strand.
Then, the phosphodiester bond adjacent to the AP site of the double-stranded probe is cleaved by AP endonuclease (in the case of class II AP endonuclease, it becomes "DNA-" and "-AP-DNA"). Separation). Since the AP endonuclease cleaves only the AP site of the double-stranded portion of DNA, the target nucleic acid and free probe DNA in a single-stranded state that has not hybridized are not decomposed.

When the probe is cleaved by the AP endonuclease, the binding force between the target nucleic acid and the probe DNA is reduced and the double-stranded structure cannot be maintained, resulting in the probe DN.
A comes off. As a result, the target nucleic acid returns to the original single strand. The target nucleic acid that has returned to the single strand is a new probe DNA
Therefore, the above reaction is continued as long as the probe DNA that has not been cleaved is present.

While such a cycle reaction in which the target nucleic acid repeatedly reacts with the probe DNA continues, the fragmented probe is accumulated in the reaction solution. Therefore, if the accumulated fragmented probe is analyzed, the target nucleic acid Presence can be detected. At this time, the fragmented probe is accumulated in the reaction solution due to the cycle reaction of the target nucleic acid, so that information is amplified and high detection sensitivity is brought about.

In the present invention, "probe hybridization / cleavage / dissociation / new probe hybridization"
In order to cause as many reaction cycles as possible in a short time, it is advantageous to use the probe at a concentration as high as possible. By having a large amount of probe present,
Hybridization of the new probe can occur more rapidly, and as a result, an improvement in the number of cycles can be expected. Therefore, it is the preferred use to utilize an excess of probe to the extent allowed for the expected target sequence. Specifically, 0.01 to 50 per 1 μl of reaction solution
When used in pmol, preferably 0.1 pmol or more, a high cycle number can be expected.

Regarding the conditions of the reaction solution, the temperature depends on the Tm of the probe. Generally, the reaction temperature is preferably about 10 ° C. below Tm. However, it is not a factor that can be determined only by Tm, so it is preferable to set it according to the probe. The salt concentration affecting Tm is generally adjusted with NaCl. Although it is affected by the reaction temperature, it is about 0 to 0.2M, preferably about 0 to 0.1M. However, it should be noted that at high concentrations, it may act inhibitory to the enzyme.

In order to increase the number of cycles of enzyme reaction, the melting temperature of double-stranded DNA (Tm value, Melting Temp
A reagent such as DMSO that lowers the rature) may be added to the reaction solution.

As a method of directly analyzing the fragmented probe accumulated in the reaction solution, for example, a method of directly detecting the fragmented probe DNA by electrophoresis or chromatography, or a signal generated by cleavage of the probe DNA There is a method in which a label that undergoes modulation is used as an index of a fragmented probe (the label will be described separately).

As a mode for detecting the fragmented probe as a single strand, for example, a nucleotide containing a molecule detectable by using terminal transferase or the like or a derivative thereof is added to the cleavage site of the cleaved probe, or T4RN at the cleavage site of the cleaved probe
Single-stranded DN containing a molecule that can be detected using A ligase
A can be attached. If a label or a binding molecule (such as biotin) for capturing is introduced into these additional molecules in advance, the fragmented probe can be easily detected and separated. These detection techniques are later described in 3'-
It will be described in detail as a method using OH.

In addition to the aspect in which the fragmented probe is detected as a single strand, the aspect in which the fragmented probe is detected as a double strand can also be adopted. The embodiment of detecting the fragmented probe as a double strand includes a step of capturing the fragmented probe or a modified product thereof.

Here, the reason why “capture” and not “hybridization” are used is that the “fragmented probe” cannot be maintained because it cannot maintain the double-stranded structure by itself, and the fragmented probe cannot be rehybridized under the same reaction conditions. This is because it is impossible to hybridize, and it is necessary to perform “capture” using a special technique. That is, although the fragmented probe accumulated in the reaction solution is insufficient in length for normal hybridization, it can be hybridized with a complementary sequence by a special accelerating technique. . For example, according to Japanese Patent Application No. 6-136189, a short nucleotide chain that cannot hybridize alone can form a stable hybrid only when the adjacent region of the complementary chain is double-stranded. (Hereinafter, referred to as "stacking hybridization (or simply CSH)"). That is, the fragmented probe generated by the cycle reaction has a single-stranded region having a base sequence complementary to the fragmented probe, and further hybridizes to a DNA having a double-stranded region near the single-stranded region. It can be captured by soybean.

By utilizing such a capture technique, it becomes possible to detect the presence of the fragmented probe by capturing the fragmented probe or its modified product which is dissociated after being cleaved.

The embodiment of the method for detecting by capturing the fragmented probe or its modified product is, for example,
The fragmented probe or its modified product dissociated after being cleaved has a single-stranded region having a base sequence complementary to the dissociated fragmented probe, and further has a double-stranded region near the single-stranded region. By capturing with a region-bearing DNA,
A mode of detecting the captured probe cleavage fragment or a modified product thereof can be shown. The details of the method of capturing the fragmented probe or its modified product dissociated after being cleaved, and the method of detecting the captured fragmented probe will be described later.

Here, as a probe detectable by such a capture technique, for example, a (1) terminal is not capable of hybridizing with a specific nucleic acid to be analyzed, but a capture DNA for capturing the probe is used. Has a hybridizable capture sequence, and (2) the capture sequence A
The portion extending to the P site has a sequence complementary to the specific nucleic acid to be analyzed, and (3) the portion extending from the AP site to the end opposite to the capture sequence has a specific sequence to be analyzed. A single-stranded DNA comprising a sequence that hybridizes with a nucleic acid but does not hybridize with the capture DNA.
Can be mentioned.

Furthermore, the present invention provides a method for analyzing a specific base at a specific site of a specific nucleic acid.

That is, a probe having a base complementary to a specific base to be detected adjacent to the AP site is used to analyze a specific base at a specific site of a specific nucleic acid in a sample. It is also possible.

A typical AP endonuclease used in the present invention is Exo III, but the present invention is not limited to this, and other enzymes can be used (examples of other enzymes will be described later).

Exo III has a 5'-AP endonuclease activity and a 3 '→ 5' exonuclease activity. In the presence of Mg ²⁺ , this 3 '→ 5
Even if the target nucleic acid does not exist due to the exonuclease activity, the probe DNA may be decomposed little by little and the specificity may be weakened. This problem can be solved by using Ca ²⁺ instead of Mg ²⁺ or coexisting with citric acid. Alternatively, if one or more phosphodiester bonds located at the 3'end of the probe DNA and 5'side of the AP site are changed to phosphorothioate bonds, the probe DNA is decomposed by 3 '→ 5' exonuclearase activity. There is no such thing.

[Aspect of Probe (Structure)] In the probe of the present invention or a method for analyzing a nucleic acid using the probe, the AP site of the probe is an apurinic acid site, an apyrimidic acid site, thymine glycol or urea. It is preferred that it is selected from the group consisting of a residue, tetrahydrofuran, propanediol, ethylene glycol, deoxyribitol, and α-anomer of D-deoxyribose. In this case, the apurinic acid site or the apyrimidic acid site is generated by enzymatic removal of the uracil residue in the probe, and the apurinic acid site or the apyrimidic acid site is generated by the thymine dimer generated by the irradiation of the probe with ultraviolet light. It also includes those produced by the enzymatic removal of.

Further, the AP site in a state where a part of the bases of the probe DNA used in the above reaction is lost is unstable because it is likely to be cleaved by β-elimination reaction on the 3'side of the AP site. As a solution, probe DNA A
Chemically modified P site can be used (AP site of structures such as tetrahydrofuran, propanediol, ethylene glycol: Takeshita, M.et, al.J.Biol.Chem.262.10).
l71 10179 (l987)). Since this chemically synthesized AP site is chemically very stable, nonspecific degradation does not occur, but A
Cleavable only by P endonuclease.

The probe according to the present invention may have one AP site or two or more AP sites.

Furthermore, the probe according to the present invention may contain one or more detectable molecules. Detectable molecules include radioisotopes, luminescent molecules, fluorescent molecules, fluorescent elements,
It can be selected from the group consisting of enzymes and molecules that bind to a particular molecule. Techniques for introducing these detectable molecules into probes are already known. Specifically, a method in which a functional linker is introduced into the basic skeleton of DNA and various molecules are bonded thereto, or an element or a molecular structure constituting the basic skeleton of DNA has a radioisotope or fluorescence. A method of substituting the

When a molecule that emits fluorescence or luminescence is used as the detectable molecule, it is possible to detect the cleavage of the probe from the modulation of the signal generated by the change in the distance between the molecules. For example, fluorescein and rhodamine cause energy transfer between molecules when they are close to each other, and when the distance between the two exceeds a certain range, energy transfer disappears and fluorescence of fluorescein becomes strong. When this phenomenon is applied to the present invention, if these molecules are introduced into a site where the positional relationship between the two changes due to the cleavage of the probe DNA, the cleavage of the probe DNA can be grasped as a fluorescence change of fluorescein or rhodamine.

DNA in which ethenoadenosine is introduced
In the base-base stacking action by adjacent bases, the fluorescence of ethenoadenosine is quenched, but when the DNA is cleaved, this quenching action is lost and fluorescence is generated (Biochemistry 30,1828-1835, 1991).

Furthermore, the one or more molecules having the binding property to the specific molecule to be introduced into the probe of the present invention are selected from the group consisting of, for example, avidin, biotin, an antigen, an antibody or a derivative thereof.

These binding molecules can be applied to the indirect introduction of the label utilizing the binding property or the physical separation by the specific binding to the solid phase. For example, a biotin-introduced probe can be captured on an avidin solid phase. Alternatively, the hapten-introduced probe binds to a labeled antibody that recognizes this hapten.

[Kit] A kit for nucleic acid analysis using the probe according to the present invention is also provided. This kit is capable of hybridizing with a specific nucleic acid in a sample, has AP sites bound with DNA on the 5'side and 3'side,
The P site is a kit for analyzing a nucleic acid, which contains a probe characterized in that it can be cleaved by an AP endonuclease only when the probe hybridizes with the specific nucleic acid, and the nucleic acid analysis method according to the present invention Perform a predetermined analysis according to.

[Terms Used in the Present Specification] <AP site> AP at the AP site is an acronym for Apyrimidine / Apurine, which is a prefix meaning "A does not have ..." Means "site lacking Pyrimidine / Purine". In this specification, AP is purine /
It is used to mean a nucleic acid that does not have a pyrimidine base (= Abasic).

The AP site is generated directly after irradiation with radiation, when alkylated or deaminated by a mutagen, or when dUTP is erroneously polymerized into DNA, after these abnormal bases are excised by DNA glycosylase. To do. This AP site can also be prepared in vitro. Since the generated AP site has a structure of 2-deoxyribose having no base, β-elimination is likely to occur due to alkali or heat and is unstable. In the main chain of these AP sites, nucleotides are linked by 3'-5 'phosphodiester bonds and sugars and phosphates are connected to each other, and the base moiety is deleted. There are also natural types, of which thymine glycol,
The urea residue is decomposed by Exo III (Biochemistry, 28, 3894-3901 (1989)).

On the other hand, non-nucleosides (an
There is also an AP site in which the nucleooside site) is the main chain. Probes having these non-natural sites, which can be easily synthesized and are decomposed by Exo III or endonuclease IV, include those of tetrahydrofuran, propanediol, ethylene glycol, deoxyribitol, or D-deoxyribose. There are α anomers and the like. These are very stable because they are not the structure of 2-deoxyribose (Takeshita, M.et.al.J.Biol.Chem.262.10171 10
179 (1987)). Therefore, these chemically synthesized AP sites are chemically very stable so that non-specific degradation does not occur, but they can only be cleaved by AP endonucleases. Therefore, these chemically synthesized AP sites are very suitable for the present invention.

<Specific structure of AP site>
The specific structure of the site is shown.

[0049]

Embedded image

Embedded image

Embedded image <AP probe> The probe (AP probe) according to the present invention has the following structure.

[0050]

[Table 1] 1) Ordinary AP site 2) Thymine glycol type AP site 3) Urea type AP site 4) Tetrahydrofuran type AP site 5) Propanediol type AP site 6) Ethylene glycol type AP site 7) Deoxyribitol type AP Site 8) Methoxyamine-type AP site 9) α-anomer of D-deoxyribose Where 1) is a structure lacking only the base portion of normal DNA, 2) to 9) are not found in natural nucleic acids. AP
It is a structure that can be recognized and cleaved by an endonuclease.

As mentioned above, the AP site (non-basic site) is used in the present specification to mean a nucleic acid site having no purine / pyrimidine base (= Abasic) in DNA. In addition to the AP site in which only the base moiety is deleted, such as the structure of 2-deoxyribose having no base, those in which the base moiety is non-natural such as thymine glycol and urea residue are also referred to in the present specification. It should be within the AP range. Note that these are decomposed by Exo III as shown in Examples (Biochemistry, 28, 3894-3901 (19
89)).

[1] Sequence Specific to a contiguous sequence of the target sequence. However, A
Hybridization is possible depending on the conditions as long as a certain degree of homology is maintained, even if the sequences other than both sides of the P site are not forced to be completely complementary sequences. A probe having a homology of 70 to 80% or more under general hybridization conditions can be used.

The number of bases constituting the probe sequence is an important condition that affects specificity. Probabilistically, the larger the number of constituent bases, the smaller the possibility of non-specific reaction due to coincidence. Therefore, in order to expect high specificity, it is effective to increase the number of bases constituting the probe. on the other hand,
If the probe is too long, it will be difficult to synthesize it.
When it is set to 00, preferably 50 or less, both sufficient specificity and required reactivity can be achieved, and at the same time, easy synthesis can be achieved.

[2] Bases adjacent to AP site are perfectly matched.
Cleavage activity cannot be obtained unless the 3'side of the AP site and the 5'side of class II are the same.

Depending on the characteristics of the AP endonuclease, in order to obtain sufficient cleavage activity, it is preferable that the 5'and 3'ends of the AP site are matched so that they hybridize with each other. Also, depending on the enzyme, the base adjacent to the AP site must be completely hybridized,
In some cases, it cannot be cut.

[3] Sequence and Reaction Conditions In order to proceed the reaction of “cleavage → dissociation” of the present invention, the relationship between the sequence of the entire probe DNA (the number of bases and the constituent base species) and the reaction conditions is an important factor. . That is, the probe according to the present invention is required to rapidly hybridize to the target sequence, and at the same time, the DNA on both sides of the AP site must be rapidly separated from the target sequence when cleaved at the AP site. Therefore, it is necessary to set reaction conditions such that a series of reaction cycles of “hybridization → cleavage → dissociation” according to the present invention can be rapidly rotated depending on the number of bases and base species constituting the sequence set based on [1]. There is.

It is generally known that the greater the number of bases, the easier the hybridization is and the more difficult it is to dissociate into single strands. Therefore, for example, when the number of bases constituting the probe is increased with the expectation of high specificity, it is advisable to select a condition that facilitates dissociation. 1 double-stranded DNA
Conditions that dissociate into single strands are set to a high temperature,
It is possible to show that the hybridization promoting component is not used. On the other hand, when specificity can be expected with a small number of bases, or when a probe with a small number of bases is used to detect a wide range of sequences by intentionally reducing the specificity, the reaction temperature is set to promote hybridization. Ingenuity such as setting it low is required.

Regarding the base species, GC contained in the double strand
It is known that the ratio of pairs affects the maintenance of structure. That is, for double-stranded DNA of the same length, the larger the GC content, the more difficult it is to dissociate into single strands. Therefore, when considering the reaction conditions, it is necessary to consider the GC content as well as the number of bases in the probe.

Other factors that influence hybridization and dissociation include salt concentration and pH.
In the present invention, an enzyme such as nuclease is also included in the reaction system, so it is necessary to provide conditions under which sufficient enzyme activity can be expected. Consider these conditions comprehensively and set the necessary conditions.

<AP endonuclease> In the present invention, AP endonuclease (AP-endonuclease, apurinic
/ apyrimidinic endonuclease) refers to an enzyme that recognizes a site lacking a base in DNA and decomposes a phosphodiester bond on the 5'side or 3'side thereof. For example, Escherichia coli has three kinds of enzymes, and Exo III and endonuclease IV make a cut at the 5'side of an abasic site, whereas endonuclease III cuts at the 3'side. Exo
The AP-endonuclease activity of III is responsible for over 85% of repair of alkylation damage in cells. Bacillus subtilis,
Similar enzymes to E. coli have been found in calf thymus and liver, plants, human placenta and cultured cells. In some cases of xeroderma pigmentosum, the activity of this enzyme is reduced (Seikagaku Encyclopedia, 2nd edition, Tokyo Kagaku Dojin p195).

When Exo III is used as the AP endonuclease, it is preferable to use it at a concentration of 0.01 to 10 U per 1 μl of the reaction solution. When the AP site is not a natural type but a synthetic one such as propanediol type AP,
Higher concentration of Exo III because it is less reactive than natural AP
You can get better results by using. As a guide, it is preferable to use about 10 times as much as the natural AP site, and specifically, a concentration of 1 U or more per 1 μl of the reaction solution is suitable.

Escherichia coli Exo III has an enzymatic activity of hydrolyzing various types of phosphodiester bonds of double-stranded DNA. That is, 3 '->5' exonuclearase activity that releases 5'-mononucleotides sequentially from the 3'end of the DNA chain, and 3'which hydrolyzes the phosphomonoester at the 3'end.
A phosphotase activity and a 5 ′ → AP endonuclease activity that hydrolyzes a phosphodiester bond on the 5 ′ side of an apurinic acid or apyrimidic acid site. Also, D
NA: RN that degrades only RNA of RNA hybrid
It also has aseH activity. Although the Mg ²⁺ presence various nuclease activity is activated to suppress the 3'→ 5'Ekusonureaze activity by the coexistence or using Ca ²⁺ in place of Mg ^2+, or citric acid, approximately It is possible to have only 5 '→ AP endonuclease activity.

Since the 3 '→ 5' exonuclease activity cannot decompose modified bonds such as phosphorothioate bonds, the phosphodiester bond at the 3'end of the probe DNA or at the 5'side of the AP site is changed to a phosphorothioate bond. On the contrary, the probe DNA is not decomposed due to the activity of 3 '→ 5'exonuclease, and 5'AP
It is possible to activate only the endonuclease activity. In this case, the phosphorothioate bond may be conferred with almost complete enzyme resistance by modifying several bonds, preferably about three bonds from the end, rather than one.

In addition to Exo III described above, endonuclease IV and endonuclease of bacteria such as Escherichia coli and Bacillus subtilis
III, enzymes with double-strand-specific AP endonuclease activity, such as plant and calf thymus and liver (BJSSander
son et al., Biochemistry, 28,3894-3901 (1989)), an enzyme derived from human placenta (APEX nuclease: L. Harrison eta)
l., Human Molecular Genetics, 1,677-1680 (l992)) and the like can be used for this reaction. Exo III and Bovine
Apurinic Endonuclease recognizes the double-stranded structure and AP
Only the site is decomposed specifically. Furthermore, cyclobutane-type pyrimidine dimer and 6-
Although it is possible to use an enzyme that decomposes 4-photoproducts, it is necessary that the AP site in the probe DNA be a cyclobutane-type virimidine dimer or 6-4 photoproducts produced by ultraviolet irradiation or the like (KKBowman et al.
al., Nucleic Acid Res. 22, 3026-3032 (l994)).

Incidentally, Japanese Patent Laid-Open No. 62-
Exo III is also used in Japanese Patent Publication No. 190086, but it is clearly different from the present invention in that it uses different activities (or substrate specificities). That is, the RNase H activity of Exo III is used in the invention described in JP-A-62-190086, but such activity is not used in the present invention (the AP site in the present invention should be cleaved by RNase H). Cannot be done), so the two are clearly distinguished.

<AP Endonuclease that can be used in the present invention> Enzymes having AP endonuclease activity are classified into two types according to their cleavage activity. One is class II that cuts the 5'side of the AP site, and the other is class I that cuts the 3'side of the AP site.

[0067]

[Chemical 4] [Use of class I enzyme] class I enzyme (3 'of AP site
3'-AP site-O as a result of cleavage by
H and 5'-P are generated. Since the 3'-AP site-OH generated here is not hydrogen-bonded to the template chain, class II
Unlike the above case, it cannot function as a primer for DNA polymerase. However, the 5'-phosphorylated DNA generated by the decomposition serves as a substrate for T4 DNA ligase and E. coli DNA ligase, so that it becomes possible to bind to a separately prepared DNA having a 3'OH end, and the degradation product is used. Only will be able to detect.

Class I enzymes also include enzymes having a glycosylase activity, such as endonuclease derived from bacteriophage T4. This has two enzyme activities at the same time, but when using a probe having an AP site in advance, a system utilizing only AP-endonuclease activity is possible. Alternatively, the following system can be constructed by positively utilizing the combined activity of the respective enzymes and allowing the probe to have a structure recognized by the enzyme even if the probe does not have an AP site in advance. That is, the bacteriophage T4 endonuclease recognizes thymine photodimer,
It becomes possible to introduce a thymine photodimer into the probe by UV irradiation or the like, form an AP site by glycosylase activity, and cleave the probe by AP-endonuclease activity of the same enzyme.

[Use of class II enzyme] As a result of cleavage by class II (5 'side cleavage of AP site), 3'-OH and 5'
A'-P-AP site is created.

In the present invention, a detection method utilizing 3'-OH generated by a class II enzyme, for example, a chain extension reaction by a DNA terminal transferase is possible. The DNA-RNA chimera probe introduced as the prior art cannot adopt this type of reaction because it has an RNA molecule at the end.

On the other hand, the 5'-P end generated by the class II enzyme can be used as a substrate for λ exonuclease or the like.
Therefore, for example, the decomposed product after the reaction can be hybridized by applying CSH to form a double strand, which can be detected using the enzymatic decomposition from the 5'-end as an index.

In the present invention, it is important that the AP endonuclease, whether class I or class II, decomposes only the probe hybridized with the target (in other words, the single-stranded probe does not decompose). It is difficult to determine whether or not it is completely specific for double strands, but since this is a matter of degree, the enzymes shown below with high double strand specificity are not suitable for the present invention. Can be used. In particular, in the case of Exo III, the single-strand break activity from the 3'end can be reduced to below the detection limit in the presence of Ca ²⁺ or citric acid.

Examples of enzymes that can be used in the present invention are shown below.

[0074]

[Table 2] class II AP endonuclease: an enzyme that cleaves the 5'phosphodiester bond at the AP site Origin Name Reference E. coli Exonuclease III Endonuclease IV J.Biol.Chem.263,8066-9071 1988 S. serevisiae Apn1 Proc.Natl.Acad.Sci.USA87, 4193-4197 1990 Hemophilus DNase Adv. Enzymol. 60,1-34 1987 influenzae (no unique name) Streptococcus ExoA J. Bacteriology 171,2278-2286 pneumoniae 1989 mouse APEX nuclease J. Biol. Chem. 266, 20797-20802 1991 bovine BAP1 (bovine AP endonuclease I) Biochemistry 28, = APE (apurinic endodeoxy 3894-3901 1989 ribonuclease) human APEX = HAP1 = APE Biochem.Biophys.Acta 1131, 287-299 1992 class I AP endonuclease: an enzyme that cleaves the 3'phosphodiester bond at the AP site E. coli Endonuclease III Biochemistry 28,4444-4449 1989 bacteriophageT4 T4 endonuclease V J.Biol.Chem. 264,2609-2614 1989 S.serevisiae Redoxyendonuclease Biochemistry 27,2629-2634 1988 M.luteus UV endonuclease Method in Enzymology 65, 185-191 1980 Appropriate enzyme selection for the AP site can be made by one skilled in the art as appropriate, eg Exo III for tetrahydrofuran, propanediol, ethylene glycol.

<Selection and Application of Target Sequence> In the present invention, the target sequence is not limited to DNA, and even RNA may be a target sequence if it is DNA by reverse transcription. Moreover, the origin of the nucleic acid is not limited. Furthermore, even if it is double-stranded, it can be used as a target sequence by denaturing it into single-stranded one.

The probe of the present invention has a special property when the flanking sequence of the abasic site is not complementary to the target. That is, when Exo III is used as the enzyme and the reaction is carried out in the presence of Ca ²⁺ , the base adjacent to the AP site 5 ′ side of the probe is not easily cleaved when it is not complementary. This feature can be used to detect mutations in gene sequences. That is, by controlling the sequence adjacent to the 5'side of the AP site in the probe, it is possible to construct a system that is cleaved only when a mutation occurs in the target sequence or only when it is normal. For example, in Examples (ApoE3 and ApoE4), when a probe in which one base on the 5'side of the AP site is changed is used, the reaction proceeds only when this portion is completely complementary to the sequence to be detected. I have confirmed that. Thus, when the probe of the present invention is used, the bases on the 3'side and / or the 5'side of the AP site have a base complementary to the specific base to be detected. It is possible to detect that there is (claim 12).

Some mutations in the gene sequence are known in advance after the mutation, and in this kind of mutation, a probe complementary to the sequence after the mutation can be prepared.

<Detection Method of Fragmented Probe> The fragmented probe (cleavage fragmented probe) produced by carrying out the present invention is basically prepared by a technique such as electrophoresis or liquid chromatography according to a conventional method. Can be detected.

{Detection method A of fragmented probe} <Method utilizing CSH phenomenon (Japanese Patent Application No. 6-136189)> In the present invention, the Tm of the cleaved probe is lowered and the double strand cannot be maintained. The phenomenon of returning to the main chain is the basis of the cycle reaction. Therefore, under normal conditions, the cleaved probe fragment cannot be hybridized again. However, even in a fragmented probe that cannot form a double strand by itself, if adjacent portions form a double strand in advance, hybridization is promoted (C
SH phenomenon; Japanese Patent Application No. 6-136189). Specifically, if a capture probe (sometimes referred to as a stacking probe) having a partially double-stranded structure having the following relationship is prepared, fragments generated by cleavage are captured one after another. It is possible to continue. In the probe of the present invention, since the end of the fragmented probe is a DNA base, it can be further ligated with a DNA ligase derived from T4 phage or E. coli. If the capture probe having the following relationship is immobilized on the solid phase separately from the AP probe, the fragmented probe can be captured on the solid phase. The detection of whether or not the fragmented probe has been captured is performed by previously labeling the probe of the present invention with a fluorescent label (other labels may be used), and detecting the fluorescence intensity of the solid phase after the reaction (performed by "other label"). In this case, the detection can be carried out easily by carrying out detection corresponding to the label.

As a label other than fluorescence, for example, biotin can be used. In this system, a biotin-labeled probe can be captured by CSH and detected by, for example, an avidinating enzyme. Alternatively, a commercially available biotinylated enzyme may be bound via avidin. Biotinylated acetate kinase and the like are commercially available as biotinylated enzymes, and extremely high sensitivity can be expected if ATP produced by acetate kinase is detected by bioluminescence reaction by luciferase.

The fragmented probe captured by CSH was ligated to the adjacent sequence to form a continuous DNA fragment.
With this, a more stable double strand can be obtained. T4 DNA ligase or the like is used for ligation. Since the 5'end of the probe obtained by ordinary chemical synthesis is not phosphorylated, this portion is not ligated with ligase. If the double-stranded chain is made more stable in this way and then washed with an alkali (for example, 0.1 N NaOH solution), the labeled components and the like that are nonspecifically adsorbed can be strongly removed, and
Since only the main strand can be left, detection with low background becomes possible.

The same effect of stabilizing a double strand can be realized by applying the technique described in, for example, Japanese Patent Publication No. 61-500688 (WO85 / 02628). In this prior art, the double chain formed by hybridization is crosslinked by a covalent bond to form a stable double chain structure. The crosslinked structure formed by the covalent bond is not removed by washing or the like.

[0083]

[Formula 5] {Formula 5} Shown above is the basic embodiment of a fragmented probe capture method that applies CSH. In addition to such an aspect, the following trapping forms can be used, for example. In any of the examples, the capture DNA can be bound to the solid phase at the DNA end or at any site where an appropriate linker is introduced.

1) Only one double-stranded structure in formula 5 is used.

2) The double-stranded structure is not a hybrid of two DNAs but a hairpin structure formed by folding back the ends of one DNA. In this case, it is possible to fold both ends to form a structure as shown in Formula 5.

This example is a characteristic example of the present invention in that it cannot be used in the conventional method in which the number of bases of the fragmented probe is not uniform.

If a probe having a structure satisfying the following conditions (1)-(3) is adopted as the probe, it is possible to capture the fragmented probe with simple single-stranded DNA without using CSH.

(1) A capture sequence that cannot hybridize with a specific nucleic acid to be analyzed but is hybridizable with a capture DNA that captures the probe is added to the end (2) and The part from the sequence end to the abasic site has a sequence complementary to the specific nucleic acid to be analyzed, (3) Furthermore, the part from the abasic site to the end opposite to the capture sequence is the analysis target. Which has a sequence that hybridizes with a specific nucleic acid, but does not hybridize with the capture DNA.

However, in such a capture system, a probe that is not fragmented can also hybridize with the capture DNA, so that the 3'-OH generated by the cleavage of the abasic site is generated.
A combination with a detection system that utilizes

{Detection method B of fragmented probe} <3′-OH of fragmented probe generated by cleavage of AP site
Method Using Ends> In the present invention, a DNA having a constant fragmentation probe length and a 3′-OH end is always produced in combination with a class II enzyme. Therefore, a special technique utilizing this is used. Can be adopted. The methods for detecting fragmented probes are listed below.

<Method 1 utilizing 3′-OH end; Method utilizing terminal transferase> A typical detection method utilizing the fact that a fragmented probe generated by a class II enzyme has a 3′-OH end is , A detection method using terminal transferase. Since this enzyme specifically recognizes 3'-OH and binds to another nucleotide chain, only 3'-OH generated by cleavage can be specifically detected. In the case of terminal transferase, it is not required that 3'-OH is hybridized with the template, and therefore, it can be reacted as a single strand.

Terminal transferase (Terminal
The method using deoxyribonucleotidyl transferase (TdT)) is specifically performed as follows. 5 of AP site
Prepare a probe in which the 3'end is labeled with biotin and the 3'end was dideoxylated, and after the reaction, the 3'-OH end generated for the first time after the reaction contains a fluorescent substance (of course, other labeling substances may be used) by TdT. Elongate the polymer of nucleic acid analogs. Then, the biotin on the 5 ′ side is used to trap the streptavidin solid phase carrier, and the fluorescence intensity of the solid phase is detected. In this system, a labeling substance is connected to the 3'side of the fragmented probe, so that very high sensitivity can be expected.

<Method 2 using the 3′-OH end; T4
Method using RNA ligase (reference; Analytical Bio
Chemistry 158, 171-178 1986)> Detection similar to the method using TdT described above can also be performed using T4 RNA ligase. In carrying out this method, the probe according to the present invention (probe having an AP site)
Dephosphorylate the 5'end of (in normal DNA synthesis,
The phosphoric acid is not attached) and its 3'end is dideoxylated, and at the same time, the 5'side of the AP site is labeled with biotin.

T4 RNA ligase efficiently binds single-stranded DNA chains to each other in the presence of hexamine cobalt chloride and polyethylene glycol. If T4 RNA ligase was added to the reaction system together with an oligonucleotide (phosphorylated at the 5'end and dideoxylated at the 3'end) containing a fluorophore (other labeling substances are acceptable), it was generated after the reaction. An oligonucleotide containing the fluorophore is bound to the 3'-OH end of the fragmented probe. After that, as in the method using TdT, the presence or absence of the target nucleic acid can be detected by detecting the fluorescence intensity trapped in the avidin solid-phase carrier using biotin on the 5 ′ side.

<Method 3 using 3′-OH end> The cycling reaction (Japanese Patent Application No. 5-337863) in which the DNA polymerase and the Exo III, which the applicant filed in the past, is combined is applied. This cycling reaction repeats a reaction cycle of "decomposing a base added by a polymerase with a nuclease". Using a nuclease-resistant primer in advance, a base (1
Base can be used). When the added base is decomposed by nuclease, the base is added again by the polymerase. This cycle reaction can be traced with high sensitivity using pyrophosphoric acid generated by the addition of a base or a decomposition product of the base as an index. Since the fragmentation probe of the present invention has a proper base sequence, such a reaction system can be applied to the detection of the fragmentation probe. In this case as well, since the fragmented probe cannot hybridize alone, it is necessary to apply CSH. Further, when applying this method, it is also necessary to modify the 3'ends of the fragmentation probe and the stacking probe so as to be Exo III resistant.

Specifically, the reaction is carried out using the probe according to the present invention in which the 3 ′ end is Exo III resistant. After the reaction, deoxynucleoside triphosphate,
The presence of the target nucleic acid can be confirmed by adding DNA polymerase and Exo III and detecting the resulting pyrophosphate or nucleoside monophosphate.

<4 'utilizing the 3'-OH end; DN
Method using A polymerase>3'of fragmented probe
-OH is used in addition to terminal transferase
It can be detected by the action of A polymerase. This enzyme, like terminal transferase, also has 3'-
Since it specifically recognizes OH and binds to other nucleotide chains, it is possible to specifically detect only 3'-OH generated by cleavage. However, in the case of terminal transferase, the reaction can be carried out as a single strand, but in the case of DNA polymerase, it is required that 3'-OH is hybridized with the template.
This method differs from the above methods 1 to 3 in that the fragmented probe generated by cleavage needs to be double-stranded.

Here, the fragmented probe cannot be used as a primer because it cannot maintain a double strand because the number of bases is insufficient. However, the previously mentioned CSH
By using, it is possible to hybridize even a short sequence that cannot form a double strand by itself and to function as a primer, so that it becomes possible to use DNA polymerase.

When detection is carried out using a DNA polymerase, the 3'side is modified beforehand by dideoxylation or phosphorylation of the 3'-OH group so as not to serve as a substrate for the polymerase, or 3'side. According to the present invention, A
Cleave the P site. Then, the original probe does not serve as a primer, but it is not modified by cleavage to produce 3 '.
A fragmented probe with -OH end was generated,
If H is used, it can function as a primer. Utilizing this feature, a system for initiating a reaction by a DNA polymerase only when cleavage occurs can be constructed, and a fragmented probe can be detected by using a nucleic acid synthesis reaction by a DNA polymerase as an index.

Specifically, a CSH stacking probe is supported on a solid phase in a system to which a DNA polymerase is added, and the fragmented probe is allowed to hybridize there. In this case, the fluorescently labeled nucleotide is used as a substrate and the fragmented probe is used as a primer to extend the DNA chain. When the AP site of the probe according to the present invention is cleaved, the fragmented probe hybridizes to the stacking probe carried on the solid phase to serve as a primer, and the DNA chain is elongated by the action of DNA polymerase. Since the elongation of the DNA chain is accompanied by the incorporation of the fluorescently labeled nucleotide, it is possible to determine whether or not the target nucleic acid is present by detecting the fluorescence of the solid phase. If the 5'side of the AP site of the probe according to the present invention is fluorescently labeled in advance, the presence or absence of the target nucleic acid can be determined only by detecting the fluorescence of the solid phase without extending the DNA chain.

{Detection method C of fragmented probe} The reaction is carried out by preparing a probe in which the 5 ′ side of the AP site is labeled with fluorescein (other labels may be used) and the 3 ′ side is labeled with biotin. After the reaction, the streptavidin solid-phase carrier is allowed to trap both unreacted and decomposed 3'side fragments of the AP site. In this case, when the AP site is not decomposed, the 5'side labeled with fluorescein is also trapped along with the 3'side labeled with biotin.
On the other hand, when the AP site is decomposed, only the biotin-labeled 3 ′ side is trapped and the fluorescein-labeled 5 ′ side remains in the solution. in this way,
In this system, a fluorescent component is detected in the liquid phase only when the probe DNA is cleaved. Therefore, by measuring the fluorescence of the fragment on the 5'side of the decomposed AP site remaining in the solution, the target nucleic acid The presence or absence of can be detected.

{Detection method of fragmented probe D} Prepare a probe in which 5'side of AP site is labeled with fluorescein and 3'side is labeled with rhodamine (or vice versa), and energy transfer occurs in the distance between each labeled product. Range. After the reaction, energy transfer does not occur in the decomposed probe, and the fluorescence intensity of rhodamine decreases due to the energy transfer (fluorescence intensity of fluorescein increases), so the amount of decomposed probe can be quantified (reference: Nucleic acids Reseac
h 22, 920-928, 1994).

[0103]

The probe according to the present invention is a known probe R
The storage stability is improved simply by replacing the NA part with the AP part. Further, it has an advantage that it can be easily synthesized.

That is, with the DNA-RNA chimera probe, only the RNA located at the terminal can be decomposed,
Although RNA in the middle part cannot be degraded, it is possible to surely cut at a specific site by using a probe having an AP site.

Since the probe of the present invention has an AP site, it can be combined with an AP endonuclease to form a DNA-RNA-DNA chimera probe and an RNase.
Many effects (specifically, for example, the following effects) that cannot be achieved by the combination of are possible.

The probe according to the present invention has a chemically stable bond and is unlikely to undergo nonspecific decomposition. The AP endonuclease used specifically recognizes double-stranded DNA and decomposes it, but hardly decomposes the single-stranded probe. Therefore, the presence of the target DNA can be specifically detected. On the other hand, DNA-RNA-
When a DNA chimera probe is used, there is a structurally unstable RNA portion, and it is easily decomposed by a nonspecific RNA degrading enzyme that easily mixes, so that the background is likely to increase. .

In the present invention, a type 1 or type 2 AP
It is also an advantage that the range of detection of the fragment after cleavage can be widened by properly using the endonuclease properly. When any of the AP endonucleases is used, the cleavage point is strictly defined, and the fragment after the cleavage is stable DNA, so clear results can be obtained. However, the degradation reaction of the chimeric probe with RNase H yields those having one or more RNA molecules at the ends. The RNA molecules at this end are particularly labile and easily degraded, so that the final product is a mixture of those with and without the RNA portion. Therefore, it is expected that detection of the final product will be difficult.

More importantly, the AP endonuclease recognizes the base adjacent to the AP site. Therefore, when adjacent bases are mismatched, the decomposition reaction does not occur, and by utilizing this, the mutation point can be detected.

[0109]

EXAMPLES The preferred examples of the present invention will be shown below, but the present invention is not limited thereto. [Example 1] AP probe (structure 1) / 5 ′ end
³² P-labeled <Target DNA sequence> As a material for the model experiment, a part of the e-antigen gene of HBV viral nucleic acid was added to M13mp18DN.
The one cloned on A was used. The probe sequence complementary to the target DNA is shown below.

[0110]

Embedded image 5′-AATGCCCCTATCTTATCAACACTTC-OH <Preparation of probe nucleic acid> As a probe nucleic acid, an oligonucleotide having the base sequence shown below was chemically synthesized and used.

[0111]

Embedded image 5'-AATGCCCCTATCUTATCAACACTTC-OH and T residues in the probe <Preparation of Probe (AP probe) with AP sites> After chemically synthesized oligonucleotides with U residues, the 5 'end with ³² P Labeled. Next, the U residue was deuracilated by treatment with uracil DNA glycosylase. By this operation, the base uracil is lost and the AP site is formed.

<Signal Amplification Reaction> The deuracilated probe DNA and the DNA complementary thereto were mixed, and Exo III
Was added and reacted (reaction solution composition: 50 mM Tri
s-HCl pH 8.0, 50 mM NaCl, 10
mM 2-mercaptoethanol, 5 mM CaC
l ₂ , 0.1 μg / μl BSA, 1 pmol / μl
Probe labeled with ³² P at the 5'end (20 pmol /
tube), 0-1 pmol target DNA, 1 U / μl
Exo III (20U / tube), final concentration 20% DMS
O, total volume of reaction solution 20 μl).

First, as a pretreatment, the above reaction solution except the enzyme (Exo III) was boiled for 5 minutes and left on ice for 1 minute.
It was centrifuged. Then add the enzyme (Exo III),
Incubation was carried out at 37 ° C for 60 minutes. After the reaction, 0.5M EDTA
5 μl and 5 μl of formamide dye were added and boiled for 5 minutes, which was electrophoresed on a polyacrylamide gel (20%) containing 7 M urea, and the fragmented probe was detected by autoradiography.

<Results> When the target DNA was present, the fragmented probe was detected on the polyacrylamide gel,
It was confirmed that the decomposition of the AP probe occurred. On the other hand, when the target DNA was not present or when DNA having no complementarity was targeted, fragmentation of the AP probe did not occur in either case. Therefore, this reaction is the target DNA
Was found to be specific to.

Degradation of probe DNA was observed even when the target DNA concentration was 0.1 pmol / μl. Furthermore, the target DN
When the A concentration was 0.1 pmol / μl or more, it was confirmed that a cycle reaction of about 500 times occurred because all probe DNAs were decomposed.

[Example 2] AP probe (structure 5) / 5
The 'end as the material of the ³² P-labeled <target DNA sequence> model experiments, M13mp18DN part of e antigen gene in HBV viral nucleic acid
The one cloned on A was used. The probe sequence complementary to the target DNA is shown below.

[0117]

Embedded image 5′-AATGCCCCTATCTTATCAACACTTC-OH <Preparation of probe having AP site (AP probe)> As a probe nucleic acid, an oligonucleotide having a base sequence shown below was chemically synthesized and used.

[0118]

Embedded image 5'-AATGCCCCTATCXTATCAACACTTC-OH: After the T residues (X AP site) in the probe was chemically synthesized oligonucleotides with AP sites having the structure of propanediol were labeled 5 'end with ³² P . The structure of propanediol is Spacer P from Prime Synthesis.
Hosphoramidite C3 was used.

<Signal Amplification Reaction> A probe DNA and a DNA complementary thereto are mixed, Exo III is added,
Reacted (reaction solution composition; 50 mM Tris-HCl
pH 8.0, 50 mM NaCl, 10 mM 2-mercaptoethanol, 5 mM CaCl ₂ , 0.1 μg /
μl BSA, 1 pmol / μl 5 ′ probe labeled with ³² P (20 pmol / tube), 0-1
pmol target DNA, 1 U / μl Exo III (20 U /
tube), final concentration 20% DMSO, total reaction solution 20
μl).

First, as a pretreatment, the above reaction liquid excluding the enzyme (Exo III) was boiled for 5 minutes and allowed to stand on ice for 1 minute.
It was centrifuged. Then add the enzyme (Exo III),
Incubation was carried out at 37 ° C for 60 minutes. After the reaction, 0.5M EDTA
5 μl and 5 μl of formamide dye were added and boiled for 5 minutes, which was electrophoresed on a polyacrylamide gel (20%) containing 7 M urea, and the fragmented probe was detected by autoradiography.

<Results> When the target DNA was present, the fragmented probe was detected on the polyacrylamide gel,
It was confirmed that the AP probe was decomposed.
On the other hand, fragmentation of the probe DNA did not occur when the target DNA was not present or when DNA having no complementarity was targeted. Therefore, it was found that this reaction was atypical for the target DNA.

Degradation of the probe DNA was observed even when the target DNA concentration was 0.1 pmol / μl. Furthermore, the target DN
When the A concentration was 0.1 pmol / μl or more, it was confirmed that a cycle reaction of about 500 times occurred because all probe DNAs were decomposed.

[Example 3] AP probe (structure 5) / 5
'P-end labeled with ³² P <target DNA sequence> As a material for model experiments, human Ap
oE3 and part of the ApoE4 gene were replaced with Ml3mp18
The one cloned on DNA was used. Target DNA
The probe sequence complementary to is shown below. The labeled human ApoE3 and E4 gene sequences have highly homologous sequences in which only 1 base out of 24 bases is different.

[0124]

Embedded image ApoE3: 5′-CGGCCGCACACGTCCTCCA-3 ′ ApoE4: 5′-CGGCCGCGCACGTCCTCCA-3 ′ <Preparation of probe having AP site (AP probe)> A
As the P probe, an oligonucleotide having the base sequence shown below was chemically synthesized and used.

[0125]

Embedded image ApoE3: 5′-CGGCCGCAXACGTCCTCCA-3 ′ ApoE4: 5′-CGGCCGCGXACGTCCTCCA-3 ′ (X: AP site) An oligonucleotide having an AP site (indicated by X) having a propanediol structure in the probe after chemically synthesized and labeled 5 'end with ³² P. The structure of propanediol is
Spacer Phosphoamidite C3 was used.

<Signal Amplification Reaction> The probe DNA and the target DNA were mixed, and Exo III was added and reacted (reaction solution: 50 mM Tris-HCl pH 8.0,
5 mM CaCl ₂ , 10 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mg / ml BS
A, 20% DMS0). After the reaction, electrophoresis was performed on a polyacrylamide gel, and the fragmented probe DNA was detected by autoradiography.

<Results> It was confirmed that each AP probe for ApoE3 or ApoE4 produces a fragmented probe only when the corresponding target gene is present. The production of fragmented probes when the target gene was replaced with a non-corresponding one was slight, and it was revealed that the present invention can recognize the difference in bases.

In all of the detection systems of Examples 1 to 3, the cleavage fragment was detected only by the ³² P label at the 5'end, but only in Example 3 the present invention can recognize a difference of 1 base. It was confirmed.

[0129] Since the AP probe was decomposed in a larger amount than the target DNA, it was confirmed that the expected cycle reaction had occurred.

Needless to say, with either probe,
No fragmentation of the probe DNA occurred when the target DNA was not present or when the DNA having no complementarity was targeted. Therefore, this reaction was found to be specific to the target DNA.

[0131]

As described above, the present invention has the following useful effects.

Since the signal can be amplified without special temperature control operation, the operation is simple and the automation is easy.

Since the product increase proceeds linearly, target D
Quantification of NA is easy.

Hardly affected by contamination.
That is, since the probe product decomposed by AP endonuclease is detected, even if the product after the reaction is contaminated, the contamination is not amplified.

Probe synthesis is easy and inexpensive.

The probe is stable and is not susceptible to non-specific degradation.

It has conditions advantageous for detection such that the length of the degradation product is constant or it has 3'-OH.

<Related Literature> 1) P. Duck et al .: Probe Amplifier System Based on
Chimeric Cycling Oligonucleotides, BioTechniques 9,
142-147 (1990) 2) ID Biomedical: Will CPR beat PCR?: The Genesis R
eport / Dx, 2-8 (1994) 3) Japanese Unexamined Patent Publication No. 61-290326, "Molecules Effective in Detection of Nucleic Acid Sequences" Inventor: Peter. Duck, Robert Bender Applicant: Miogenics Incorporated 4) Japanese Patent Publication No. 2-504110 “Nucleic acid sequence detection method”
Inventor: Peter Duck, Robert Bender Applicant: Miogenics Incorporated 5) M.Takeshita et al.:Oligodeoxynucleotides Conta
inig Synthetic Abasic Sites, Model Substrates for D
NA polymerases and apurinic / apyrimidinic endonucle
ases, J. Biol. Chem. 262, 10171-10179 (l987) 6) CC Richardson and A. Kornberg: A Deoxyribonucle
ic acid Phosphatase Exonuclease from Escherichia co
li, I.Purification of the enzyme and characterizati
on of the phoshatase activity, J. Biol. Chem. 239,242-
250 (1964) 7) C, C. Richardson, IR Lehman and A. Kornberg: A Deo
xyribonucleic acid Phosphatase Exonuclease from Es
cherichia coli, II. Characterzation of the exonu
clease activity, J. Biol. Chem. 239, 251-258 (1964) 8) SG Rogers and B. Weiss: Exonuclease III of E.co
li K 12, and AP Endonuclease, Methods In Enzymology,
65,201-211 (1980) 9) S.Liungquist: Endonuclease IV from E.coli, Meth
ods In Enzymology, 65,212-216 (1980) 10) G. Vesnaver et al .: Influence of abasic and an
ucleosidic sites onthe stability, conformation, and
melting behavior of a DNA duplex, Correlations of t
hermodynamic and structural data, Proc.Natl.Acad.Sc
i.USA, 86,3614-3618 (1989) 11) B. Sanderson et al .: Mechanism of DNA cleavage
and Substrate Recognition by a Bovine Apurinic En
donuclease, Biochemisty 28, 3894-3901 (1989) 12) L. Harrison et al .: Human apuricic Endonucleas
e gee, structure and genomicmapping, Human Mol. Geneti
cs, 1, 677-680 (1992) 13) Nucleic acid detection method JP-A-6-169800 Inventor: Hideki Kanbara et al., Applicant: Hitachi, Ltd.

[Brief description of drawings]

FIG. 1 is a diagram for explaining a state of a cycle reaction by a probe according to the present invention.

Claims (29)

[Claims] 1. A hybridizable nucleic acid in a sample, which has at least one abasic site to which DNA is bound on the 5'side and the 3'side. Can be cleaved by AP endonuclease only when hybridized with the specific nucleic acid,
A probe characterized in that only the cleaved probe can be dissociated from the specific nucleic acid. 2. A probe used in the analysis of a specific nucleic acid in a sample, characterized in that the following reactions occur in sequence repeatedly. A probe having a structure in which DNAs are bound to the 3'side and the abasic site, and can be cleaved by an AP endonuclease only when the probe hybridizes with the specific nucleic acid. (1) a reaction in which the specific nucleic acid in the sample hybridizes with the probe, (2) a reaction in which the abasic site of the probe hybridized with a double-stranded DNA-specific AP endonuclease is cleaved, (3 ) A reaction in which the cleaved probe dissociates from the specific nucleic acid. 3. The abasic site of the probe comprises an apurinic acid site, an apyrimidic acid site, thymine glycol, a urea residue, tetrahydrofuran, propanediol, ethylene glycol, deoxyribitol, and D-deoxyribose α-anomer. The probe according to claim 1, which is selected from the group. 4. The probe according to claim 1 or 2, wherein the probe having an abasic site contains a detectable molecule. 5. The detectable molecule is selected from the group consisting of a radioisotope, a luminescent molecule, a fluorescent molecule, a fluorescent element, an enzyme, and a molecule capable of binding to a specific molecule. The probe described in. 6. The detectable molecule is selected from the group consisting of a luminescent molecule, a fluorescent molecule, and a fluorescent element, and the optical signal generated from these detectable molecules is modulated by the cleavage of the probe. The probe according to 4. 7. The probe according to claim 6, wherein there are two or more abasic sites. 8. The probe according to claim 5, wherein the molecule having a binding property with a specific molecule is selected from the group consisting of avidin, biotin, an antigen, an antibody or a derivative thereof. 9. The probe according to claim 1, wherein the hydroxyl group at the 3 ′ end of the probe is modified in a predetermined manner. 10. The probe according to claim 9, wherein the 3′-terminal hydroxyl group of the probe is modified so as not to be extended or added by terminal transferase, ligase, or a predetermined polymerase. 11. A specific nucleic acid in a sample is analyzed by detecting the cleaved probe accumulated in the system, which is characterized in that the following reactions (1) to (3) are repeated in order. Way for. (1) A specific nucleic acid in a sample, which can hybridize with the specific nucleic acid, and is located on the 5'side and 3'of the abasic site.
It has a structure in which DNA is bound to the side, and the abasic site is A only when the probe hybridizes with the specific nucleic acid.
A reaction which hybridizes with a probe characterized by being cleavable by P endonuclease, (2) 2
A reaction in which the abasic site of the probe hybridized with the double-stranded DNA-specific AP endonuclease is cleaved, and (3) a reaction in which the cleaved probe is dissociated from the specific nucleic acid. 12. AP present in the abasic site of the probe
3'side of cleavage site by endonuclease, and /
Alternatively, the 5'side base has a base complementary to a specific base to be detected.
The method described in. 13. The method according to claim 11, wherein the AP endonuclease is Escherichia coli exonuclease III (Exo III). 14. The cleavage reaction of the probe is carried out in the presence of Ca ²⁺ ion or citric acid.
The method described in. 15. The α-base of the non-basic site is an apurinic acid site, an apyrimidic acid site, thymine glycol, a urea residue, tetrahydrofuran, propanediol, ethylene glycol, deoxyribitol, D-deoxyribose.
The method according to claim 11, characterized in that a probe selected from the group consisting of anomers is used. 16. The method according to claim 11, wherein the cleaved probe is separated and detected by electrophoresis or chromatography. 17. The method according to claim 11, characterized in that a probe containing a detectable molecule is used. 18. The detectable molecule is a radioisotope,
18. The method according to claim 17, wherein the method is selected from the group consisting of a luminescent molecule, a fluorescent molecule, a fluorescent element, an enzyme, and a molecule having a binding property with a specific molecule. 19. The detectable molecule is selected from the group consisting of a luminescent molecule, a fluorescent molecule, and a fluorescent element, and the optical signal generated from these detectable molecules is modulated by cleavage of the probe. The method according to 4. 20. The method according to claim 19, wherein there are two or more abasic sites. 21. Capturing a probe fragment dissociated after being cleaved by AP endonuclease, or a modified product thereof or an undecomposed probe.
The method according to 1. 22. A probe fragment dissociated after being cleaved by AP endonuclease, or a modified product thereof or an undecomposed probe, having a single-stranded region having a base sequence complementary to the dissociated probe, 22. The method according to claim 21, further comprising capturing with a DNA having a double-stranded region near the single-stranded region. 23. The captured probe is used as a primer,
The method according to claim 22, wherein the nucleotide synthesis reaction is carried out by a DNA polymerase. 24. A DN having a cleaved probe, a single-stranded region having a base sequence complementary to the cleaved probe, and further having a double-stranded region near the single-stranded region.
23. The method according to claim 22, which comprises reacting with DNA polymerase, exonuclease, and at least one deoxynucleotide triphosphate after being captured by A, and detecting the resulting pyrophosphate or nucleotide monophosphate. 25. As a probe, at (1) end, a capture sequence that cannot hybridize with a specific nucleic acid to be analyzed but is capable of hybridizing with a capture DNA that captures the probe is added, (2) And, the portion from the end of the capture sequence to the abasic site has a sequence complementary to the specific nucleic acid to be analyzed, (3) Furthermore, from the abasic site to the end opposite to the capture sequence. The entire portion is composed of a sequence that hybridizes with a specific nucleic acid to be analyzed but does not hybridize with the capture DNA 1.
Single-stranded DNA is used, and as the capture DNA, single-stranded DNA having a region that hybridizes with a labeled probe containing a detectable molecule is adjacent to the sequence side of the portion that hybridizes with the probe, 22. The method of claim 21, comprising detecting the labeled probe captured on the capture DNA. 26. A single-stranded region having a base sequence complementary to a labeled oligonucleotide having a predetermined sequence and a base sequence complementary to the cleaved probe is adjacently provided,
Further, a DNA having a double-stranded region continuous with the single-stranded region
23. The method according to claim 22, wherein the cleaved probe and the labeled oligonucleotide are captured by. 27. The method according to claim 11, wherein a nucleotide detectable by terminal transferase is added. 28. A nucleotide having a molecule that can be detected by a ligase is bound.
The method according to 1. 29. DNA capable of hybridizing with a specific nucleic acid in a sample and having DNA on the 5 ′ side and the 3 ′ side of an abasic site.
A kit for analyzing a nucleic acid, which comprises a probe having a structure in which the abasic site is bound, and the abasic site is cleavable by an AP endonuclease only when the probe hybridizes with the specific nucleic acid.