JPH06153998A - Method for detecting nucleic acid hybrid body and probe used therefor - Google Patents

Abstract

PURPOSE:To obtain a method for detecting a nucleic acid hybrid, utilizing hybridization consisting of a simplified process without requiring separation of B/F and capable of obtaining good measuring sensitivity. CONSTITUTION:Analysis of a sample is carried out using a reagent substance capable of detecting a double helix structure in a target single-stranded nucleic acid.probe.hybrid to detect whether target nucleic acid exists in the sample or not.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for detecting and identifying a desired nucleotide sequence of nucleic acid (DNA or RNA) of viruses, microorganisms, animals and plants, humans, etc., or detecting the presence or absence of mutation in various nucleotide sequences, and Regarding the probe used.

[0002]

2. Description of the Related Art A large number of various mutant genes have been found out by the development of nucleic acid analysis techniques, and various genetic diseases based on gene mutations are being elucidated. It has been clarified that some of these genes have partially deleted bases in the gene and some have point mutations in the bases, which cause mutations in the protein and cause various symptoms. ing. At present, these genetic diseases are usually found by enzyme-based assays and immunological methods using antibodies after the appearance of symptoms, but from the viewpoint of early treatment, serious symptoms are present. It has been pointed out that it is important to detect the presence or absence of a mutation on a gene before the occurrence of.

RFLP is one of the effective methods.
(Restriction fragment length polymorphism). This method is, for example, a DNA obtained by cleaving all human genes with a restriction enzyme.
The fragment was developed by agarose gel electrophoresis, fixed on a filter using Southern blotting, and then hybridized with a probe composed of DNA (or RNA) labeled with a radioactive isotope or the like to obtain normal DNA. Sample DNA
The presence or absence of genes associated with these diseases is detected based on the difference in the cleavage patterns of the above.

Further, the DNA diagnosis is not necessarily used for human genes, but can also be used for identification of infected bacteria.

Conventionally, a method for identifying bacterial species based on similarity based on the morphological and biochemical properties of the isolated bacteria has been used. In this method, there are problems that it takes a long time for culturing, the determination of the properties is different depending on the test method, and the identification result is different depending on which property is emphasized.

Therefore, in recent years, especially in the field of detection and identification of causative bacteria in bacterial infections, DNA-DNA
Attempts have been made to use a hybridization method or a DNA-RNA hybridization method. This method uses bacteria to generate nucleic acid (DNA or RNA)
Extract, focusing on a specific part of the bacterial nucleic acid,
A nucleotide sequence having a high homology with the nucleotide sequence of that portion is examined by a hybridization method to determine whether or not it exists in the test nucleic acid sample of interest, and whether or not a problematic bacterium exists in the sample. This is the method of judgment.

[0007] These are the basic technologies, hybridise-
In general, the method called “ion” is composed of the following steps. (1) A step of cutting DNA and developing it by gel electrophoresis. (2) A step of adsorbing each of the developed DNA fragments to a nitrocellulose filter (Southern blot). (3) A step of reacting the nitrocellulose filter obtained in (2) with a probe to form a hybrid. (4) A step of detecting a DNA fragment forming a hybrid.

For example, in a hybridization reaction between DNAs, a labeled probe DNA and a target DN are labeled.
A and A form a hybrid by hydrogen bonding at the complementary sequence portions.

The probes used for these hybridization reactions have changed with the times. In the earliest days, long DNA fragments were labeled with radioisotopes by nick translation reaction. Long DNA with the development of DNA synthesizer
Synthetic oligonucleotides have been used instead of, and labeling substances are shifting from dangerous radioactive substances to safer biotin-avidin system and further to various chemiluminescent systems.

In the hybridization reaction,
In order to form a hybrid accurately between complementary sequences, it is necessary to optimally select the reaction temperature and ionic strength. That is, if the temperature is too high, the probe cannot bind to a nucleic acid having a complementary sequence, and if the temperature is too low, the probe nonspecifically binds to the nucleic acid. In addition, the salt concentration of the solution should be reduced for better accuracy, or
It is important to raise the temperature of the solution to remove labile hydrogen bonds and wash away non-specifically bound or mismatched probes. Therefore, appropriate reaction conditions,
It takes a lot of trial and error to set the cleaning conditions.

In the genetic diagnosis, the hybridization reaction and the washing conditions are required to be more accurate in removing the mismatch at the single base pair level.

In the hybridization reaction, when the target nucleic acid is used by being immobilized on a carrier such as nitrocellulose, there is an advantage that washing for removing nonspecific binding of the probe can be easily performed after the reaction. However, the complicated operation makes it difficult to automate the inspection. Moreover, it has a drawback that it takes time, so it is not suitable for mass processing of samples.

Therefore, it is expected that the method of detecting a hybrid in a solution without immobilizing nucleic acid will open up the possibility of automation, and various attempts have been made. The biggest problem in the method without immobilizing nucleic acid is how to distinguish between probe bound to target nucleic acid and excess probe not bound (B / F).
Separation). Further, in this case as well, similar to the hybridization reaction using the above-mentioned immobilized nucleic acid, appropriate reaction conditions for removing nonspecific adsorption and mismatch of the probe, and setting washing conditions are important subjects. Become.

As a method for detecting a hybrid of a target nucleic acid and a probe without performing B / F separation, several detection methods using a fluorescence depolarization method have been proposed (JP-A-2-295496). Japanese Patent Laid-Open No. 2-759
58, etc.). In these methods, a fluorescent-labeled single-stranded DNA probe is brought into contact with DNA in an analysis sample to form a double-stranded DNA, and fluorescence polarization before double-strand formation and double-strand formation after double-strand formation are performed. This is a method for detecting whether or not a DNA in a sample has a base sequence corresponding to the base sequence of the probe by measuring a mutation with fluorescence polarization. In this method, the principle of detection is that the fluorescent substance bound to the single-stranded probe becomes difficult to move due to the double strand, and fluorescence anisotropy increases.

However, in these methods, the sample contains contaminants such as proteins, which are probe DNA.
Non-specifically adsorbing onto the syrup causes an increase in the background for detection of hybrids, and therefore a complicated work of completely removing these contaminants in advance is required. In addition, the non-specific adsorption of the probe DNA and the pseudo-hybrid due to the base mismatch require an operation for removing it in advance as in the case of other solution systems. Furthermore, although it is true that B / F separation is not necessary, it is necessary for measuring the mutation of fluorescence that the probe concentration is approximately the same as the target DNA concentration.

[0016]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As described above, B /
When F separation is required, it includes many operations such as B / F separation (removal of excess probe), non-specific adsorption and mismatch removal, whether or not the target nucleic acid is immobilized and the solution system in which it is not immobilized. , Quite complicated. Moreover, since the optimum conditions for these operations differ depending on the length of the probe or each base sequence, it is necessary to examine and set the conditions in each case. In particular, the position of the mismatched base on the probe is also an important factor affecting the stability of the hybrid, and it may occur that the mismatched hybrid cannot be removed depending on its position. In consideration of the characteristics, more complicated work of setting the conditions of the hybridization reaction according to each case is required.

Further, even in the method for measuring the mutation of fluorescence which does not require the above-mentioned B / F separation, it is complicated to prevent the non-specific adsorption or mismatch from occurring or to remove the non-specific adsorption or mismatch generated. Processing is required. Moreover, the presence of impurities may affect the measurement sensitivity,
Since it is essential that the probe concentration is approximately the same as the target nucleic acid concentration, it is necessary to secure a sufficient amount of sample, and there is a problem that it may not be applicable to analysis of a sample that can be taken in a small amount.

The present invention has been made in view of the above problems in the prior art, does not require B / F separation, is composed of more simplified steps, and can obtain good measurement sensitivity. It is intended to provide a method for detecting a target nucleic acid using hybridization and a probe used therefor.

Another object of the present invention is to provide a method for detecting a target nucleic acid which can accurately detect only a desired hybrid even in the presence of a mismatched hybrid, and a probe used therefor.

[0020]

The method for detecting a nucleic acid hybrid of the present invention comprises a probe and a target obtained when a target nucleic acid is present in the sample solution by adding a probe to the sample solution and reacting them. Hybrid of 2 with nucleic acid
It is intended to detect a heavy helical structure.

The detection method of the present invention detects a hybrid based on a principle completely different from the conventional method. That is, the method of the present invention detects the formation of a double helix structure associated with hybrid formation, and does not require B / F separation. Furthermore, in the present invention, by setting conditions that can detect only an accurate double-helical structure, it becomes possible to detect the formation of a desired nucleic acid hybrid as it is even if non-specific adsorption or mismatch occurs, thus improving the measurement accuracy. It is possible to The present invention is applicable to DNA-DNA hybridization, DN
It is applied when a nucleic acid forms a regular double helix structure such as A-RNA hybridization.

The present invention will be described in detail below.

The phenomenon of hybridization has heretofore been merely understood from the viewpoint of hydrogen bonding between nucleic acid bases complementary to each other. This is because it was common to carry out the hybridization reaction after immobilizing nucleic acid (DNA or RNA). However, in the case of a hybridization reaction in a solution, if a nucleic acid forms a double strand with a certain length, it is expected to have a double helix structure. The present inventors have noticed that the higher-order structure and chemical properties of the nucleic acid are different when the nucleic acid is single-stranded and double-stranded (hybrid),
The detection system was established and the present invention was completed.

In the double helix structure, the nucleobase part forms a base pair by hydrogen bonding, and the phosphate part and the sugar part are spirally wound in an outward direction. Nucleobases are stacked on top of each other and are stabilized by stacking and are located at the center of the helix axis. As the double helix structure, A, B, C and Z types and their modifications are known. Each structure differs not only in base sequence but also in pitch length, spiral symmetry, groove width, groove depth, etc., depending on the ion species and salt concentration used during annealing, and even when the same sequence is used. It is said that the double helix structure changes depending on the conditions. Generally, DNA is said to have a B-type structure, in which case the pitch length is 33.8 angstroms.
It is said that the number of nucleic acid base pairs per pitch is 10 bases.

The present invention uses a reagent substance capable of causing a detectable change by utilizing the double-helix structure of the hybrid, and forms a double-helix structure by measuring a chemical change of the reagent substance. Is a method of detecting.

As the reagent substance, (a) a substance which reacts (interacts) with the double helix structure itself to cause a change,
(B) It is possible to use, for example, two or more kinds of reagent substances that interact through a double helix structure and that detect a change based on the interaction.

When the reagent substance of (a) is used, the sample solution and the probe are reacted to form a double helix having a hybrid of the probe and the target nucleic acid, which is formed when the sample contains the target nucleic acid. The structure is detected by changes in reagent substances or nucleic acid constituents. In this case, the reagent substance may be allowed to coexist in the reaction system of the sample solution and the probe, or may be added after completion of these reactions. The change due to the reaction of the reagent substance with the double helix structure is detected by measuring the change of the reagent substance itself, if the change itself is measurable. If it induces a change in, the other change is measured.

In this method, when the reagent substance is allowed to coexist in the reaction, the reagent substance and the double helix are combined by binding the reagent substance to the probe and by using an intercalator for the nucleic acid base as the reagent substance. It is preferable because the reaction with the structure can be facilitated.

The reagent substance that interacts with the double helix structure is, for example, a chemical substance that reacts with a substance such as a nucleic acid forming the double helix structure and can be detected by itself or on the double helix structure side. Those that cause a change or structural mutation, or those that cause a detectable change by reacting with a third substance added to the reaction system in the presence of a double helix structure can be used.

As this interaction, for example, an interaction based on charge transfer between the double helix structure and the reagent substance can be used. When charge transfer is used as the interaction, a substance that can be an electron donor or an electron acceptor with respect to the double helix structure is used as the reagent substance. This interaction is, for example, a change in the chemical structure, a change in the electronic state, or a change in the chemical structure of the reagent substance, the double helix structure, or the third substance capable of interacting with these substances before and after the formation of the hybrid. It is detected as a change in the signal originating from the substance.

As the interaction based on this charge transfer,
As in the case of the reagent substance (b) described below, the so-called th
In case of rough space and through b
The case of ond can be considered, and for example, charge transfer via stacking of nucleic acid base pairs and charge transfer based on the proximity effect of an electron donor and an electron acceptor accompanying a change to a double helix structure can be used.

When charge transfer between the double-helix structure and the reagent substance does not occur easily, a mediator that mediates the charge transfer or a substance called a sensitizer intervenes between them. May be.

In the present invention, it is necessary that the reagent substance is arranged at a position where it can react with the double helix structure so that these interactions occur. The method of arranging the reagent substance at the position where it can react with the double helix structure is as follows: when it enters between nucleic acid base pairs like an intercalator, when it is embedded in the groove of the double helix structure, and when it is a double helix structure. It can be used when it is arranged so as to be close to the structure. In either case, it is essential for the present invention that it be specifically arranged in the double helix structure of the hybrid formed by the single-stranded probe and the target nucleic acid.

Among these, as in the case of (b) described later, the intercalator is most advantageous in the case of utilizing the charge transfer via stacking.

The method of catching the change in the interaction between the double helix structure and the reagent substance as the change in the electron acceptor can be classified into several types according to the detecting means.

For example, when a spin labeling agent is used, the transferred charges can be detected as a spectrum change by ESR or the like by the spin elimination method or the like. It can also be regarded as the appearance or change of a new absorption spectrum like the charge transfer absorption band. A system in which a solution is colored or discolored as a result of charge transfer can be directly observed by the eye, and is more effective as a simple system. A light emitting system such as fluorescence or phosphorescence can also be used. In this case, it is possible to use a reaction in which fluorescence or phosphorescence is newly generated or a reaction in which a light-emitting substance disappears as a result of an interaction.
In addition, a method in which an electron acceptor chemically changes to another substance as a result of charge transfer and is detected can also be used. At this time, it is also possible to add a third substance to the changed substance and to cause chemiluminescence due to the chemical reaction of both substances for detection. When a protein such as an enzyme or an antibody is used as the third substance, a bioluminescence detection method can be used.

As a detection method, in addition to the change in the electron acceptor, the interaction may be detected by the change in the electron donor. And, basically, most of the methods used in the case of electron acceptors can be directly applied. When a fluorescent substance is used as an electron donor, it is possible to detect a direct change such as fluorescence quenching by using a substance whose quantum yield of fluorescence decreases with charge transfer, and Furthermore, a method of visualizing it in combination with several reactions can also be used.

When the reagent substance is an electron donor for the double helix structure, it is activated by light to emit electrons to start charge transfer, and the third substance is an electron donor. There may be a substance that stimulates a reagent substance to generate electrons.

Further, when the reagent substance is an electron acceptor for the double helix structure, it may be activated so that the electron is withdrawn from the electron acceptor by the activation thereof.
And as the initiator, as in the case of the electron donor,
In addition to light, any initiator may be used.

As described above, as the third substance, a mediator which mediates charge transfer, or
A substance called a sensitizer may intervene. Then, these substances may interact with the double helix to promote charge transfer to the electron donor and electron acceptor which are not directly bound to the double helix.

As the reagent substance (a), for example, a reagent substance such as riboflavin that binds only to a nucleic acid having a double helix structure and has a function of extracting an electron from the partner can be used. In addition to riboflavin, N, N'-dimethyl-2,
Oxidizing agents such as 7-diazapyrrhenium ion are available.

The reagent substance (a) can be used in an unbound state or bound to a probe as long as it causes a detectable change due to a reaction specific to the double helix structure.

The above-mentioned method of using the reagent substance (b) is caused by the interaction of two or more reagent substances with the structural change from single strand to double strand, that is, double helix. The hybrid is detected by measuring the change. Examples of the interaction here include charge transfer. When charge transfer is used as the interaction, the two or more kinds of reagent substances include at least a set of an electron donor and an electron acceptor. The interaction between the two is, for example, the change in the chemical structure, the change in the electronic state, or the change in the electronic state of the electron acceptor, the electron donor, or the third substance capable of interacting with these substances before and after the formation of the hybrid. , Is detected as a change in the signal derived from the changed substance.

The relationship between the electron acceptor and the electron donor is determined by the energy-state relationship between the two. Therefore, in the present invention, substances generally defined as electron donors or electron acceptors are not used as defined, but rather in individual combinations of two or more reagent substances, electron donors and electron donors are used. A substance that can serve as a receptor is appropriately selected and used. For example, while anthracene is a typical electron donor whose redox potential is measured,
It is well known that its properties have been investigated as electron acceptors.

As the interaction between the electron donor and the electron acceptor, the so-called through space and t
It is considered to be the case of a rough bond. The former includes, for example, a case where an electron donor and an electron acceptor interact through stacking of nucleobase pairs, and a proximity effect between the electron donor and the electron acceptor accompanying the change to the double helix structure. And the like. The latter (thro
In the case of "ugh bond", the transfer of charges including the base, the phosphate moiety or the sugar moiety constituting the nucleic acid is considered. In either case, the form is not limited as long as the interaction is derived from the formation of a double helix.

Through stacking between nucleobases means that when the distance between the electron acceptor and the electron acceptor placed in a position capable of reacting with the double helix structure is so large that they cannot interact with each other originally, The emitted electrons are transferred to adjacent nucleic acid base pairs one after another through an electron cloud spreading on the nucleic acid base pairs, and finally reach the electron acceptor. On the contrary, a mechanism is also established in which the electron acceptor pulls out an electron from the nucleic acid base pair, which is carried out in a chained manner, and finally the electron is taken from the electron donor. That is, the mediator in charge transfer is nucleic acid base pair.

On the other hand, in the case of being based on the proximity effect of the electron donor and the electron acceptor, the distance between the electron donor and the electron acceptor is close to the extent that these interactions are possible due to the formation of the double helix structure. That is the case. For example, both an electron donor and an electron acceptor are bound to the probe, and when the probe is in a single-stranded state, they do not interact with each other and hybridize with the target nucleic acid to form a double helix structure. The formation of a double helix structure can be detected depending on whether or not these interactions have occurred, by setting such interactions to occur when they are formed in close proximity to each other.

When charge transfer between the electron donor and the electron acceptor via the double helix structure is unlikely to occur, a mediator or a sensitizer that mediates the charge transfer between them. You may intervene the substance called.

As described above, in the present invention, it is necessary that the electron donor and the electron acceptor are arranged at a position where they can react with the double helix structure so that their interaction occurs. The method of arranging the reagent substance at the position where it can react with the double helix structure is as follows: when it enters between nucleic acid base pairs like an intercalator, when it is embedded in the groove of the double helix structure, and when it is a double helix structure. It can be used when it is arranged so as to be close to the structure. In either case, it is essential for the present invention that it be specifically arranged in the double helix structure of the hybrid formed by the single-stranded probe and the target nucleic acid.

Among these, the intercalator is
Most advantageous when utilizing charge transfer via stacking. So the intercalator is generally
It is a planar compound having an electron spread, and is oriented in a position parallel to the nucleic acid base pairs at the same distance as the distance between the nucleic acid base pairs on the extension line of the stacking of the nucleic acid base pairs. For example, if an intercalator is used as the electron donor and an electron acceptor is arranged on the opposite side of the double helix structure, the electron released from the electron donor is sent to the adjacent nucleobase pair, It can flow straight to the electron acceptor via the electron cloud of each nucleobase pair. Or
On the contrary, if an intercalator is used as an electron acceptor and the electron acceptor is arranged on the opposite side of the double helix structure, the electron hole on the electron acceptor draws an electron from the adjacent nucleic acid base pair, In some cases, electron withdrawal occurs between other nucleic acid base pairs one after another, and finally electron is withdrawn from the electron donor to carry out charge transfer. Considering these points, in the case of charge transfer through stacking, at least one of an electron donor and an electron acceptor is preferably an intercalator, and when both are intercalators, further, It is more preferable because the charge transfer efficiency can be increased. It is known that the intercalator stabilizes the double helix structure and raises its melting temperature, and that the electron donor or electron acceptor is an intercalator
It is also advantageous in that it stabilizes the hybrid between the protein and the target nucleic acid.

The method of catching the change in the interaction between two or more kinds of reagent substances due to the double helix formation as the change in the electron acceptor can be classified into several kinds according to the detecting means.

For example, when a spin labeling agent is used, the transferred charges can be detected as a spectrum change by ESR or the like by the spin elimination method or the like. It can also be regarded as the appearance or change of a new absorption spectrum like the charge transfer absorption band. A system in which a solution is colored or discolored as a result of charge transfer can be directly observed by the eye, and is more effective as a simple system. A light emitting system such as fluorescence or phosphorescence can also be used. In this case, it is possible to use a reaction in which fluorescence or phosphorescence is newly generated or a reaction in which a light-emitting substance disappears as a result of an interaction.
In addition, a method in which an electron acceptor chemically changes to another substance as a result of charge transfer and is detected can also be used. At this time, it is also possible to add a third substance to the changed substance and to cause chemiluminescence due to the chemical reaction of both substances for detection. When a protein such as an enzyme or an antibody is used as the third substance, a bioluminescence detection method can be used.

As a detection method, in addition to the change in the electron acceptor, the interaction may be detected by the change in the electron donor. And, basically, most of the methods used in the case of electron acceptors can be directly applied. When a fluorescent substance is used as an electron donor, it is possible to detect a direct change such as fluorescence quenching by using a substance whose quantum yield of fluorescence decreases with charge transfer, and Furthermore, a method of visualizing it in combination with several reactions can also be used.

In the present invention, in addition to the case where the electron donor is activated by light to emit an electron to start the charge transfer, as the third substance, the electron donor is stimulated to generate an electron. Material may be present.

Further, the electron acceptor may be activated so that electrons are extracted from the electron acceptor by being induced thereby. The initiator may be some kind of initiator other than light, as in the case of the electron donor.

As described above, in addition to the electron donor and the electron acceptor, the third substance is called a mediator that mediates charge transfer or a sensitizer. A substance may intervene. Then, these substances may interact with the double helix to promote charge transfer to the electron donor and electron acceptor which are not directly bound to the double helix.

Examples of the form using the electron donor and the electron acceptor include the following. a) Both of them are present in the reaction system when the double helix structure is formed by the hybridization of the probe and the target nucleic acid. b) Both of them are added to the reaction system after the formation of the double helix structure by the hybridization of the probe and the target nucleic acid. c) One of these is allowed to exist in the reaction system at the time of forming the double helix structure by the hybridization of the probe and the target nucleic acid, and then the other is added.

The electron donor and the electron acceptor have an interaction of 2 when they are added to the reaction system in a non-bonded state.
If it occurs specifically in the heavy helix structure, it can be used as it is in the unbound state.

However, when one or both of the electron donor and the electron acceptor coexist freely in the reaction system, the interaction between the two does not occur regardless of the presence or absence of the target nucleic acid. If it occurs and the background rises and the S / N ratio deteriorates, it is preferable to use one or both of these bound to the probe. In addition, if the concentration in the reaction system is selected so that such a rise in background does not occur, it is possible to use it in an unbound state by selecting these concentrations. The attachment of the electron donor or electron acceptor to the probe is optionally carried out via a linker such as (CH ₂ ) _n , in which case these positions are chosen so that these interactions are most efficient. Consider relationships.

In the most desirable form, both are
In this case, since the positional relationship between the interacting reagent substances becomes clear, the interaction can be controlled by these positional relationships in the probe. It is advantageous. In this case, the distance between the electron donor and the electron acceptor on the probe is appropriately selected according to the types of the electron donor and the electron acceptor to be used. For example, when the proximity effect is used, the electron donor is used. The distance between the body and the electron acceptor is preferably 20 to 120 angstroms, and more preferably 50 to 80 angstroms. Further, in the case of performing charge transfer through the double helix structure, these distances are preferably 20 to 120 angstroms, and more preferably 50 to 80 angstroms. The position of binding these to the probe depends on the length of the probe, but it is advantageous to separately bind to both ends of the probe from the viewpoint of easy binding.

The length of the probe is appropriately selected in each case so that good hybridization with the target nucleic acid is possible and a stable double helix structure is obtained. If both the electron donor and the electron acceptor are bound to the probe, and if they are close to each other and can interact even in the absence of the double helix structure, consider the distance between them. Although the probe length is determined by the above, the length is, for example, 8 bases or more, preferably 12 bases or more.

However, in order to stabilize the double helix structure, in addition to the probe length, the base sequence itself, the salt concentration of the reaction system and the ionic strength greatly influence. G-C base pairs are A-
Since the number of hydrogen bonds is larger than that of T base pairs, a more stable double helix structure is formed in a sequence having a large number of GC. Also, K
When the molar concentration of Cl is increased from 0.01M to 1M, D
The melting point temperature of NA is said to rise by 30 ° C. The presence of the intercalator also greatly contributes to stability. Therefore, a probe having a length of less than 8 bases can be used by appropriately using these stabilizing factors.

It is desirable that the change of the electron acceptor of the electron donor due to the interaction is irreversible. That is, if the electron donor or the electron acceptor used for detection changes irreversibly, the change can be accumulated and detected, which is advantageous in terms of sensitivity.

[0064]

The present invention will be described in more detail with reference to the following examples. Example 1 [1] Preparation of 20-mer oligonucleotide probe to which TEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidine) which is a spin labeling agent was bound (1) 4-amino Hexylamino-2,2,6,6,-
Synthesis of tetramethylpiperidine-N-oxyl (4-aminohexylamino-TEMPO) 0.5 mmole of 4-oxo-TEMPO and 5 mmole of hexamethylenediamine dihydrochloride were added to methanol 30
Soluble in sodium cyanide 0.4
mmole and molecular sieve 3A were added, and at room temperature 2
These were reacted by stirring for 4 hours. The reaction solution was then filtered through a glass filter to remove the molecular sieves and subsequently the solvent was removed from the filtrate under reduced pressure. Next, 30 ml of 1N hydrochloric acid was added to the obtained residue to dissolve it, and the mixture was extracted with chloroform. After the chloroform phase was washed with water, chloroform was distilled off under reduced pressure. Water was added to the obtained residue and the insoluble material was filtered off, and the filtrate was again distilled under reduced pressure to remove the solvent to obtain a red oily substance.

(2) Synthesis of Oligonucleotide A 20-mer oligonucleotide having a base sequence partially complementary to M13mp18 DNA (single strand) as a target DNA was synthesized by using 381A DNA automatic synthesis group manufactured by ABI. . The 5'-terminal dimethoxytrityl group was removed on an automatic synthesizer. The base sequence is as follows.

5'-GTTTGAAAAACGACGGC
CAGT-3 ′ (3) Synthesis of spin-labeled oligonucleotide probe Oligonucleotide (1 synthesized in the above step (2)
μmole) was transferred to a gas tight syringe while still attached to the CPG support. The following reactions were carried out in a syringe. Dioxane 1m in which 50 mg of carbonyl-N, N'-diimidazole (CDI) was dissolved in CPG support
1 was added and left at room temperature for 1 hour. It was washed with dioxane and dried under reduced pressure, followed by 4-aminohexylamino-TEMP.
DMSO solution of O (0.2 M 0.4 ml) was added 55
It was left at 24 ° C. for 24 hours. It was washed with DMSO, dioxane and methanol in this order and dried under reduced pressure.

The spin-labeled oligonucleotide was cleaved with concentrated aqueous ammonia and deprotected according to a conventional method and purified by RPLC.

[2] TEMPO probe and M13mp1
Hybridization reaction with 8 DNA 0.2 μM of TEMPO-introduced oligonucleotide probe prepared in the above [1] and M13mp18
0.2 μM of DNA (Takara Shuzo) was added to 1 mM phosphate buffer solution (pH 7.0) / 145 mM NaCl / 5 mM
The mixture was heated to 80 ° C. in KCl, then gradually cooled and cooled to room temperature to prepare a probe-target DNA hybrid. Next, fluorescein (manufactured by Kodak Co.) was added to the reaction solution so that the final concentration was 10 μM, and the ESR spectrum shown below was measured. Also, M13
Perform the same operation as above without using mp18DNA
A sample (probe alone) for measuring the SR spectrum was obtained.

[3] Measurement of ESR spectrum ESR measurement was performed at 20 minute intervals for each sample.
And continued for 100 minutes. Then, the intensity ratio and the change in line width with time were followed. The ESR was manufactured by JEOL Ltd., and a flat cell of artificial quartz was used for the measurement.

The ESR and light irradiation device were set as follows.

[0071]

[Table 1] Light irradiation device Monochrome meter 490nm Power supply 88.5V-89V / 22A Fig. 1 shows changes with time of ESR signal intensity ratio and changes of line width. As can be seen from FIG. 1, in the case of the probe alone, no change was observed in the intensity ratio and the line width even when irradiated with light. Also, fluorescein / probe / M13mp1
Also in the case of 8, no change was observed in the intensity ratio and the line width when light irradiation was not performed.

On the other hand, fluorescein / probe /
When M13mp18 DNA was irradiated with light (490 nm), the ESR intensity ratio decreased with time. Further, since no change was observed in the line width, it can be said that the intensity change in ESR is not due to the chemical change. Therefore, it was confirmed that the spin of TEMPO was canceled as a result of the charge transfer from fluorescein to TEMPO bound to the probe through the double helix structure formed by the probe and M13mp18 DNA. That is, B / F
This means that the probe-target DNA hybrid could be detected without separation. Example 2 Except that the following sequence was used as the base sequence of the probe,
The same experiment as in Example 1 was performed.

5'-GTTGTAAAAGGACGGC
CAGT-3 ′ The sequence of this probe is the one obtained by converting the 10th base from the 5 ′ end of the probe sequence used in Example 1 to C to G, and differs from that of Example 1 by 1 base. , M1
It is designed to mismatch with 3mp18 DNA.

Probes having this mismatch sequence 0.
M13mp18DNA (0.2 μM) was added to 2 μM, and annealed in the same manner as in Example 1, fluorescein was further added, and changes in the ESR signal intensity ratio and the line width with time were examined.

As a result, the change in strength seen in Example 1 was not observed, and the strength remained the same as in the case of the probe alone. This means that in the case of a hybrid of mismatched oligonucleotide probe and DNA,
It can be said that the correct double strand was not formed and the charge did not move.

Example 3 Synthesis of Oligonucleotide Probe with Both End Labels (5'-TEMPO, 3'-FITC) (1) Synthesis of 3'Amino Group-Coupled Oligonucleotide 3'-Amino Modifier C manufactured by Glen Research
A 20-mer oligonucleotide having a base sequence partially complementary to the single-stranded DNA of M13mp18DNA as a model of the target DNA used in Example 1 with PG (1 μmol) as a carrier was used as a 381A DNA automatic synthesis group manufactured by ABI. Synthesized in. The 5'-terminal dimethoxytrityl group was removed on an automatic synthesizer. (2) Synthesis of Spin Label Oligonucleotide Probe The oligonucleotide synthesized in (1) above (1 μmo
l) was transferred to a gas tight syringe while still attached to the CPG support. The following reactions were carried out in a syringe. CP
1 ml of dioxane in which 50 mg of carbonyl-N, N'-diimidazole (CDI) was dissolved was added to G support, and the mixture was allowed to stand at room temperature for 1 hour. It was washed with dioxane and dried under reduced pressure, and then the DMSO solution of 4-aminohexylamino-TEMPO shown in Example 1 (0.2 M 0.4 ml).
Was added and the mixture was left at 55 ° C. for 24 hours. It was washed with DMSO, dioxane and methanol in this order and dried under reduced pressure.

Then, the spin-labeled oligonucleotide was cleaved with concentrated aqueous ammonia and deprotected according to a conventional method and purified by RPLC to obtain TEMPO at the 5'end.
A spin-labeled oligonucleotide labeled with was obtained. (3) Synthesis of both-end labeled oligonucleotide probe Since the 3-amino-2-hydroxy group is bound to the 3'end of the spin-labeled oligonucleotide synthesized in (2) above, this amino group and FITC (fluorescein) are combined. Isothiocyanate, Sigma) was attached by the following procedure.

0.2 μmol (700 μl aqueous solution) of spin-labeled oligonucleotide synthesized in (2) above and 1
100 μl of M sodium carbonate buffer (pH 9) was mixed, 2 mg of FITC in DMF was added to the resulting solution, and the mixture was reacted at 35 ° C. for 24 hours. It was treated with a gel filtration column NAP-25 manufactured by Pharmacia to remove a large excess of FITC, followed by purification by RPLC and TEMP at the 5'end.
A probe in which O was bound and FITC was bound to the 3'end was obtained.

(4) Measurement of ESR spectrum The probe obtained in (3) above and M13mp18 DNA (0.2 μM each) were annealed according to a standard method to obtain ES.
The change in the intensity ratio of the R signal and the change in the line width were examined in the same manner as in Example 1. No change in intensity was observed in the probe alone or in the probe / target DNA / hybrid body at the time of non-irradiation, whereas in the probe / target DNA / hybrid body, the excitation wavelength of fluorescein was 490n.
Only when m light was applied, the signal decreased with time, and charge transfer was confirmed (Fig. 2). Furthermore, the degree of spin elimination is larger than that in the case where fluorescein is not bound to the probe (Example 1), and electron transfer occurs more effectively when an electron donor and an electron acceptor are bound to both ends. It was confirmed.

Example 4 An oligonucleotide (having the same sequence as in Example 2) having a base sequence different from M13mp18DNA by one base in the middle of the probe was synthesized, and TEMPO was added to both ends thereof in the same manner as in Example 3. Was combined with fluorescein.

This probe and M13mp18 DNA (0.2 μM each) were annealed according to a standard method to obtain ES.
The change in the intensity ratio of the R signal and the change in the line width were examined in the same manner as in Example 1. When the probe / target DNA / hybrid is irradiated with light of 490 nm, which is the excitation wavelength of fluorescein, as in the case of the probe alone or the probe / target DNA / hybrid without irradiation of light, ES
No change in the intensity ratio of the R signal and the line width was observed.

Example 5 A 20-mer oligonucleotide having the same nucleotide sequence as in Example 1 was synthesized by an automatic DNA synthesizer. Using this oligonucleotide as a probe, M13mp18D
A hybrid with NA was formed. Solution is 10 mM
Phosphate buffer and probe have final concentrations of 3 μM, 6 μM, 1
The concentrations of 0 μM, 20 μM, and M13mp18 DNA were adjusted to a final concentration of 10 μM. Further, riboflavin was added to this probe-target DNA hybrid to a final concentration of 100 μM. Probe / M1
1 ml of the 3 mp18 DNA / riboflavin mixed solution was irradiated with light having an excitation wavelength of riboflavin of 470 nm for 5 minutes.

200 μl of the solution was added to Nuclease P ₁
And then E. E. coli was digested with alkaline phosphatase to decompose the probe and DNA into nucleosides. This degradation product was analyzed by the method of Kasai et al.
n-75, p841-844, (1984)) and separated by HPLC to give 8-hydroxyguanosine (8-O).
The amount of H-G) was quantified. As a control experiment, probe /
Similar experiments were performed for each of the riboflavin solution and the M13mp18 DNA / riboflavin solution.

As a result, as shown in FIG. 3, 8-OH-G was purified depending on the concentration of the probe, and the maximum value was
It was revealed that the ratio of probe to target DNA was 1: 1. Probe / riboflavin solution, M13m
8-OH-G was not detected in the p18 DNA / riboflavin solution. That is, 8-OH-G was produced only when the probe and DNA formed a hybrid.

Example 6 A 20-mer oligonucleotide having the same nucleotide sequence as in Example 1 was synthesized by an automatic DNA synthesizer. Using this oligonucleotide as a probe, M13mp18D
Hybridized with NA. The solution was 10 mM phosphate buffer, and the probe had a final concentration of 3 μM, 6 μM, 10
The concentrations of μM, 20 μM, and M13mp18 DNA were adjusted to a final concentration of 10 μM. Furthermore, this professional
Riboflavin was added to the target DNA hybrid to a final concentration of 100 μM. Probe / M13
1 ml of the mp18DNA / riboflavin mixed solution was irradiated with light of 470 nm, which is the excitation wavelength of riboflavin, for 5 minutes.

As a control experiment, the same experiment was carried out for each of the probe / riboflavin solution and the M13mp18DNA / riboflavin solution.

100 μl of each sample after light irradiation was salified
Mon Testes DNA (50 μg) was added and fixed on a microplate, and 8-OH-G was prepared by the ElISA method.
Was reacted with the antibody. The result of quantification by the color development of alkaline phosphatase as a secondary antibody is shown in FIG. Salm
When evaluated based on 50 μg of on Testes DNA, a probe / riboflavin solution, M13mp18DN
A / Salmon Testes for riboflavin solution
Only a slight color development similar to that of DNA was observed, whereas in the probe / DNA complex, color development was observed depending on the probe concentration. Then, when the probe and DNA were equimolar, the maximum was obtained.

Example 7 A 20-mer oligonucleotide having the same nucleotide sequence as in Example 1 was synthesized by an automatic DNA synthesizer. Using this oligonucleotide as a probe, M13mp18D
Hybridized with NA. The solution was 10 mM phosphate buffer, and the probe had a final concentration of 3 μM, 6 μM, 10
The concentrations of μM, 20 μM, and M13mp18 DNA were adjusted to a final concentration of 10 μM. Furthermore, this professional
The final concentration of riboflavin in the DNA / DNA hybrid is 1
It was added to be 00 μM. Probe / M13mp1
1 ml of the 8 DNA / riboflavin mixed solution was irradiated with light of 470 nm, which is the excitation wavelength of riboflavin, for 5 minutes.

As a control experiment, the same experiment was carried out for each of the probe / riboflavin solution and the M13mp18DNA / riboflavin solution.

The riboflavin absorption intensity (450 nm) was measured for each sample (FIG. 5). Riboflavin only, probe / riboflavin, M13mp18DNA /
In the riboflavin solution, the absorption intensity did not change and
D-probe / M13mp18DNA /
It was confirmed that in the riboflavin solution, the absorption intensity decreased and riboflavin was changed to another substance as a result of charge transfer.

[0091]

The method for detecting a target nucleic acid of the present invention comprises a B / F
It has the advantage that no separation is required. As a result, many operations such as removal of excessive probe, complicated treatment for removing non-specific adsorption, and examination of the conditions, which were indispensable in the conventional method, became unnecessary.

Furthermore, by selecting a reagent substance so that a signal change is observed only in an accurate hybrid, an accurate double-helical structure is formed even if a mismatch occurs in the reaction system. It is possible to detect only hybrids.

[Brief description of drawings]

FIG. 1 (A) is a graph showing the change over time in the ESR signal intensity ratio obtained in Example 1. (B)
3 is a graph showing the change over time of the ESR signal line width obtained in Example 1. In addition, □ shows the case where the reaction system contains the probe, target DNA and fluorescein and is irradiated with light, and ■ the case where the reaction system contains the probe, target DNA and fluorescein and is not irradiated with the light, ◆
Represents the case where the reaction system contains a probe and fluorescein and is irradiated with light.

FIG. 2 is a graph showing the change over time in the ESR signal intensity ratio obtained in Example 3. □, ■ and ◆ are shown in Fig. 1
Is the same as.

FIG. 3 is a graph showing the relationship between the amount of 8-hydroxyguanosine produced in Example 5 (ratio to G) and the amount of probe used.

FIG. 4 is a graph showing the relationship between the amount of 8-hydroxyguanosine produced in Example 6 (color reaction by ELISA method) and the amount of probe used.

FIG. 5 is a graph showing the interaction between the riboflavin and the probe / target DNA / hybrid obtained in Example 7.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical indication location // A61B 10/00 (72) Inventor Yoshinori Tomita 3-30-2 Shimomaruko, Ota-ku, Tokyo Kya Non-Incorporated (72) Inventor Keisuke Makino 33-24 Kamitakano Saimyoji Temple, Sakyo-ku, Kyoto-shi, Kyoto Prefecture (24) (72) Inventor Akira Murakami 363-1 Iwakura-nakacho, Sakyo-ku, Kyoto-shi, Kyoto Prefecture

Claims (46)

[Claims] 1. A process of adding a probe to a sample solution to react them, and detecting a double helix structure of a hybrid of the probe and the target nucleic acid obtained when the target nucleic acid is present in the sample solution. In the method for detecting a nucleic acid hybrid having a process, the presence of a target nucleic acid by allowing two or more kinds of reagent substances that cause a detectable change due to an interaction through a double helix structure to exist in the sample solution. The double helix structure is detected by measuring a change based on the interaction between the two or more kinds of reagent substances, which occurs through the double helix structure of the hybrid of the probe and the target nucleic acid formed in the above. And a method for detecting a nucleic acid hybrid. 2. The method according to claim 1, wherein the interaction between two or more reagent substances is based on charge transfer between these reagent substances. 3. The method according to claim 2, wherein the two or more reagent substances include at least an electron donor and an electron acceptor. 4. The method according to claim 3, wherein the charge transfer is performed through stacking of base pairs constituting a double helix structure. 5. The charge transfer according to the proximity effect between an electron donor and an electron acceptor, which accompanies the formation of a double helix structure.
The method described in. 6. The electron acceptor is EP by accepting an electron.
The method according to claim 3, which is a substance that causes a change in R (electron spin resonance) spectrum. 7. The method of claim 3, wherein changes due to the interaction between the reagent substances are optically detectable. 8. The method of claim 3, wherein the change due to the interaction between the reagent substances is chemically detectable. 9. The method according to claim 3, wherein the reagent substance is an intercalator for nucleobases forming a double helix structure. 10. The method according to claim 3, wherein the interaction between the reagent substances is initiated by irradiation with light. 11. The method according to claim 2 or 3, wherein at least one of the reagent substances is bound to the probe. 12. The method according to claim 2 or 3, wherein the change due to the interaction between the reagent substances is irreversible. 13. A process of adding a probe to a sample solution to react them, and detecting a double helix structure of a hybrid of the probe and the target nucleic acid obtained when the target nucleic acid is present in the sample solution. And a probe formed when a target nucleic acid is present, in which a reagent substance that causes a detectable change due to an interaction with a double helix structure is present in the sample solution, A method for detecting a nucleic acid hybrid, which comprises detecting a double helix structure by measuring a change caused by an interaction between the double helix structure of a hybrid with a target nucleic acid and the reagent substance. 14. The method of claim 1, wherein the interaction is charge transfer based. 15. The method of claim 14, wherein the reagent substance is an electron donor or electron acceptor for the double helix structure. 16. The method according to claim 15, wherein the charge transfer is performed through stacking of base pairs forming a double helix structure. 17. The method according to claim 15, wherein the charge transfer is due to a proximity effect between an electron donor and an electron acceptor associated with the formation of a double helix structure. 18. The electron acceptor is E by accepting an electron.
The method according to claim 15, which is a substance that causes a change in PR (electron spin resonance) spectrum. 19. The method of claim 15, wherein the interaction-based change is optically detectable. 20. The method of claim 15, wherein the interaction-based change is chemically detectable. 21. The reagent substance is an intercalator for a nucleobase constituting a double helix structure.
The method according to 5. 22. The method according to claim 15, wherein the interaction is initiated by irradiation with light. 23. The method according to claim 14 or 15, wherein the reagent substance is bound to the probe. 24. The method according to claim 14 or 15, wherein the interaction-based change is irreversible. 25. A probe for detecting a target nucleic acid having a sequence for hybridizing with a target nucleic acid, wherein the probe interacts with a double helix structure formed by hybridization of the target nucleic acid. At least one of two or more reagent substances that causes a detectable change
A probe for detecting a target nucleic acid, which comprises a species bound thereto. 26. The interaction between two or more reagent substances is based on charge transfer between these reagent substances.
The target nucleic acid detection probe according to item 5. 27. The probe for detecting a target nucleic acid according to claim 26, wherein the two or more kinds of reagent substances contain at least an electron donor and an electron acceptor. 28. The probe for detecting a target nucleic acid according to claim 26, wherein the charge transfer is performed through stacking of base pairs forming a double helix structure. 29. The probe for detecting a target nucleic acid according to claim 26, wherein the charge transfer is based on a proximity effect of an electron donor and an electron acceptor via a double helix structure. 30. The electron acceptor is E by accepting an electron.
The target nucleic acid detection probe according to claim 26, which is a substance that causes a change in PR (electron spin resonance) spectrum. 31. The probe for detecting a target nucleic acid according to claim 26, wherein a change based on the interaction between the reagent substances is optically detectable. 32. The probe for detecting a target nucleic acid according to claim 26, wherein a change due to an interaction between reagent substances can be chemically detected. 33. The reagent substance is an intercalator for a nucleobase constituting a double helix structure.
The probe for detecting a nucleic acid hybrid according to item 6. 34. The probe for detecting a target nucleic acid according to claim 26, wherein the interaction between the reagent substances is initiated by light irradiation. 35. The probe for detecting a target nucleic acid according to claim 26, wherein a change based on an interaction between the reagent substances is irreversible. 36. A probe for detecting a target nucleic acid having a sequence for hybridizing with a target nucleic acid, wherein the probe interacts with a double helix structure formed by hybridization with the target nucleic acid. A probe for detecting a nucleic acid hybrid, comprising a reagent substance bound to produce a change detectable by the action. 37. The probe of claim 36, wherein the interaction is charge transfer based. 38. The probe according to claim 37, wherein the reagent substance is an electron donor or an electron acceptor for the double helix structure. 39. The probe according to claim 38, wherein the charge transfer is performed through stacking of base pairs forming a double helix structure. 40. The probe according to claim 38, wherein the charge transfer is due to a proximity effect between an electron donor and an electron acceptor accompanied by formation of a double helix structure. 41. The electron acceptor is E by accepting an electron.
The probe according to claim 38, which is a substance that causes a change in PR (electron spin resonance) spectrum. 42. The probe of claim 38, wherein the interaction-based change is optically detectable. 43. The probe of claim 38, wherein the interaction-based change is chemically detectable. 44. The reagent substance is an intercalator for a nucleobase constituting a double helix structure.
8. The probe according to item 8. 45. The probe according to claim 38, wherein the interaction is initiated by irradiation with light. 46. The probe according to claim 37 or 38, wherein the interaction-based change is irreversible.