JPH03151900A - Method for detecting nucleic acid - Google Patents

Abstract

PURPOSE:To enable rapid and simple detection with a good accuracy by subjecting a nucleic acid to be detected in a sample, probe nucleic acid and immobilizing nucleic acid having a part to be hybridized to hybridizing reaction. CONSTITUTION:Probe nucleic acids are selected to provide a probe nucleic acid 2 having a nucleotide chain length of <=1/10 based on that of a nucleic acid 1 to be detected, a part (B) to be hybridized with an immobilizing nucleic acid 3 on the 5'-terminal side of it self and a part (A) to be hybridized with a nucleic acid 1 to be detected on the 3'-terminal side. A sample containing the nucleic acid is subsequently reacted with the nucleic acid 2 to afford a hybrid, which is then subjected to labeling treatment to provide a labeled hybrid (a). The resultant ingredient (a) and the immobilizing nucleic acid 3 are subjected to hybridizing reaction in a solution such as formamide so as to afford formation of double strands with the nucleic acid 3 in the part (B) of the nucleic acid 2. Thereby, a hybrid (b) bound to the solid phase is obtained. The nucleic acid is then detected utilizing the label of the ingredient (b).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for detecting nucleic acids using a nucleic acid hybridization reaction.

[Prior Art] Various methods are known as methods for detecting whether or not a nucleic acid to be detected (nucleic acid to be detected) is present in a sample.

For example, a method in which a labeled probe nucleic acid is reacted with a sample immobilized on a solid phase, and the presence or absence of formation of a target nucleic acid/probe nucleic acid hybrid on the solid phase is detected by a label applied to the probe nucleic acid; react with the probe nucleic acid,
There is a method of detecting the presence or absence of formation of a target nucleic acid/probe nucleic acid hybrid in the resulting reaction mixture.

In one example of the former method, the sample is first fractionated by electrophoresis,
By blotting, a replica of the electrophoretic pattern of the sample is formed on the nitrocellulose filter, and this replica is reacted with a radioactive probe nucleic acid. At this time, if the nucleic acid to be detected is present in the sample, a hybrid is formed between the nucleic acid to be detected and the radioactive probe nucleic acid.

Thereafter, unhybridized radioactive probe nucleic acids are washed and removed, and then the radioactively labeled region of the hybrid formed on the nitrocellulose filter is detected by autoradiography or the like.

In one example of the latter method, a sample and a radioactive probe nucleic acid are reacted in a solution, and a pipe lid that is formed when the sample contains a nucleic acid to be detected and unreacted substances are separated by column treatment. Detection is performed by measuring the labeling activity of the radioisotope in the formed hybrid.

Alternatively, there is a method in which a probe nucleic acid is immobilized on a column, a sample is caused to flow therethrough, the resulting hybrid is extracted, and then dissociated into single strands, which are then collected and detected.

[Problems to be Solved by the Invention] The method using a sample immobilized on a solid phase as described above wastes a large amount of sample, takes a long time to perform the detection operation, and the preparation of the solid phase is complicated and requires many steps. There are drawbacks.

In addition, in a method in which a sample and a probe nucleic acid are reacted in a liquid phase and the resulting hybrid and unreacted substances are separated by column treatment, if the nucleotide chain of the nucleic acid to be detected is relatively short, the separation accuracy by column treatment deteriorates. There is a drawback.

Furthermore, in the method of using a probe nucleic acid immobilized on a column, the column can only be used for a specific purpose, that is, when detecting a specific nucleic acid base sequence, and thus lacks versatility. In addition, when detecting multiple nucleobase sequences,
There is a problem in that probe nucleic acids corresponding to each base sequence must be created and immobilized on separate columns.

The present invention was made as a result of intensive studies to solve the problems in various conventional detection methods using hybridization methods, and is a method that can quickly and easily detect the presence of a nucleic acid as a specific detection target. The purpose is to provide

[Means for Solving the Problems] The detection method of the present invention includes a sample containing a nucleic acid to be detected, a probe nucleic acid, a portion bound to a solid phase, and a portion for immobilization that hybridizes with the probe nucleic acid. Using a nucleic acid, a) a process of forming a hybrid of a nucleic acid to be detected, a probe nucleic acid, and a nucleic acid for immobilization, and b) a process of forming a hybrid of a nucleic acid to be detected and a probe nucleic acid using a hybrid label bound to a solid phase. It is characterized in that it includes a detecting process and an obtaining process using a nucleic acid for immobilization.

The present invention can be used to detect various nucleic acids such as DNA derived from living organisms and DNA obtained by genetic manipulation, and the type of nucleic acid to be detected is not limited.

As the probe nucleic acid, any nucleic acid can be used as long as it has a base sequence that can specifically hybridize with the nucleic acid to be detected and the nucleic acid for immobilization, but relatively short nucleotide chains that can be easily synthesized with a synthesizer can be used. oligonucleotides are readily available.

The nucleotide chain length of the pronib nucleic acid is preferably 1/10 or less of the nucleotide chain length of the nucleic acid to be detected.

In addition, in the present invention, when labeling the double-stranded portion of the probe nucleic acid and the detected nucleic acid, the requirements necessary for labeling are not required for the probe nucleic acid itself.

For example, in the case of labeling using the nick translation method or the labeling enzyme binding method, the nucleic acid to be labeled needs to have a certain length of nucleotide chain, but such a requirement is not required for the probe nucleic acid used in the present invention. Not done.

Therefore, a short nucleotide chain that is easy to obtain (prepare) and can realize highly sensitive detection as described above can be used as a probe nucleic acid.

However, in the present invention, when a label is applied to the double-stranded portion of the detection nucleic acid and the probe nucleic acid, the structure and nucleotide chain length of the probe nucleic acid are adjusted such that the double-stranded portion is easily labeled. It is desirable to select.

For example, as described below, one nucleotide strand of the double-stranded portion of the detection nucleic acid and the probe nucleic acid is used as a primer, the nucleotide strand is extended from the end, and a labeling substance is incorporated into the extended portion. When the probe nucleic acid is used as a brimer in the labeling method, the double strand forming part is configured such that the nucleotide strand of the detected nucleic acid hybridized with the probe nucleic acid can function as a template when one end of the brimer is extended. That is, it is necessary to have a configuration in which the nucleotide chain of the nucleic acid to be detected exists in a single-stranded state in the terminal extension direction of the probe nucleic acid.

Therefore, in the case of this labeling method that utilizes such a probe nucleic acid as a primer, it is desirable to determine the composition and nucleotide chain length of the probe nucleic acid in consideration of the structure of the double-stranded portion to be formed. .

Note that, depending on the case, the sample nucleic acid may be used as a primer.

The structure of the probe nucleic acid includes, for example, a B portion that can hybridize with the immobilizing nucleic acid at the 5' end, and an A portion that can hybridize with the nucleic acid to be detected at the 3' end. Examples include those that can form a linked structure of each nucleic acid as shown.

As the nucleic acid for immobilization, any nucleic acid can be used as long as it has a base sequence that can hybridize with the probe nucleic acid and can bind to a solid phase, but it can be easily synthesized using a synthesizer like the probe nucleic acid. The length of the immobilizing nucleic acid is preferably determined by taking into consideration the structure of the probe nucleic acid and the nucleotide chain length. Note that when it is desired to perform a dissociation treatment on the immobilized nucleic acid hybridized with the probe nucleic acid and reuse the immobilized nucleic acid, it is preferable to use an immobilized nucleic acid having a configuration that takes dissociation operations into consideration.

As a configuration for immobilizing the nucleic acid for immobilization onto a solid phase, a configuration incorporating a substance that selectively and specifically binds to the substance immobilized on the solid phase described below can be used. Avidin is a substance that selectively binds to avidin, and biotin is a substance that selectively binds to it and is capable of binding to nucleotide chains. chain,
Any material can be used as long as it can be introduced into a solid phase.

Hybridization between a sample, a probe nucleic acid, and a nucleic acid for immobilization can be performed according to a conventional method.

Note that the conditions for the hybridization reaction vary depending on the probe nucleic acid used, the nucleotide chain length and base sequence of the immobilization nucleic acid, so the operating conditions for hybridization should be selected appropriately depending on the desired purpose. good.

The hybridization reaction can be carried out in a temperature-controlled piperidization solution containing formamide, a suitable salt, and Denhardt's solution.

For immobilization of nucleic acids for immobilization, various gels,
A method can be used in which the substance is physically or chemically bound to a carrier such as nitrocellulose, and then a reaction is caused in the amount of the substance bound selectively and specifically.

In the method of the present invention, a sample and a probe nucleic acid are reacted, and the resulting hybrid is selectively labeled.

As a method for this labeling, for example, one of the strands constituting the double-stranded part of the hybrid is used as a primer, and when the end is extended to make a single-stranded part into a double-stranded part, the new A method of incorporating a labeling substance into the synthesized extended portion can be used.

In step a of the method of the present invention, for example, the sample and the probe nucleic acid are reacted under conditions necessary for the formation of a double strand between the A portion of the probe nucleic acid and the nucleic acid to be detected, and then the resulting reaction mixture is This can be carried out by reacting the immobilization nucleic acid with the B portion of the probe nucleic acid and the immobilization nucleic acid under conditions necessary for forming a double strand.

By this operation, when the sample contains the nucleic acid to be detected, a hybrid of the nucleic acid to be detected, the probe nucleic acid 2 and the nucleic acid for immobilization 3, as shown in FIG. 1, for example, is formed.

This hybridization reaction includes a method in which a sample is reacted with a probe nucleic acid and a nucleic acid for immobilization simultaneously in a hybridization solution, a method in which a sample and a probe nucleic acid are hybridized, and the resulting reaction mixture is mixed with a nucleic acid for immobilization. A method of hybridization, a method of hybridizing a probe nucleic acid and a nucleic acid for immobilization, and a method of hybridizing a sample to the resulting reaction mixture can be used. In the above, the nucleic acid for immobilization may be immobilized on a solid phase.

That is, the probe nucleic acid and the immobilization nucleic acid may be reacted first, and then the obtained reaction mixture and the sample may be reacted.

The present invention can be carried out, for example, by carrying out step a in a liquid phase, and subjecting the resulting reaction mixture to the necessary treatments for immobilizing the nucleic acid for immobilization contained in the reaction mixture onto a solid phase. At this time, if a hybrid of the nucleic acid to be detected, the probe nucleic acid, and the nucleic acid for immobilization is formed in the reaction mixture, this hybrid is also immobilized on the solid phase via the nucleic acid for immobilization, for example, as shown in FIG. A state of fixation to the stationary phase 5 can be obtained.

Furthermore, a treatment for immobilizing the nucleic acid for immobilization onto a solid phase can also be carried out by introducing the treatment before the reaction for forming double strands. Specific methods include a method in which a sample and a probe nucleic acid are simultaneously reacted with an immobilized nucleic acid immobilized on a solid phase, a method in which a probe nucleic acid is reacted with an immobilized nucleic acid immobilized on a solid phase, and then a sample is reacted with the immobilized nucleic acid. A method for reacting, a method for reacting a sample and a probe nucleic acid, and a method for reacting the resulting reaction mixture with a nucleic acid for immobilization immobilized on a solid phase, a method for reacting a nucleic acid for immobilization with a probe nucleic acid, and a method for reacting the resulting reaction mixture for immobilization. There is a method in which a nucleic acid-probe nucleic acid hybrid is immobilized on a solid phase and then a sample is reacted with the hybrid.

The above steps in the method of the present invention can be carried out, for example, by selectively labeling a hybrid of a nucleic acid to be detected and a probe nucleic acid, and detecting the hybrid by a method depending on the label.

For this labeling, as mentioned above, one strand of the double-stranded region is used as a brimer, and the other strand is used as a template, and when the end of the strand that will become the brimer is extended, a new strand is formed. A method of incorporating a marker into the extension part can be used.

FIG. 2 shows the case where the 3' end of the probe nucleic acid, which constitutes the A portion, is used as a primer.

Specifically, for example, d required for extension of one end of the brimer.
A nucleotide such as ATP, dCTP, dGTP, or dTTP is reacted with a hybrid to be labeled in the presence of an enzyme for forming a nucleotide chain, and a labeled nucleotide is used as a template to form a new nucleotide. A method of incorporating a label into the nucleotide chain to be used can be used.

Examples of the labeled nucleotide include those labeled with radioisotopes (RI), which are generally used for labeling probes, such as biotin, dinitrophenyl nucleotide derivatives, etc. that induce fluorescence, luminescence, or color development. Necessary enzymes and compounds labeled with non-radioactive labeling substances (nonRI) can be used.

As the enzyme for nucleotide chain formation, E. coli DNA polymerase 1% Klenow fragment of DNA polymerase T;
Various DNA polymerases such as T4 DNA polymerase and reverse transcriptase can be used.

Labeling in the present invention can be performed while the hybrid is immobilized on a solid phase via an immobilization nucleic acid. The labeling process is preferably introduced after the nucleic acid to be detected and the probe nucleic acid form a hybrid.

Furthermore, according to this method, a non-hybridized nucleic acid does not have a portion that functions as a primer for forming a new double-stranded portion and a portion that serves as a template for forming an extended portion. A double-stranded reaction that incorporates the labeling substance does not occur.

Note that after the labeling reaction is completed, the labeled hybrid and the label that is not incorporated into the hybrid can be separated, for example, by the following method. That is, when a hybrid is formed between the probe nucleic acid immobilized on the solid phase via the immobilization nucleic acid and the sample, the hybrid is also immobilized on the solid phase. Wash in this state to wash out and remove the label that has not been incorporated into the hybrid.

In addition, even if the reactions of the sample, probe nucleic acid, and immobilization nucleic acid are variously changed as described above, the above-mentioned method can be applied after the hybrid is finally immobilized on the solid phase via the immobilization nucleic acid. Labels that are not incorporated into the hybrid can be separated by a similar washing process.

Detection of the label incorporated into the hybrid can be carried out, for example, by the second
As shown in the figure, a method for detecting a label incorporated into a hybrid immobilized on a solid phase using a method according to the label, a nucleic acid for immobilization of a hybrid immobilized on a solid phase, and a probe nucleic acid. The label incorporated into the target nucleic acid/probe nucleic acid hybrid separated from the solid phase can be detected by a detection method, etc. in a manner appropriate for the label. In this method, the solid phase bound to the immobilization nucleic acid obtained by dissociating the bond between the hybrid probe nucleic acid immobilized on the solid phase and the immobilization nucleic acid can be repeatedly reused for the next detection reaction. . At that time, if multiple types of probe nucleic acids are used, in which the B part is formed in common with the immobilization nucleic acid to be reused, and the A part is different depending on the nucleic acid to be detected, it is possible to immobilize the immobilization on the solid phase. The used nucleic acid can be repeatedly reused for the detection of different target nucleic acids.

Furthermore, in the method of the present invention, the amount of the label incorporated into the hybrid can be measured to quantify the nucleic acid to be detected.

[Example 1 A gene having a part of the base sequence of plasmid pUc19 was detected.

A corresponding to part of the base sequence of plasmid puc 19
An oligonucleotide with the following structure, which has a B part with a complementary base sequence to the immobilization nucleic acid described later, and a base sequence that maintains the distance between A and B, is used as a DN.
A synthesis device (Applied Biosystems,
381 A type) and designated as probe nucleic acid 1.

5' A portion Next, as an oligonucleotide for preparing a nucleic acid for immobilization, an oligonucleotide having the following base sequence was synthesized using a DNA synthesizer.

A portion of these synthesized oligonucleotides was sampled and its purity was examined by electrophoresis on a 20% polyacrylamide gel containing 7M urea. As a result, 95%
Since the purity was above, it was decided to use it in the following reaction without further purification.

Furthermore, biotin was introduced into the oligonucleotide for preparing a nucleic acid for immobilization obtained as described above by the following procedure to obtain a nucleic acid for immobilization.

Synthetic oligonucleotide at 50μ℃ (50μg), mixed reagent solution ((1,46M potassium cacodylate, 0.)2MT
ris-OHpH6,9,3,3mMCoC1g,0,
33mM dithiothreitol) 100μJ, 4.0mM
Biotinylated UTP (manufactured by BRL) 40μβ, 1.0m
Mix 1 u4 of M dTTP, 100 ufi of H2O and 15 un (approximately 90 units) of TdT,
The reaction was carried out at 0°C. After 10 minutes, 0.2M
EDTA at 4 μC was added to the reaction system to stop the enzymatic reaction, followed by phenol treatment and ethanol precipitation. The resulting precipitate was dried and dissolved in HaO100LLI2.

Next, plasmids pUC19 and p13R32 were used as samples.
2 and a mixture thereof (1:1), each sample was digested with EcoRI according to a conventional method, and the resulting digest was heat-treated to convert double-stranded DNA into single-stranded DNA, and from each sample The following operations were performed using the three types of single-stranded DNA mixtures obtained individually.

Put 20 μg of the single-stranded DNA mixture, 2 μg of the previously prepared immobilization nucleic acid, and 2 μg of probe nucleic acid into a test tube, and add IO.
X annealing solution (100mM Tri 5-HCl
pH8, 0, 60mM MgC1□, 60mM β
-Add 10u12 of mercaptoethanol, 500mM NaCl), and then add distilled water to make the whole 100μi
It was prepared so that The resulting solution was heated to 65°C, maintained at that temperature for 10 minutes, and then slowly cooled to room temperature over about 1 hour. FIG. 2 schematically shows the reaction state at this time.

Next, 10 LL of IOX annealing solution (1,1mM dATP, 1mM dCT
P and 1 mM dG T P at 107% each. After adding tn, 100 uA of p"-TTP was added, and Sarani distilled water was added to make the total volume 200 μρ to prepare a labeling solution. To this solution, Kle of DNA polymerase I was added.
Five units of the now fragment were added and reacted for 1 hour under water cooling. FIG. 3 schematically shows the state at this time.

After the reaction, this reaction solution was mixed with a solid phase in which 7-garose gel was activated with cyanogen bromide and avidin was bound thereto. After being mixed slowly for 20 minutes, the mixture was briefly centrifuged at room temperature and the supernatant was discarded.

The resulting precipitate was washed twice with TE buffer and the radiation counts were measured several times using a scintillation counter. When a mixture of puc l 9 and pUc19 and pBR322 was used, the intensity was 10' to ' cpm. The value in the pBR322-only sample was less than twice background.

In addition, the above precipitate was added to TE buffer, heated at 80°C for 10 minutes, quickly centrifuged, and the supernatant was discarded.
The precipitate was washed with TE buffer and radioactivity was measured. As a result, no radioactivity was detected in the precipitate, confirming that the label-incorporated portion was dissociated by this heat treatment.

Next, when the precipitate obtained after this heat treatment was reused in the same operation as described above, the nucleic acid could be successfully manipulated. Therefore, it was confirmed that the precipitate was one in which the nucleic acid for immobilization was bound to cyanogen bromide-activated agarose through avidin-biotin binding, and that it could be repeatedly reused.

[Effects of the Invention] Conventionally, in order to prepare a nucleic acid fragment according to the base sequence of the target gene to be detected and use it as an immobilized probe, it was necessary to perform an immobilization process.

Furthermore, if the type of gene to be detected changes, it is necessary to prepare new nucleic acid fragments accordingly and to immobilize them. In other words, each time the gene to be detected changes, it is necessary to form an immobilized probe accordingly.

However, in the present invention, by using an immobilization probe and a probe nucleic acid having a complementary sequence to a part of the immobilization probe, even if the type of gene nucleic acid to be detected changes, only the probe nucleic acid needs to be prepared. Since there is no need for any treatment, nucleic acids can be detected quickly and easily.

Furthermore, in the present invention, since the hybrid into which the label has been incorporated and the label which has not been incorporated are accurately separated between the solid phase and the liquid phase, accurate detection operations are possible.

Furthermore, in the present invention, the immobilized nucleic acid bound to a solid phase using an avidin-biotin bond or the like can be reused by performing a dissociation treatment from the probe nucleic acid.

Therefore, by using the immobilized nucleic acid bound to this solid phase, it is possible to omit the process of binding the nucleic acid to the solid phase using a chemical reaction etc. in a new detection operation, and the operation is extremely simplified. .

[Brief explanation of the drawing]

Figure 1 is a schematic diagram showing the configuration of a nucleic acid to be detected, a probe nucleic acid, and a nucleic acid hybrid for immobilization. Figure 2 is a schematic diagram showing the state immobilized on the hybrid solid phase shown in Figure 1.
The figure is a schematic diagram showing the process of immobilized hybrid labeling shown in FIG. 2. 1: Nucleic acid to be detected 2 Niprobe nucleic acid 3: Nucleic acid for immobilization 4: Label 5: Solid phase

Claims (1)

[Claims] 1) Using a sample containing a nucleic acid to be detected, a probe nucleic acid, a nucleic acid for immobilization having a portion bound to a solid phase, and a portion hybridizing with the probe nucleic acid, a) a nucleic acid to be detected; a step of forming a nucleic acid/probe nucleic acid/immobilized nucleic acid hybrid; and b) a step of detecting the presence or absence of formation of a target nucleic acid/probe nucleic acid hybrid by using a label on the hybrid bound to a solid phase. Characteristic nucleic acid detection method. 2) The method for detecting a nucleic acid according to claim 1, wherein a double-stranded region between the nucleic acid to be detected and the probe nucleic acid is selectively labeled. 3) The method for detecting a nucleic acid according to claim 1, wherein the nucleic acid to be detected, the probe nucleic acid, and the nucleic acid for immobilization are reacted simultaneously. 4) The method for detecting a nucleic acid according to claim 1, wherein the nucleic acid to be detected and the probe nucleic acid are reacted, and the resulting reaction mixture is reacted with the nucleic acid for immobilization. 5) The method for detecting a nucleic acid according to claim 1, wherein the probe nucleic acid and the nucleic acid for immobilization are reacted, and the resulting reaction mixture is reacted with the nucleic acid to be detected. 6) The method for detecting a nucleic acid according to claim 1, comprising the step of dissociating the nucleic acid to be detected/probe nucleic acid hybrid from the immobilized nucleic acid.