JP2001269197A - Immobilized oligonucleotide probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for detecting a sequence-specific nucleic acid useful for a biological research and a clinical diagnosis. SOLUTION: The method for detecting the sequence-specific nucleic acid and a reagent kit for detecting the nucleic acid are characterized by inserting a spacer having <=10 nucleotides to a binding site of a hybridization region in a capture probe and a solid support when used as the capture probe by immobilizing oligonucleotide on the solid support.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to nucleic acid chemistry and the detection of specific nucleic acid sequences. More specifically, the present invention relates to a method for immobilizing a nucleic acid probe, a stable measurement reagent containing the immobilized probe, and a hybridization assay performed using the immobilized probe. INDUSTRIAL APPLICABILITY The present invention is useful in clinical diagnostics, forensics, microbial environmental research, food and drug quality assurance, and molecular biology research techniques.

[0002]

2. Description of the Related Art In a hybridization assay, a sequence is detected by hybridization using a labeled nucleic acid (hereinafter, also referred to as a probe) complementary to a specific sequence. Hybridization assays are a classic technique in the research field (Current protocols in
molecularbiology (John Wiley & Sons, Inc), MolecularC
loning 2nd Ed. (Cold Spring Harbor Press)), but the method described in these references involves immobilizing a sample containing a nucleic acid to be detected on a nitrocellulose membrane or the like and reacting a nucleic acid probe in a solution. . Falkow et al., In U.S. Pat.No. 4,358,535, describe the use of specific DNA probes for diagnosing infectious diseases, which also use samples (e.g., blood, cells, saliva, etc.) with membrane filters (e.g., nitro (Cellulose)
Lysing the cells and fixing the nucleic acids by chemical denaturation and heating. However, the operation of immobilizing a nucleic acid sample is troublesome, and is not suitable for clinical diagnosis in which a large number of samples are examined.

Have solved this problem by immobilizing a nucleic acid probe (Gene, 21: 77-8).
5, 1983). The target sequence in the sample is captured by hybridization with an immobilized sequence-specific nucleic acid probe, and the captured target sequence is detected with another labeled sequence-specific nucleic acid probe. This is a so-called sandwich hybridization assay (JP-A-58-40099). According to this method, the assay is completed only by the hybridization reaction, and the operation is simplified, which facilitates automation. Furthermore, Gingeras et al. Report that the reaction time can be reduced by using relatively short oligonucleotides as hybridization probes (Nucleic Acids Research, 15: 5373-539).
0, 1987).

[0004] In the above-mentioned method, there is a problem how to stably bind the nucleic acid probe to the solid support.
This bonding mode is roughly classified into a method using a non-covalent bond such as a hydrophobic bond (Japanese Patent Application Laid-Open No. 5-271272) and a method using a covalent bond. In the method using a covalent bond, a method in which an amino group is introduced into a solid support, and a phosphate group is generated at the end of the nucleic acid probe to be bonded (Analitical Bioche
mistry, 198: 138-142, 1991) or a method in which a polycarbodiimide resin is applied and bonded to an amino group introduced into a nucleic acid via a carbodiimide group or an imino group originally possessed by the nucleic acid (JP-A-8-23975). Gazette). In general, the use of a covalent bond is more practically advantageous because it provides more stable results and shelf life of the product.

The present inventors have introduced an amino group into an oligonucleotide and applied the method described in Japanese Patent Application Laid-Open No. H8-23975 to bind to and capture a polycarbodiimide-coated polystyrene microtiter plate. We have developed a method to use as a probe. However, this method has a problem that the efficiency of hybridization is low.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a detection system for a target nucleic acid by nucleic acid hybridization which is excellent in hybridization efficiency.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in view of the above circumstances, and as a result, have found that steric hindrance caused by binding to a solid support reduces the efficiency of hybridization. Then, they have found that it is possible to promote the efficiency of hybridization by separating the binding portion and the hybridizable region of the probe by a spacer region, and have completed the present invention. That is, the present invention has the following configuration. (1) A solid support on which at least one kind of oligonucleotide probe is immobilized, wherein the probe has about 10 complementary to a specific nucleotide sequence to be detected.
A hybridizing region consisting of a nucleotide sequence of 〜50 bases and a spacer region that cannot hybridize to said specific nucleotide sequence to be detected, said spacer region being bound at one end to said solid support. A solid support which is bonded to the hybridizing region at the other end, and wherein the spacer region comprises an oligonucleotide having 10 bases or less. (2) The solid support according to (1), wherein the spacer region comprises an oligonucleotide having 4 to 10 bases. (3) The solid support according to (1) or (2), wherein the solid support is a microtiter plate. (4) The solid support according to any one of (1) to (3), wherein the probe is an oligodeoxyribonucleotide or a derivative thereof. (5) detecting at least one oligonucleotide probe having a reactive group comprising a hybridizing region composed of a nucleotide sequence of about 10 to 50 bases complementary to the specific nucleotide sequence to be detected; Immobilizing to a solid support via a spacer region which is not capable of hybridizing to the specific nucleotide sequence to be performed, wherein the spacer region comprises 1
A method comprising an oligonucleotide having 0 or less bases. (6) The method according to (5), wherein a polycarbodiimide-coated microtiter plate is used as the solid support and the probe is bound to the end of the probe by an addition reaction of a carbodiimide group. (7) A reagent kit for detecting a nucleic acid, comprising the solid support according to any one of (1) to (4). (8) The nucleic acid detection reagent kit according to (7), comprising a means for detecting a specifically bound target nucleic acid. (9) A method for detecting the presence of a nucleic acid sequence in a sample, comprising the following steps (a) and (b). (A) bringing the sample into contact with any of the solid supports (1) to (4) under conditions permitting hybridization of the complementary nucleic acid sequence (b) determining whether hybridization has occurred

[0008]

DETAILED DESCRIPTION OF THE INVENTION To aid in understanding and describing the present invention, the following terms are defined below. "Label" or "label" refers to any atom or molecule that can be used to provide a detectable, preferably quantifiable, signal and can be attached to a nucleic acid or protein. The label is fluorescence, luminescence, absorption,
Provides a signal that can be detected by radioactivity, magnetism, mass, enzyme activity, and the like. Suitable labels include fluorescent materials, chromogenic materials, radioactive atoms (especially ³² P or ¹²⁵ I),
Electron density reagents, enzymes, and ligands with specific binding partners are included.

When an enzyme is used, its activity is typically
Is detected by For example, peroxidase (hereinafter, peroxidase)
HRP) is a spectrophotometer for diaminobenzidine
Due to its ability to convert to a blue pigment
Can be detected. If the same label has several differences
The above description may be
It also means classifying each label into a separate class
Not. For example,^{12 Five}I as radioactive label or
It can function as an electron density agent. HRP is an enzyme
Or an antibody such as a monoclonal antibody (MAb)
Can function as an antigen against In addition,
Various labels can be combined for the desired effect.
Wear. For example, by labeling MAb and avidin, the present invention
Can be used for the implementation of Biochi probe
Labeled with¹²⁵Label by I
Avidin, and label with HRP
Can be detected by the applied anti-biotin MAb.
Alternatively, dsDNA (or hybridized RNA)
And use the labeled MAbs to label the nucleic acids.
Direct detection of the presence of hybridization without
Can be issued. Other aspects are possible
Yes, and is not limited to the above embodiment.

An "oligonucleotide" is a molecule consisting of two or more deoxyribonucleotides or ribonucleosides. Oligonucleotides can also be nucleotide analogues, such as phosphorotinates and alkyl phosphonates, and derivatized (i.e., labeled)
An oligonucleotide can be included. Artificially synthesized oligonucleotides include primers, probes,
Used for detection controls and unlabeled blocking oligomers, etc., the size of the oligonucleotide depends on the final function or application.

A "probe" is a structure or molecule for detecting or quantifying a target substance. In the art, it often refers to a labeled or immobilized nucleic acid molecule that specifically binds to a nucleic acid sequence to be detected by hybridization. Also in the present invention, the expression “probe” simply refers to “nucleic acid probe”. Nucleic acid probes include those in which natural nucleic acid molecules are labeled or immobilized and those in which artificially synthesized nucleic acid molecules (oligonucleotides) are labeled or immobilized. The nucleic acid molecule is preferably single-stranded, but may be double-stranded. In the case of a double strand, a strand that has been treated to separate the strand before use for hybridization is usually used. In the present invention, an artificially synthesized oligonucleotide is used as a probe. The probe must be long enough to hybridize, but too long is not preferred because it will induce nonspecific reactions. Therefore, an appropriate length is determined depending on many factors, such as hybridization reaction conditions.

"Sequence-specific hybridization"
Means that there are stringent hybridization conditions that require exact complementarity between the probe and the sample target sequence for hybridization to occur. This condition is readily recognized by those skilled in the art and depends on the length of the probe (chain length) and the base composition. In general,
To obtain conditions under which the probe will not substantially hybridize in the absence of an exact match, alter the hybridization conditions such as the temperature, pH, ionic strength, and concentration of the chaotropic agent of the hybridization solution. Or, change the position of the probe or the chain length itself. For hybridization of the probe to the bound DNA, standard conditions (0.9M N
Tm (° C.) = 4 × (NG + NC) + 2 × (NA + NT) −
5 are known. Where NG, NC, NA and NT are
G (guanine), C (cytosine),
The number of A (adenine) and T (thymine) bases (J. Mei
nkoth et al., 1984, Analyt. Biochem., 138: 267-284). However, those skilled in the art will recognize that this formula only gives an approximate value for the optimum temperature and should be verified experimentally to obtain the true optimum temperature. Under hybridization conditions at a salt concentration of about 2 × SSC and a temperature of about 50 ° C., when used as a nucleic acid probe that can be identified even by a difference of one base, it is preferable that the nucleic acid probe has a chain length of about 20 to 30 nucleotides.

[0013] The present invention relates to at least one oligonucleotide having a reactive group comprising a hybridizing region composed of a nucleotide sequence of about 10 to 50 bases complementary to a specific nucleotide sequence to be detected. A method in which a probe is immobilized on a solid support via a spacer region that cannot hybridize to the specific nucleotide sequence to be detected. The present invention is characterized in that the oligonucleotide constituting the spacer region has 10 bases or less. Preferably it is 4 to 10 bases, more preferably 4 to 6 bases.

A method for introducing a spacer into an immobilized probe has already been described in Japanese Patent No. 2897959. However, the method described here requires 2
It requires a spacer region consisting of an extremely long oligonucleotide of 100 to 800 bases, at least 150 bases or more. Chemically synthesizing a nucleic acid consisting of several hundred bases not only increases the cost but also significantly reduces the yield and purity. For this reason, some contrivances such as connection after synthesis are made, but the manufacturing process becomes complicated. In contrast, in the method of the present invention, a spacer of only 10 bases or less is sufficient. In this case, even if it is added at the time of chemical synthesis of the probe oligonucleotide, the cost does not increase much, and the process of producing the probe is not much different from the case where there is no spacer. That is, a spacer sequence is simply added before or after a region to be hybridized at the time of synthesis. As a result of the study, although the efficiency of hybridization is clearly improved by the addition of the spacer,
In most cases, the effect was almost the same with 4 to 10 bases. Therefore, it is considered that 10 bases are sufficient for the spacer of the present invention. The arrangement of the spacer in the present invention is also studied. As a result, it was found that there was no problem with any sequence as long as it had no effect on the hybridizing region. In actual operation, the polyT sequence is used as a spacer for reasons such as the cost of an oligonucleotide synthesis reagent unless otherwise specified.

[0015] The mode of bonding to the solid support in the present invention is also studied. The present inventors have confirmed the same effect by a method using a non-covalent bond such as a hydrophobic bond.
For example, using an alkaline buffer (about pH 10),
Oligonucleotides having an amino linker can be immobilized on a high-adsorption microtiter plate with a hydrophobic bond, but also in this case, by adding a spacer region between the 5′-terminal linker portion and the hybridizing region, The efficiency of hybridization was clearly improved. Again, the effect was almost maximal with 4 nucleotides, showing the same trend as with covalent bonds. If the binding portion to the solid support is clear, the effect of the present invention is exerted regardless of the type of binding.

Further, the present invention provides at least one kind of oligonucleotide probe (hereinafter, also simply referred to as a probe).
Is an immobilized solid support, wherein the probe is complementary to a specific nucleotide sequence to be detected.
A hybridizing region constituted by a nucleotide sequence of 0 to 50 bases and a spacer region which cannot hybridize to the specific nucleotide sequence to be detected, wherein the spacer region is attached to the solid support at one end. A solid which is bonded to the hybridizing region at the other end, and wherein the spacer region comprises an oligonucleotide having 10 bases or less, preferably 4 to 10 bases, and more preferably 4 to 6 bases. It is a support.

Further, the present invention provides a reagent kit for detecting a nucleic acid, comprising the above solid support, and more particularly, a reagent kit for detecting a stable nucleic acid, comprising a solid support having an oligonucleotide covalently bonded via a spacer arm. About. Further, it is preferable to include a means for detecting a specifically bound target nucleic acid. The means for detecting the specifically bound target nucleic acid refers to a detection probe consisting of a label indirectly or directly bound to the target nucleic acid, or a labeled oligonucleotide, such as a radioisotope, a fluorescent substance, a luminescent substance, and an enzyme. It is. Examples of the binding mode of the label include a method of labeling a probe that hybridizes to a region different from a probe (capture probe) immobilized on a solid support on a target nucleic acid (so-called sandwich hybridization), and a method for nucleic acid amplification. There is a method of directly labeling a target nucleic acid by incorporating a labeled primer or a labeled mononucleotide. There is also a method of labeling after hybridization via a bond between an antibody and an antigen or between biotin and avidin. The nucleic acid detection reagent kit can be widely applied to clinical diagnosis, forensic medicine, environmental investigation of microorganisms, quality assurance of foods and drugs, and research techniques of molecular biology.

The present invention relates to the form of a capture probe bound to a solid support, and the method of detection is not particularly limited. Further, a target nucleic acid that hybridizes to a capture probe bound to a solid support may be directly labeled.
(JP-A-62-265999). Alternatively, a labeled detection probe for detecting a target nucleic acid bound to a capture probe may be used as in the above-mentioned sandwich hybridization assay (JP-A-58-4009).
No. 9). Examples of the label include a radioisotope, a fluorescent substance, an electron density reagent, an enzyme and the like, and it goes without saying that an indirect label using an avidin / biotin bond, an antibody or the like, or an intercalator may be used.

The material and shape of the solid support are not particularly limited, but it is preferable to use a microtiter plate from the viewpoint of ease of assay. The material of the microtiter plate is not particularly limited, but is preferably made of polystyrene. The most common shape of the microtiter plate is 9.
A plate with six holes is mentioned, but one row or one
Also includes a strip-shaped one having 8 or 12 holes corresponding to a row. Further, with the development of the dedicated device, there is essentially no problem even if the solid support is made of a material corresponding thereto or has a special shape. For example, using a glass or plastic plate-shaped solid support, it is easy to apply it to a dimensionally stable solid support (so-called DNA chip) having individual regions for immobilizing different probes on a solid phase. It can be analogized to Their use improves the physical format and increases the reliability and economy of hybridization and detection.

An important aspect of the present invention, with respect to the structure of oligonucleotide probes attached to a solid support, has provided a method that is particularly suitable for mass production and that allows for maximum probe persistence and hybridization efficiency. It is in. The present invention allows for time separation between preparation of immobilized probes and their use due to the permanence of the covalent bond, and rapid detection of target nucleic acid sequences in test samples as needed This allows for the preparation of storage stable detection reagents, ie, hybridization capture solid supports, that can be used for By interposing a spacer region between the solid support and the probe, the efficiency of hybridization is greatly facilitated and improved.

[0021]

The effects of the present invention will be further clarified by exemplifying embodiments of the present invention. The present invention is not particularly limited by the examples.

Example 1 Synthesis and Immobilization of Amino Linker Oligonucleotide An oligonucleotide having an amino linker arm was synthesized and bound to the inner surface of a polycarbodiimide-coated polystyrene microtiter plate to obtain a capture probe. At this time, the spacer region of the present invention was introduced to prepare a sample for comparative study.

(1) Synthesis of Oligonucleotide for Capture Probe Having Amino Linker Arm Sequence Listing / SEQ ID NO: 1 by a phosphoramidite method using a DNA synthesizer 392 manufactured by PerkinElmer.
Oligonucleotides having the sequence shown in (Mycobacterium tuberculosis Mycobacterium tuberculosis (Mycobacter
ium tuberculosis) An oligonucleotide for a capture probe for detecting 16S rRNA gene (hereinafter referred to as a capture probe) was synthesized. At this time, Japanese Patent Application Laid-Open No. 60-500717
Uridine having an amino linker arm at the 5-position prepared by chemical synthesis from deoxyuridine by the synthesis method described in Japanese Patent Application Laid-Open Publication No. H11-209,097 was introduced into the above oligonucleotide. This uridine can replace any T residue in the oligonucleotide, but is added here at the 5 'end, and the T residue as a spacer is 0, 4, 5, 5,
Those having 10, 15 bases added were synthesized.

[0024]

Embedded image

The synthesized linker oligonucleotide was subjected to a deprotection treatment with aqueous ammonia at 50 ° C. overnight, and then purified by Pharmacia FPLC using an anion exchange column.

(2) Bonding to the inner surface of a polycarbodiimide-coated plate The various amino linker oligonucleotides synthesized in (1) were placed on the inner surface of a polycarbodiimide-coated polystyrene microtiter plate (NS plate DN; manufactured by Nisshinbo Industries, Ltd.). (See Japanese Unexamined Patent Publication No.
No. 23975). The carbodiimide group of the solid support reacts with the amino group of the oligonucleotide to form a covalent bond. First, an amino linker oligonucleotide was added to a 2M NaCl solution at a concentration of 5 mM, and 200 μL of NS was added.
Dispensed into plate DN. The solution was kept at 37 ° C. for 1 hour and washed with distilled water. The obtained capture probe binding plate was vacuum-dried and refrigerated in a dry state.

Example 2 Performance Evaluation of the Capture Probe of the Present Invention in Directly Detected Nucleic Acid Performance of the capture probe of the present invention was evaluated by synthesizing an enzyme-labeled oligonucleotide complementary to the capture probe and detecting it as a target. .

(1) Synthesis of Oligonucleotide Having Amino Linker Arm Sequence Listing / Sequence No. 2 was prepared by the phosphoramidite method using DNA synthesizer 392 manufactured by PerkinElmer.
Were synthesized (oligonucleotides for capture probe target). At this time, uridine having an amino linker arm at the 5-position, which was prepared by chemical synthesis from deoxyuridine by the synthesis method described in JP-T-60-500717, was introduced into the oligonucleotide. Uridine having this amino linker arm can substitute any T residue in the oligonucleotide, but in this case has been substituted with the 5 'terminal T residue.
The synthesized linker oligonucleotide was subjected to deprotection treatment with ammonia water at 50 ° C. overnight, and then purified by Pharmacia FPLC using an anion exchange column.

(2) Labeling of Oligonucleotide with Enzyme (Alkaline Phosphatase) The oligonucleotide for the capture probe target synthesized in the above (1) is reacted with alkaline phosphatase (hereinafter also referred to as ALP) via its amino linker arm. The binding is described in the literature (Nucleic Acids Res .; Vol. 14, No. 6).
115, 1986). Linker oligonucleotide (OD260 = 1.5) was added to 0.2 M NaHCO ₃
The resultant was dissolved in 12.5 μL, and 25 μL of 10 mg / mL disuccinimidyl suberate (DSS) was added thereto, followed by reaction at room temperature for 2 minutes. The reaction solution was treated with 1 mM CH ₃ COONa (pH 5.
Excess DSS was removed by gel filtration with a Sephadex G-25 (Pharmacia) column (1 cmφ × 30 cm) equilibrated in step (0). The linker oligonucleotide in which the terminal amino group was activated was further added with a twice molar amount of alkaline phosphatase (Boehringer Mannheim) in 100 mM Na.
HCO ₃ , dissolved in 3M NaCl) and 1 at room temperature
The reaction was carried out for 6 hours to obtain an alkaline phosphatase-labeled oligonucleotide. The obtained labeled oligonucleotide was purified using an anion exchange column with FPLC manufactured by Pharmacia. The fractions containing the labeled oligonucleotide were collected and concentrated by ultrafiltration using Centricon 30K (manufactured by Amicon). This is used as a capture probe target oligonucleotide.

(3) Hybridization in a microtiter plate 5 × SSC containing the above-mentioned (2) ALP-labeled oligonucleotide for a capture probe target at a concentration of 1,0.1 fmol / μL or no addition thereof (pH 7.0), 0.1
% Scruff AG (Nippon Seika Co., Ltd.), 0.5% P
VP, 10 mM MgCl ₂ , 1 mM ZnCl ₂ , 0.1% N
Inject 100 μL of aN ₃ solution into various capture probe binding plates and overlay with liquid paraffin to prevent evaporation.
Shake at 0 ° C. for 30 minutes (100, 10, 0 f, respectively)
mol / assay). Thereafter, 2 × SSC (pH 7.0), 0.1
% Scruff AG, kept at 50 ° C. for 10 minutes, and further washed with 1 × SSC. After draining the cleaning liquid,
Lumiphos 480 (manufactured by Lumigen), a luminescent substrate of ALP
After injecting 0 μL and keeping the mixture at 37 ° C. for 15 minutes, the amount of luminescence (luminescence signal) was measured with a photon counter (Hamamatsu Photonics) in a dark room. All of these steps are automatically performed by a DNA probe automatic measurement system (Journal of the Japan Society of Clinical Laboratory Automation; Vol. 20, p. 728, 1995), and the required time is about 1 hour. The intensity of the luminescence signal indicates the intensity of the hybridization reaction.

(4) Results Compared to the case where the capture probe has no spacer (0 bases),
When there are four or more bases, the luminescence signal is twice or more. Since both 100 fmol / assay and 10 fmol / assay were improved at the same ratio, the total amount (capacity) of the bound capture probes did not change. It is considered that this has improved. In addition, since there is not much change from 4 bases to 15 bases, 4 bases are sufficient in the present detection system. The results demonstrated the effect of the spacer of the present invention.

[0032]

[Table 1]

Example 3 Performance Evaluation of the Capture Probe of the Present Invention in Sandwich Hybridization An oligonucleotide having a sequence complementary to the capture probe and the labeled probe was synthesized, and the oligonucleotide was detected as a target for sandwich hybridization to capture the present invention. The performance of the probe was evaluated.

(1) Synthesis of Oligonucleotide An oligonucleotide having the sequence shown in SEQ ID NO: 3 (detection of 16S rRNA gene of Mycobacterium tuberculosis) was carried out by a phosphoramidite method using PerkinElmer DNA synthesizer type 392. Labeled probe (hereinafter, referred to as labeled probe) oligonucleotide) and sequence listing
Oligonucleotide having the sequence shown in SEQ ID NO: 4
(Sandwich hybridization target oligonucleotide (hereinafter, referred to as target oligonucleotide)) was synthesized. When synthesizing an oligonucleotide for a labeled probe, uridine having an amino linker arm at the 5-position prepared by chemical synthesis from deoxyuridine by the synthesis method disclosed in JP-A-60-500717,
It was introduced into the oligonucleotide described in Sequence Listing / SEQ ID NO: 3. This uridine may be any T in the oligonucleotide.
The residue may be substituted, but was replaced here with the 11th T residue from the 5 'end. The synthesized oligonucleotide is
Each was subjected to deprotection treatment with ammonia water at 50 ° C. overnight, and then purified by Pharmacia FPLC using an anion exchange column.

(2) Labeling of Oligonucleotide with Alkaline Phosphatase For the labeled probe oligonucleotide synthesized in the above (1), ALP via its amino linker arm was used.
Binding with Literature (Nucleic Acids Research .; Chapter 14)
Volume, p. 6115, 1986). 0.2 M linker oligonucleotide (OD260 = 1.5)
NaHCO ₃ was dissolved in 12.5 μL, and 10 mg /
Then, 25 μL of mL disuccinimidyl suberate (DSS) was added and reacted at room temperature for 2 minutes. The reaction solution was 1 mM CH ₃ CO
Excess DSS was removed by gel filtration using a Sephadex G-25 (Pharmacia) column (1 cmφ × 30 cm) equilibrated with ONa (pH 5.0). The linker oligonucleotide in which the terminal amino group was activated was further added with a twice molar amount of alkaline phosphatase (Boehringer Mannheim)
Was dissolved in 100 mM NaHCO ₃ and 3 M NaCl) at room temperature for 16 hours to obtain an ALP-labeled oligonucleotide. The obtained labeled oligonucleotide was purified by Pharmacia FPLC using an anion exchange column. The fractions containing the labeled oligonucleotide were collected and concentrated by ultrafiltration using Centricon 30K (manufactured by Amicon). This is used as a labeled probe for sandwich hybridization.

(3) Sandwich Hybridization in a Microtiter Plate An aqueous solution containing the target oligonucleotide at a concentration of 10, 1 fmol / μL or pure water (for blank) is mixed with an equal volume of 0.6N NaOH. Denature, 30 μL for each test, 200 mM citrate-phosphate buffer (pH 6.
0) 2% Scruff AG (Nippon Seika Co., Ltd.), 75
The mixture was mixed with 80 μl of a solution of 0 mM NaCl and 0.1% NaN ₃ , immediately poured into the capture plate of Example 1, and overlaid with liquid paraffin to prevent evaporation, and shaken at 50 ° C. for 30 minutes (respectively, 100, 10.0 fmol / assay). By this reaction, the target oligonucleotide is captured by the capture probe.

Next, the labeled probe of the above (2) was used at 10 fm.
5 x SSC (pH 7.0), containing 0.1%
Scruff AG, 0.5% PVP, 10 mM MgC
l ₂ , 1 mM ZnCl ₂ , 0.1% NaN ₃ solution 100
After replacing with μL, liquid paraffin was similarly overlaid to prevent evaporation, and the mixture was shaken at 50 ° C. for 30 minutes. by this,
The labeled probe specifically binds to the captured target oligonucleotide. Thereafter, 2 × SSC (pH 7.0),
It was replaced with 0.1% Scruff AG, kept at 50 ° C. for 10 minutes, further replaced with 1 × SSC and washed. After draining the washing solution, Lumiphos 480 (Lumigen), a luminescent substrate for ALP
After injecting 100 μL and keeping the mixture at 37 ° C. for 15 minutes, the amount of luminescence (luminescence signal) was measured with a photon counter (Hamamatsu Photonics) in a dark room. All these steps involve DNA
The measurement is automatically performed by an automatic probe measurement system (Journal of the Japan Society of Clinical Laboratory Automation; vol. 20, p. 728, 1995), and the required time is about 2 hours. The intensity of the luminescence signal indicates the intensity of the hybridization reaction.

(4) Results Compared with the case where the capture probe has no spacer (0 bases),
When there are four or more bases, the luminescence signal is about three times higher. 100 fmol / assa as in the case of directly labeled nucleic acid
y Since both 10 fmol / assay are improved at a similar ratio, the total amount (capacity) of the bound capture probes does not change, but the addition of the spacer improves the hybridization efficiency itself. It is thought that it did. In addition, since there is not much change from 4 bases to 15 bases, it is sufficient that the length of the spacer is 4 bases in the present detection system. The fact that the tendency is very similar to that in the case of detection using the directly labeled nucleic acid of Example 2 also strongly suggests that the hybridization efficiency of the capture step using the capture probe has been improved. The results demonstrated the effect of the spacer of the present invention.

[0039]

[Table 2]

[0040]

As described above, in the present invention, 1
By immobilizing the probe on the solid support using a spacer consisting of an oligonucleotide having 0 or less bases, it has become possible to improve the hybridization efficiency of the immobilized probe on the solid support at extremely low cost. Further, the present invention is extremely effective as an improvement technique of a hybridization reaction widely used in clinical diagnosis and biological research, and greatly contributes to the development of various reagents and kits using these nucleic acid detection techniques. It is.

[0041]

[Sequence list] SEQ ID NO: 1 Sequence length: 20 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..20 Characteristics Method determined: S Other characteristics: It has a sequence complementary to the sequence of the Mycobacterium tuberculosis 16S rRNA gene. Sequence ACATGCATCC CGTGGTCCTA 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..20 Features Method determined: S Other characteristics: Sequence complementary to the sequence of Mycobacterium tuberculosis 16S rRNA gene. Sequence TAGGACCACG GGATGCATGT 20

SEQ ID NO: 3 Sequence length: 18 Sequence type: Nucleic acid number of strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..18 Features Method determined: S Other characteristics: Sequence complementary to the sequence of Mycobacterium tuberculosis 16S rRNA gene. Sequence CCCACACCGC TAAAGCGC 18

SEQ ID NO: 4 Sequence length: 39 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..18 Features Method determined: S Other characteristics: Sequence complementary to the sequence of Mycobacterium tuberculosis 16S rRNA gene. Sequence TAGGACCACG GGATGCATGT TGCGCTTTAG CGGTGTGGG 39

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) G01N 35/02 C12N 15/00 ZNAA F-term (reference) 2G042 AA01 BD12 BD20 CB03 DA08 FB05 HA07 2G058 AA09 CC09 EA11 GA01 4B024 AA01 AA11 CA01 HA12 4B063 QA01 QA18 QQ42 QR32 QR35 QR55 QR63 QR84 QS03 QS34 QX02

Claims (9)

[Claims] 1. A solid support on which at least one oligonucleotide probe is immobilized, said probe comprising a nucleotide sequence of about 10 to 50 bases complementary to a specific nucleotide sequence to be detected. A hybridizing region and a spacer region that cannot hybridize to the specific nucleotide sequence to be detected, wherein the spacer region binds to the solid support at one end and hybridizes at the other end. A solid support which is bound to a region and wherein the spacer region comprises an oligonucleotide having 10 bases or less. 2. The solid support according to claim 1, wherein the spacer region comprises an oligonucleotide having 4 to 10 bases. 3. The solid support according to claim 1, wherein the solid support is a microtiter plate. 4. The solid support according to claim 1, wherein the probe is an oligodeoxyribonucleotide or a derivative thereof. 5. At least one oligonucleotide probe having a reactive group comprising a hybridizing region composed of a nucleotide sequence of about 10 to 50 bases complementary to a specific nucleotide sequence to be detected is used. A method of immobilizing on a solid support via a spacer region that cannot hybridize to the specific nucleotide sequence to be detected, wherein the spacer region comprises an oligonucleotide having 10 bases or less. how to. 6. The method according to claim 5, wherein a polycarbodiimide-coated microtiter plate is used as the solid support, and the probe is bound to the end of the probe by an addition reaction of a carbodiimide group. 7. A reagent kit for detecting a nucleic acid, comprising the solid support according to any one of claims 1 to 4. 8. The reagent kit for detecting a nucleic acid according to claim 7, further comprising a means for detecting a specifically bound target nucleic acid. 9. A method for detecting the presence of a nucleic acid sequence in a sample, comprising the following steps (a) and (b): (A) bringing the sample into contact with the solid support according to any one of claims 1 to 4 under conditions permitting hybridization of a complementary nucleic acid sequence. (B) determining whether hybridization has occurred