JPH0787999A - Solid-phase support for detecting nucleic acid - Google Patents

Abstract

PURPOSE:To provide a solid-phase support which can detect nucleic acid in high accuracy wherein a specific nuclelc acid derivative of a single strand is adsorbed on its surface and a nuclelc acid of a relatively low molecular weight is efficiently adsorbed on the solid phase, as it keeps the ability to form its hybridoma. CONSTITUTION:The objective homo-phase support on whose surface a single strand nucleic acid derivative is adsorbed comprising an aliphatic hydrocarbon bearing an amino group of the formula: H2N-(CH2)n-(n is 2-20) and a single strand nucleic acid of 1 to 100 base pairs having a base sequence which can form a hybridoma with a specific nucleic acid. It is preferred that the nucleic acid in the sample to be tested is labeled and the labeled nucleic acid and the single-strand nucleic acid supported on the above-described solid-phase support are placed under such conditions as they can form the hybrid and the nucleic acid is detected by knowing whether the hybrid is formed or not with the label.

Description

Detailed Description of the Invention

[Background of the Invention]

TECHNICAL FIELD The present invention relates to a solid phase carrier having a single-stranded nucleic acid derivative adsorbed thereon and a method for detecting nucleic acid using the same.

[0002]

2. Description of the Related Art A basic hybridization method for detecting a specific base sequence of a nucleic acid is a solid hybridization method in which a nucleic acid carried on a carrier is hybridized with a nucleic acid in a solution.
There are liquid-hybridization methods and liquid-liquid hybridization methods that hybridize nucleic acids in solution (Anal.
Biochem., 169, 1-25 (1988)). To date, various improvements have been made to the above methods in order to make the detection of nucleic acids simpler, faster, and more sensitive (Anal. Biochem., 169,
1-25 (1988)).

Among them, various methods suitable for mechanization of detection operation include various methods using a microplate as a solid phase carrier, and in the antibody field, detection is generally automated using a microplate. There is. Therefore, even in the detection of nucleic acids, it is possible to automate using the same equipment by using a microplate. Therefore, the method using a microplate as a solid-phase carrier is very excellent when processing a large amount of sample. It can be said that it is a thing.

As an example of immobilizing nucleic acid on a microplate, a method of adsorbing (noncovalently binding) nucleic acid and stabilizing the binding by ultraviolet irradiation has been reported.
(FEBS Letters, 183 (2), 379-382 (1985)). However, when nucleic acid fragments of various lengths were non-covalently bound to a microplate, it was reported that when the nucleic acid fragments were 300 bp or less, the hybridization efficiency became poor, and 49 bp caused almost no hybridization. (JC
Lin. Microbiol, 28, 1469-1472 (1990)). Therefore, this method is limited to long-chain nucleic acids having a relatively large molecular weight, and short-chain nucleic acids (oligonucleotides having a relatively small molecular weight, which are required for highly accurate analysis such as detecting a difference of several bases). Nucleotide) (Proc. Ncad. Sci., USA, 82,278- (198
Application to 3)) cannot be expected.

On the other hand, in recent years, various methods have been proposed for supporting an oligonucleotide on a microplate, and the main method is to immobilize polythymidylate on the microplate (Molecular and Cellular Probe).
s, 3.189-207 (1989)), a method for chemically binding a nucleic acid to a microplate (Anal. Biochem., 198, 138-142 (199).
1)), a method for improving the adsorption form on a microplate by adding a hydrophobic moiety such as a naphthyl group to an oligonucleotide (JP-A-5-500479), and a long-chain nucleic acid obtained by repeatedly binding oligonucleotide units. Adsorption to microplate as a sample (Anal.Bioche
m., 209, 63-69 (1993)).

However, these methods require complicated preparation of the nucleic acid probe and leave room for further improvement in detection accuracy.

[Outline of the Invention]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a solid phase carrier in which a nucleic acid, particularly a short nucleic acid having a short molecular weight, is adsorbed on a solid phase while retaining its hybridizing ability. . A further object of the present invention is to provide a highly accurate method for detecting a nucleic acid using the solid phase carrier having the nucleic acid adsorbed as described above.

The present inventors have studied the adsorption of nucleic acid on a solid phase, and as a result, by introducing an aliphatic hydrocarbon chain having an amino group into the nucleic acid, the adsorption rate of the nucleic acid on the solid phase carrier is improved. It has now been found that it is possible to easily adsorb even a short nucleic acid having a low molecular weight onto a solid phase carrier.

[0009]

The solid phase carrier according to the present invention comprises an aliphatic hydrocarbon chain having an amino group and a single-stranded nucleic acid having a base sequence capable of hybridizing with a specific nucleic acid. It is obtained by adsorbing the single-stranded nucleic acid derivative on the surface thereof.

Further, the method for detecting a nucleic acid according to the present invention comprises (1)
A nucleic acid in a test sample is labeled, and (2) the nucleic acid labeled in step (1) and the single-stranded nucleic acid carried on the solid phase carrier according to any one of claims 1 to 5, Placing under conditions capable of hybridisation, and (3) comprising detecting the presence or absence of hybridisation by means of a label.

[Detailed Description of the Invention] Single-stranded nucleic acid derivative and solid phase carrier to which it is adsorbed The single-stranded nucleic acid used in the present invention comprises (a) an aliphatic hydrocarbon chain having an amino group, (b) A single-stranded nucleic acid having a base sequence capable of hybridizing with a specific nucleic acid.

In the present invention, the aliphatic hydrocarbon chain means a linear or branched saturated or unsaturated carbon number of 2 or more, preferably about 2 to 20. Also,
The substitution position of the amino group in the aliphatic hydrocarbon chain is not particularly limited, and it may be not only at the end of the hydrocarbon chain but also in the hydrocarbon chain. The number of amino group substitutions is not particularly limited as long as it is 1 or more, but about 1 to 20 is preferable, and 1 to 5 is particularly preferable.

A preferred specific example of the aliphatic hydrocarbon chain having an amino group according to the present invention is NH ₂ — (CH ₂ )
n- (here, n represents an integer of 2 to 20) can be mentioned.

In the present invention, the single-stranded nucleic acid has a base sequence capable of hybridizing with a specific nucleic acid. The base sequence is not particularly limited as long as it hybridizes with a specific nucleic acid, and can be freely selected according to the purpose. Also, the number of bases is not particularly limited, but in the present invention, even if the base pair is particularly short (for example, 5 to 20 base pairs or less), the solid-phase carrier while maintaining its hybridizing ability. It is advantageous because it can be adsorbed on. When the solid phase carrier to which the single-stranded nucleic acid derivative according to the present invention is adsorbed is used for detecting a specific nucleic acid, for example, an oligonucleotide capable of specifically hybridizing with the specific nucleic acid can be used. .

The term "nucleic acid" as used herein means DNA or RN.
A, refers to molecules such as modified nucleotides or combinations thereof. In addition, hybrid formation between a single-stranded nucleic acid derivative and a specific nucleic acid is not limited to the case where the single-stranded nucleic acid and the specific nucleic acid are paired over the entire region thereof, but the single-stranded nucleic acid is partially formed in the specific nucleic acid. It also includes the case where they are paired.

The single-stranded nucleic acid can be prepared using an automatic nucleic acid synthesizer. When the single-stranded nucleic acid is DNA, for example, denatured double-stranded nucleic acid and complementary separation can be used as the single-stranded nucleic acid [Nuclei
c Acids Res., 13, 5457-5468 (1985)]. Also, DNA
It may be synthesized using a polymerase and separated from the template [Anal. Biochem., 162, 130-136 (1987).
]. In addition, M containing the gene to be detected
Single-stranded nucleic acid obtained from 13 phages, or composite vector of phage and plasmid (for example, pUC11
8, pBSM13 +, PUCf1 etc.) can also be used [Methods in Enzymolog
y, 153, 3-34 (1987)]. When the single-stranded nucleic acid is RNA, not only naturally-occurring RNA but also those synthesized in vitro using RNA polymerase or the like may be used. In addition, when a base sequence other than a sequence capable of specifically hybridizing with a specific nucleic acid contained in these single-stranded nucleic acids is inconvenient for the hybridization reaction, Messin
g et al. [Methods in Enzymology, 101, part C, 20
(1983)], the part can be removed.

The binding position between the aliphatic hydrocarbon chain having an amino group and the single-stranded nucleic acid is not particularly limited, and the binding position is not limited to the 5'end or the 3'end of the nucleic acid but also in the nucleic acid chain (for example, Phosphodiester bond site or base site).

This single-stranded nucleic acid derivative is disclosed in Japanese Patent Publication No. 3-74.
239, U.S. Pat. No. 4,667,025, U.S. Pat.
It can be prepared according to the method described in 9737. In addition to this method, for example, a commercially available reagent for introducing an amino group (eg, Aminolink II (trade name) (manufactured by Applied Biosystems), Amino Modifier (trade name))
(Manufactured by Clontech) or the like, or 5'of DNA
A well-known method for introducing an aliphatic hydrocarbon chain having an amino group into the terminal phosphoric acid (Nucleic Acids Res., 11 (18), 6513-
(1983)).

In the present invention, this single-stranded nucleic acid derivative is used by being adsorbed on a solid phase carrier. The single-stranded nucleic acid derivative according to the present invention is excellent in adsorption to the solid phase carrier, and is adsorbed while the base sequence therein retains its hybridizing ability. Specifically, as will be apparent from the examples described below, even if the single-stranded nucleic acid derivative has a dozen base pairs, a large amount of the single-stranded nucleic acid can be adsorbed on the solid phase carrier. It can hybridize with a particular nucleic acid. Therefore, the solid-phase carrier to which the single-stranded nucleic acid derivative is adsorbed can be used for highly accurate detection of a specific nucleic acid, for example, detection of a point mutation of a target nucleic acid.

The adsorption of the single-stranded nucleic acid derivative on the solid-phase carrier is
It can be easily carried out by bringing them into contact with each other. Further, according to a preferred embodiment of the present invention, in order to improve its adsorption efficiency, for example, when the solid phase carrier is a microtiter plate, MgCl ₂ or N ₂ is used.
It is preferred to add aCl. Furthermore, the adsorption efficiency can be improved by performing ultraviolet irradiation.

In the present specification, the term "adsorption" refers to a state in which the single-stranded nucleic acid derivative and the solid phase carrier are physically supported, and is excluded when they are supported by a chemical bond.

Polystyrene is a preferred material for the solid phase carrier. The solid phase carrier may be variously modified for the purpose of increasing the adsorption amount of proteins and the like, and examples thereof include those modified by irradiation with ⁶⁰ Co.

The form of the solid phase carrier is not particularly limited and may be a preferred form depending on the application. Specifically, a microtiter plate, a bar,
Examples include fine particles, beads, and plate-shaped materials.

Method for detecting nucleic acid (labeling of nucleic acid in test sample) "Nucleic acid in test sample"
It does not necessarily have to be purified. Further, not only nucleic acids obtained from all living organisms such as bacteria, viruses and higher animals and plants, but also synthetic nucleic acids obtained by amplifying these according to well-known methods, or synthetic nucleic acids complementary to these may be used.

Labeling of nucleic acids can be performed as follows. If the amount of nucleic acid is too small to detect,
Various methods for amplifying specific nucleotide sequences (Bio / TECHNOLOG
Y, 8,291 (1990)) can be used to synthesize and label nucleic acids.

For example, as a method for amplifying a nucleotide sequence, P
When the CR method (Science, 230, 1350-1354 (1985)) is used, a labeled synthetic nucleic acid can be obtained by using a labeled primer or a labeled mononucleotide triphosphate. When the amplification method utilizing Qβ replicase (Bio / TECHNOLOGY, 6, 1197, (1988)) is used, a labeled synthetic nucleic acid can be obtained by using a labeled mononucleotide triphosphate. In an amplification method other than these nucleic acid amplification methods, the target labeled synthetic nucleic acid can be obtained by pre-labeling the mononucleotide triphosphate or the oligonucleotide incorporated by the extension reaction or the amplification reaction.

As a method for labeling and detecting nucleic acid in a test sample other than these, for example, a method of introducing a biotin derivative into a nucleic acid by a photoreaction and binding the enzyme-labeled streptavidin to biotin to detect it (Nucleic Acid Re
s., 13,745, (1983)), a method for detecting a nucleic acid using a sulfonated and enzyme-labeled antisulfonated antibody (Proc. Natl.
Acad.Sci.USA, 81, 3466-3470, (1984)) and the like.

The labeling substance used may be radioactive or non-radioactive as long as it can be detected after hybridization. From the viewpoint of handling and disposal, it is preferable to use a non-radioactive label.

Examples of non-radioactive labels that can be used in the method of the present invention include biotin, 2,4-dinitrophenyl group, haptens such as digoxigenin, fluorescein and its derivatives [eg, fluorescein isothiocyanate (FITC)]. , Rhodamine and its derivatives [eg, tetramethylrhodamine isothiocyanate (TRITC), Texas red, etc.], 4-fluoro-7-nitrobenzofuran (NBDF), dansyl and other fluorescent substances or acridine and other chemiluminescent substances. . In the case of labeling the oligonucleotide with these, any known means (Japanese Patent Laid-Open No. 59-93).
No. 6098, JP-A-59-93099). When labeling nucleotide triphosphates, known means [Proc. Natl. Acad.S
Ci. USA, 80, 4045 (1983), JP-A-63-152364] or a commercially available product can be used.

(Hybridization) Hybridization between a nucleic acid in a sample and a single-stranded nucleic acid derivative is carried out by a conventional method (for example, BDHames and SJHiggins, Nucleic Acid Hybr).
idization, A Practical Approach, the method described in IRL Press (1985), etc.). Although the washing operation after hybridization can be performed according to the conventional method, it is necessary to change the washing condition from the conventional condition in the case of performing highly accurate detection such as detecting a difference in base sequence over several base pairs. is there. For example Nucleic Acid R
It is preferable to utilize the conditions as described in ec., 16, 4637-4650 (1988).

According to the present invention, even a dozen or more base pairs can be supported on a solid phase carrier, so that highly precise detection of a specific nucleic acid can be performed by using the method for detecting a nucleic acid according to the present invention. You can

(Detection of Label) The detection of the label may be appropriately selected and determined according to the type of the label of the nucleic acid. Here, when the label present on the target nucleic acid is directly detectable, that is, when the label is, for example, a radioisotope, a fluorescent substance, a dye, etc., the detection operation is performed with the labeled nucleic acid bound to the solid phase. After the label is bound to the nucleic acid or released from the labeled nucleic acid in the solution, the detection operation is performed by a method according to the label.

Further, when the label is indirectly detectable, that is, when the label is a ligand for a specific binding reaction such as biotin or hapten, it is generally used for their detection. The detection operation is carried out using a receptor (for example, avidin or antibody) to which a label that directly generates a signal or an enzyme that catalyzes a reaction that generates a signal is bound.

It should be noted that these receptors may be added in advance in the hybridization step. In this case,
Part of the detection step, ie the specific binding reaction between the ligand and the receptor, can be carried out simultaneously with the hybridization step, which makes the whole step even simpler.

[0035]

EXAMPLES The present invention will be explained in more detail by the following examples, but the present invention is not limited thereto.
In the following examples, oligonucleotide synthesis was performed by Applied Biosystems, Inc. automatic synthesizer model 3
Using 81A, the general method (Oligonucleotide Synt
hesis, IRL Perss (1984)) and used after deprotection and purification.

Example 1 Preparation of Oligonucleotides Oligonucleotides having a sequence complementary to the HLA-DRB gene as shown in FIG. 1 (probe A, probe B,
Probe C) was synthesized. In addition, the aminolink II was added to the 5'-end of the oligonucleotide having the same sequence as each.
(Trade name) (Applied Biosystems Inc.) was added to separately synthesize oligonucleotides (Probe D, Probe E, Probe F) into which NH ₂ (CH ₂ ) ₆ − was introduced. The probes A and D are completely complementary to DRB5 * 0101, and the probes B and E are DRB1 * 020.
1 is completely complementary to 1 and probes C and D are DRB1 *
It is perfectly complementary to 0901.

Example 2 Oligonucleotide to solid phase carrier
Adsorption of the oligonucleotide obtained in Example 1 with 10 mM
Tris.HCl pH 7.6, 1 mM EDTA solution to a concentration of 2 ng / μl, to which an equal volume of immobilization buffer (1.5 M NaCl, 0.3 M Tris.HC
l pH 8.0, 0.3M MgCl ₂ ) was added and mixed, and a microplate (Sumitomo Bakelite Co., H-pl)
100 μl / well) was added to each well. The plate was sealed with a seal and left at 37 ° C. for 16 hours.
Then, remove the liquid and air-dry at 37 ℃ for 30 minutes, 200μ
l wash buffer 1M NaCl, 2mM MgCl
₂ , 0.1 mM Tris.HCl pH 9.3, 0.
Wash 3 times with 1% Tween 20).

Example 3 Marked HLA-DRB inheritance
Preparation of offspring Using 1 μg of DNA extracted from human peripheral blood as a template, the following biotin-labeled primer (DRBAMP-A,
DRBAMP-B) DRBAMP-A; biotin-CCCCACAGCACGTTTCTTG DRBAMP-B; biotin-CCGCTGCACTGTGAAGCTCT was used to amplify the HLA-DRB gene by PCR. For the reaction, a reagent of GeneAmp (trade name) manufactured by Cetus was added according to the protocol, and the total amount of the reaction solution was 100 μl. The reaction solution was heated at 95 ° C. for 5 minutes with a thermal cycler (PJ-2000) manufactured by Perkin Elmacitas, and then a cycle of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 60 seconds was repeated 30 times.

[0039] microplate having adsorbed oligonucleotides complementary to HLA-DRB gene obtained in Example 4 Hybridization Example 2, the hybridization solution (5xSSC, 200 [mu] g salmon sperm DNA / ml) was added, further embodiments 5 μl of amplified product of HLA-DRB gene labeled with biotin prepared in Example 3
Was denatured by heating on a boiling water bath for 5 minutes, quenched, and then added.
After leaving it at 50 ° C for 60 minutes, the solution was removed, and the plate was heated to 50 ° C, and 200 µl of 3M Tetramethl was added.
ammmonium Chloride, 50mM Tris ・ HCl pH
Washed 7.5 times with 2 mM EDTA. Further, add 200 μl of 0.1 M Tris.H to the microplate.
Cl pH 7.5, 0.1 M NaCl, 2 mM Mg
_{Cl 2, 0.05% (v /} v) Triton X-10
After washing 3 times with 0, a streptavidin-alkaline phosphatase solution (streptavidin-alkaline phosphatase manufactured by BRL was added to 0.1M Tris.HC.
pH 7.5, 0.1 M NaCl, 2 mM MgC
₁₂ , 0.05% (v / v) Triton X-100
(Diluted to 1/2000) with 100 μl / well, and left at 25 ° C. for 15 minutes. After removing the reaction solution,
200 μl of 0.1 M Tris.HCl pH7.
5, 0.1 M NaCl, 2 mM MgCl ₂ , 0.0
It was washed 3 times with 5% (v / v) Triton X-100, and p-nitrophenyl phosphate solution (1 M diethanolamine pH 9.8, 0.5 mM MgCl ₂ , 4 m) was used.
100 μl / well of gp-nitrophenylphosphoric acid / ml) was added and the mixture was reacted at 25 ° C. for 1 hour, and the absorbance was measured at 405 nm. The results are shown in Tables 1 and 2.

As is clear from Tables 1 and 2, the microplate on which the unmodified oligonucleotide was adsorbed showed weak color development as a whole, and no color formation was observed even for a genotype having a high homology in the sequence. On the other hand, NH ₂ (C
In the microplate on which the H ₂ ) ₆ -added oligonucleotide is adsorbed, a color specific to a genotype having high sequence homology is observed.

Table 1 NH ₂ (CH ₂ ) ₆ -Oligonucleotide Sample Microplate Genotype Probe D Probe E Probe F DRB5 * 0101 0.829 0.187 0.321 DRB1 * 1201 0.177 0.565 0.5. 176 DRB1 * 0901 0.291 0.229 1.045

Table 2 Unmodified oligonucleotides Samples Microplate Genotype Probe A Probe B Probe C DRB5 * 0101 0.191 0.143 0.179 DRB1 * 1201 0.132 0.211 0.187 DRB1 * 0901 0. 186 0.196 0.296

Example 5 Effect of UV Irradiation The oligonucleotide prepared in Example 1 was adsorbed on a microplate in the same manner as in Example 2. However, before washing with a washing buffer, Stratalinker (trade name) 24
00 (manufactured by Stratagene), 500,000μ
UV irradiation of J was performed. Hybridization was performed using this plate in the same manner as in Example 4. The results are shown in Tables 3 and 4.
As shown in.

As is clear from Tables 3 and 4, in the microplate on which the usual oligonucleotide was adsorbed even when binding was carried out by ultraviolet irradiation, the overall color was weak, and even the genotype with high sequence homology was colored. Can't be seen. On the other hand, in the microplate on which the NH ₂ (CH ₂ ) ₆ -added oligonucleotide is adsorbed,
Genotype-specific coloring with high sequence homology is observed. Further, comparing the case where the oligonucleotide to which NH ₂ (CH ₂ ) ₆ − is adsorbed without ultraviolet irradiation (Table 1) and the case where ultraviolet irradiation is performed (Table 3), the ultraviolet irradiation is more The color is slightly strong, and the adsorption of the oligonucleotide to the microplate is more stable.

Table 3 NH ₂ (CH ₂ ) ₆ -Oligonucleotide + UV Irradiation Sample Microplate Genotype Probe D Probe E Probe F DRB5 * 0101 1.160 0.201 0.351 DRB1 * 1201 0.212 0. 660 0.198 DRB1 * 0901 0.333 0.177 1.089

Table 4 Unmodified oligonucleotide + UV irradiation sample Microplate Genotype probe A probe B probe C DRB5 * 0101 0.265 0.179 0.174 DRB1 * 1201 0.186 0.188 0.128 DRB1 * 0901 0.221 0.144 0.286

Comparative Example 1 Aliphatic hydrocarbon and aromatic carbon
Synthesis of oligonucleotide having hydrogen halide (1) Oligonucleotide having n-hexyl group n-hexyl alcohol (623 μl, 5 mmol) 10
ml tetrahydrofuran and 1140 μl (4.5 mm
ol) N, N-diisopropylethylamine,
Next, 1 g (4.5 mmol) of 2-cyanoethyl-N, N-diisopropyl-chlorophosphoramidite (manufactured by SIGMA) was added under a nitrogen gas atmosphere, and the mixture was stirred at room temperature for 30 minutes. Precipitated salt was removed by centrifugation (3000 xg, 5 minutes), and the supernatant was evaporated and concentrated. To this, 30 ml of ethyl acetate was added and mixed. Then 30
The operation of adding 5 ml of a 5% sodium hydrogen carbonate aqueous solution and separating with a separating funnel was repeated three times, and further, the operation of adding 30 ml of saturated saline and separating with a separating funnel was performed once.
The collected organic layer was evaporated and concentrated. The operation of adding pyridine to this and azeotroping under reduced pressure was repeated 4 times, and the operation of adding toluene and azeotroping under reduced pressure was repeated 3 times.
Repeated times. The resulting reaction product was dried under reduced pressure for 16 hours to obtain 1090 mg of oily 2-cyanoethyl N, N-diisopropyl-hexylphosphalamidite.

100 mg / ml of this with anhydrous acetonitrile
5'of the oligonucleotide (probe A, probe B, probe C) using an automated synthesizer model 381A manufactured by Applied Biosystems.
Oligonucleotides (probe G, probe H, probe I) having CH ₃ (CH ₂ ) ₅ − introduced into the terminal were synthesized. The synthesized oligonucleotides were analyzed by reverse-phase high performance liquid chromatography, and oligonucleotides with the same base sequence (probe A, probe B, probe C)
It was confirmed that the elution time was later than that.

(2) Oligonucleotide having CH ₃ (CH ₂ ) ₁₁ -The n-hexyl alcohol was added to 1-dodecanol (1121).
(1 μl, 5 mmol), except that 1234 mg of oily 2-cyanoethyl N,
N-diisopropyl-dodecylphosphoramidite was obtained.

100 mg / ml of this with anhydrous acetonitrile
Prepared as a oligonucleotide (probe A, probe B, probe C) _CH 3 at the 5 'end of the _{(CH 2) 11} by using an automatic synthesizer model 381A of Applied Biosystems - was introduced oligo Nucleotides (probe J, probe K, probe L) were synthesized.
The synthesized oligonucleotide was analyzed by reverse phase high performance liquid chromatography, and it was confirmed that the elution time was delayed as compared with the oligonucleotides having the same base sequence (probe A, probe B, probe C).

(3) Oligonucleotide having β-naphthyl- (CH ₂ ) ₂ — The same operation as in (1) above except that n-hexyl alcohol was replaced with 2-naphthalene ethanol (861.2 mg, 5 mmol). Done, 1234 mg of oily 2-cyanoethyl N, N-diisopropyl-2- (2-naphthalene)
An ethyl phosphoramidite was obtained.

100 mg / ml of this with anhydrous acetonitrile
And β-naphthyl- (CH ₂ ) ₂ -at the 5'end of the oligonucleotide (probe A, probe B, probe C) using an automated synthesizer model 381A manufactured by Applied Biosystems. The synthesized oligonucleotides (probe M, probe N, probe O) were synthesized. The synthesized oligonucleotides were analyzed by reverse-phase high performance liquid chromatography, and oligonucleotides having the same base sequence (probe A, probe B, probe C)
It was confirmed that the elution time was later than that.

Comparative Example 2 Hybridization Each oligonucleotide obtained in Comparative Example 1 was immobilized on a microplate by the same procedure as in Example 5.
Using this, hybridization was performed using HLA-DRB gene typing as a model in the same manner as in Example 4.

The results are as shown in Tables 5, 6 and 7. As can be seen by comparing Tables 5, 6 and 7 with Table 3,
Even if an oligonucleotide probe having an amino group-free aliphatic hydrocarbon and an aromatic hydrocarbon added to the 5'end and having a hydrophobic property is adsorbed on a microplate, sufficient color cannot be obtained. It can be seen that strong coloring cannot be obtained even for genes with high homology in sequence.

Table 5 CH ₃ (CH ₂ ) ₅ -Oligonucleotide Samples Microplate Genotype Probe G Probe H Probe I DRB5 * 0101 0.280 0.115 0.115 DRB1 * 1201 0.142 0.158 0. 136 DRB1 * 0901 0.158 0.153 0.171

Table 6 CH ₃ (CH ₂ ) ₁₁ -Oligonucleotide Samples Microplate Genotype Probe J Probe K Probe L DRB5 * 0101 0.551 0.169 0.148 DRB1 * 1201 0.203 0.185 0. 148 DRB1 * 0901 0.164 0.167 0.190

Table 7 β-Naphthyl- (CH ₂ ) ₂ -Oligonucleotides Sample Microplate Genotype Probe M Probe N Probe O DRB5 * 0101 0.429 0.155 0.158 DRB1 * 1201 0.148 0.285 0.138 DRB1 * 0901 0.158 0.148 0.246

[0058]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTCATACTTA TCCTGCTG 18

SEQ ID NO: 2 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GGTTACTGGA GAGACACT 18

SEQ ID NO: 3 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GTATCTGCAC AGAGGCAT 18

SEQ ID NO: 4 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics 5'C Phosphate The sequence CTCATACTTA TCCTGCTG 18 in which NH _2- (CH ₂ ) ₆ -is bound to

SEQ ID NO: 5 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acids Synthetic DNA Sequence Features 5'G G Phosphate the _{NH 2 - (CH 2) 6} - sequences are attached GGTTACTGGA GAGACACT 18

SEQ ID NO: 6 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate the _{NH 2 - (CH 2) 6} - sequences are attached GTATCTGCAC AGAGGCAT 18

SEQ ID NO: 7 Sequence length: 100 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Human (Homo sapiens) Sequence characteristics human leukocyte antigen Partial sequence of HLA-DRB gene GGGGACACCC GACCACGTTT CTTGTGGCAG CTTAAGTTTG AATGTCATTT CTTCAATGGG 60 ACGGAGCGGG TGCGGTTGCT GGAAAGATGC ATCTATAACC 100

SEQ ID NO: 8 Sequence length: 100 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Human (Homo sapiens) Sequence characteristics Human leukocyte antigen Partial sequence of HLA-DRB gene GGGGACACCA GACCACGTTT CTTGGAGTAC TCTACGGGTG AGTGTTATTT CTTCAATGGG 60 ACGGAGCGGG TGCGGTTACT GGAGAGACAC TTCCATAACC 100

SEQ ID NO: 9 Sequence length: 100 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Human (Homo sapiens) Sequence characteristics Human leukocyte antigen Partial sequence of HLA-DRB gene NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NTTCAACGGG 60 ACGGAGCGGG TGCGGTATCT GCACAGAGGC ATCTATAACC 100

SEQ ID NO: 10 Sequence length: 100 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Human (Homo sapiens) Sequence characteristics Human leukocyte antigen Partial sequence of HLA-DRB gene GGGGACACCC GACCACGTTT CTTGCAGCAG GATAAGTATG AGTGTCATTT CTTCAACGGG 60 ACGGAGCGGG TGCGGTTCCT CGCAAGGAGC ATCTATAACC 100

SEQ ID NO: 11 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'terminal C with a spacer Sequence bound by biotin via CCCCACAGCA CGTTTCTTG 19

SEQ ID NO: 12 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'terminal C with a spacer Sequence bound via biotin via CCGCTGCACT GTGAAGCTCT 20

SEQ ID NO: 13 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics 5'C Phosphate the _{CH 3 - (CH 2) 5} - sequences are attached CTCATACTTA TCCTGCTG 18

SEQ ID NO: 14 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate CH _3- (CH ₂ ) ₅ -is bound to the sequence GGTTACTGGA GAGACACT 18

SEQ ID NO: 15 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acids Synthetic DNA Sequence Features 5'G G Phosphate the _{CH 3 - (CH 2) 5} - sequences are attached GTATCTGCAC AGAGGCAT 18

SEQ ID NO: 16 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics 5'C Phosphate The sequence CTCATACTTA TCCTGCTG 18 in which CH _3- (CH ₂ ) ₁₁ -is bound to

SEQ ID NO: 17 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate The sequence GGTTACTGGA GAGACACT 18 in which CH _3- (CH ₂ ) ₁₁ -is bound to

SEQ ID NO: 18 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate The sequence GTATCTGCAC AGAGGCAT 18 in which CH _3- (CH ₂ ) ₁₁ -is bound to

SEQ ID NO: 19 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Characteristics 5'C Phosphate Β-naphthyl- (CH ₂ ) ₂
The sequence that is bound by CTCATACTTA TCCTGCTG 18

SEQ ID NO: 20 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate Β-naphthyl- (CH ₂ ) ₂
The sequence to which is attached GGTTACTGGA GAGACACT 18

SEQ ID NO: 21 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 5'G G Phosphate Β-naphthyl- (CH ₂ ) ₂
The sequence that is bound by GTATCTGCAC AGAGGCAT 18

[Brief description of drawings]

FIG. 1 is a nucleotide sequence of a single-stranded nucleic acid derivative (probe A to
It is a figure showing the relation between O) and the base sequence of the nucleic acid of a test sample.

Claims (9)

[Claims] 1. A single-stranded nucleic acid derivative comprising an aliphatic hydrocarbon chain having an amino group and a single-stranded nucleic acid having a base sequence capable of hybridizing with a specific nucleic acid is adsorbed on its surface. A solid support. 2. An aliphatic hydrocarbon chain having an amino group is NH
The solid phase carrier according to claim 1, which is represented by _2- (CH ₂ ) n- (where n represents an integer of 2 to 20). 3. The solid phase carrier according to claim 1, wherein the single-stranded nucleic acid has 1 to 100 base pairs. 4. The solid phase carrier is polystyrene.
4. The solid phase carrier according to any one of 3 to 3. 5. The solid phase carrier according to any one of claims 1 to 4, wherein the form of the solid phase carrier is a microtiter plate. 6. A single-stranded nucleic acid derivative comprising an aliphatic hydrocarbon chain having an amino group and a single-stranded nucleic acid having a base sequence capable of hybridizing with a specific nucleic acid, which is a solid-phase carrier. A single-stranded nucleic acid derivative used by being adsorbed. 7. An aliphatic hydrocarbon chain having an amino group is NH
_The single-stranded nucleic acid derivative according to claim 6, which is represented by _2- (CH ₂ ) n- (where n represents an integer of 2 to 20). 8. The single-stranded nucleic acid derivative according to claim 6, wherein the single-stranded nucleic acid has 1 to 100 base pairs. 9. (1) A nucleic acid in a test sample is labeled, and (2) the nucleic acid labeled in step (1) and the solid phase carrier according to any one of claims 1 to 5 are carried. Single-stranded nucleic acid,
A method for detecting a nucleic acid, which comprises placing under hybridizing conditions and (3) detecting the presence or absence of hybridization with a label.