JPS60169495A - Modified dna and use thereof - Google Patents

Abstract

NEW MATERIAL:A polydeoxynucleotide having hapten linked directly or through a ligand to the phosphoric acid moiety at the 5'-terminal. USE:A detection reagent for viral genes or abnormal chromosomes by the method for nonradioactive nucleic acid hybridization. PREPARATION:A modified DNA obtained by reacting a hepten formation reagent, e.g. 2,4-dinitrophenylethylenediamine, with the phosphoric acid moiety at the 5'-terminal of a poly DNA, e.g. hepatitis B viral DNA. The resultant modified DNA is then hybridized with a polynucleotide in a sample immobilized in a cell or support, and an antidinitrophenyl antibody labeled with fluorescence or an enzyme to introduce a label into the sample. The optical response caused by optical excitation or addition of a substrate is sensed to detect specific base sequence in the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel modified DNA and its uses.

Experiments using radioactive isotopes are widely used as sensitive methods in biochemical research. However, careful consideration must be given to the risk of radiation exposure and waste disposal, and conducting these experiments requires enormous amounts of money and special space. Furthermore, the half-lives of 32P and 125F are short, and reagents containing them cannot be stored, resulting in the inconvenience of having to prepare them on-the-go.

On the other hand, currently in molecular biology, nucleic acid hybridization methods using deoxynucleotides (hereinafter sometimes abbreviated as DNA) or RNA fragments that have complementary base sequences are often used to detect or identify specific genes. ing. Currently, this method mainly uses 32
However, due to its short lifespan, it is limited to basic research and is not used as a means of clinical testing in hospitals.

It is especially important in clinical practice to easily and quickly detect specific DNA sequences such as virus genes or abnormal chromosomes that are worth diagnosing. It is desired to directly detect the virus genome inside.

Recently, non-radioactive immunodiagnostic methods have been shown to be fully comparable to radioactive immunodiagnostic methods, but the present inventors have combined fluorescence immunoassay and enzyme immunoassay techniques with nucleic acid hybridization methods. Believing that it would be possible to develop a specific genetic diagnostic method with a 16-degree level, we conducted extensive research and completed the present invention.

That is, the present invention provides a polydeoxynucleotide in which a hapten is bound to the 5'-terminal phosphate moiety directly or via a ligand, and a polydeoxynucleotide in which the polydeoxynucleotide is immobilized in cells or on a support. Provided is a method for detecting a specific base sequence in a polynucleotide, which comprises hybridizing with a polynucleotide, introducing a fluorescent or enzyme label into the hybrid using an immunological method, and detecting a photoresponse caused by photoexcitation or addition of a substrate. It is something.

Regarding the above-mentioned modified polydeoxynucleotide, the hapten may be any low molecular weight hapten as long as it has antigenicity, but due to the ease of obtaining antibodies against the hapten, dinitrobenzene derivatives such as 2,4-dinitrophenyl, Biotin derivatives such as biotinoyl and iminobiotinoyl, and aldosterone. 17-β-estradio-μ
, steroids such as testonuterone, and hydantoin derivatives such as diphenylhydantoin.
,4-dinitrophenyl is preferred.

Although the hapten may be directly bound to the 5'-terminal phosphate moiety of the polydeoxynucleotide, it is preferably bound to the 5'-terminal phosphate moiety of the polydeoxynucleotide via a ligand. The ligand has the formula -A-Z-
(wherein A is a bond or a formula -X-(CH2)n-(X
represents O, NH or a bond, n represents an integer from 1 to 8)
, 2 represents O or NH, the hapten is bonded to A, and the 5'-terminal phosphate portion of the nucleotide is bonded to 2]. Among them, as a ligand, the formula, N
It is preferable that il (CH2)nNH (n has the same meaning as above), and preferably that n is 2.

The above-mentioned polydeoxynucleotide (hereinafter sometimes abbreviated as polyDNA) is a polynucleotide that is complementary to a specific base sequence of a polynucleotide such as a gene or virus to be detected. It is JDNA. The polyDNA has 8 or more bases, preferably 12 to 1,000 bases.

Regarding the polyDNA mentioned above, Bo! JDNA with a short chain length can be easily obtained in large quantities by known chemical synthesis. Those with a short chain length are inferior in terms of the ability to form a high grid body, so those with a high COG content are desirable. In general, probes with a relatively short chain length are appropriate when specifically detecting a specific part of the genome, while probes with a long chain length are useful when identifying and detecting a wide region of the genome.
It tends to lack specificity.

The relatively long chain length indicates the cloned DN.
A is cut out as a double strand by a restriction enzyme, and the fragments with different chain lengths obtained here may be separated and used by gel/I/selectivity electrophoresis, but they can be haptenized as a mixture. can also be used to advantage.

When the denatured DNA of the present invention is used to detect the presence of hepatitis B virus, for example, po! As JDNA, for example, TCTTATGTAAG complementary to the base sequence existing between the surface antigen protein gene and the core protein gene.
ACCT or polyDNA with an average size of 200 base pairs obtained by digesting hepatitis B virus DNA with restriction enzyme 5au3A.

The denatured DNA of the present invention can be obtained by reacting complete Bo9DMA with a haptenizing reagent containing a siligant, as desired, or by leaving a part of the polyDNA containing a hapten. It can be produced by condensing with JDNA or DNA.

Specifically, it can be produced by condensing a haptenizing reagent such as 2,4-dinitrophenylethylenediamine or biotin with polyDNA.

When reacting the amino group of the halogenation reagent with the phosphoric acid residue of polyDNA, triphenylphosphine and 2
The reaction can be carried out in the presence of a dehydration catalyst such as , 2'-dipyryldiμ disulfide, and is usually carried out in a polar solvent such as N,N-dimethylformamide, dimethyl sulfoxide, water, or a mixture thereof. The reaction temperature is -10 to 10°C.

Power of Habtenizing Reagent: When reacting a phoxyl group, it is preferable to use it as an acid halide.

The denatured DNA of the present invention thus obtained is extracted,
It can be separated and purified by standard chemical operations such as column chromatography, recrystallization, and reprecipitation.

The modified DNA of the present invention has low toxicity and can be safely used, for example, in the following applications.

Detection of a specific base sequence in a polynucleotide using the denatured DNA of the present invention can be carried out, for example, by the following method.

The polynucleotide to be detected is immobilized on a support such as a sinitrose filter or the like by a conventional method, and if desired, denatured with dialkali, heat, etc. to obtain a single polynucleotide.

The nucleotide-immobilized filter and the haptenized DNA (I) MA probe) are hybridized in a buffer solution.

The above-treated filter was washed with a saline solution containing Tris-HCl, human apmin, etc., and rabbit anti-dinitrophenyl-dou serum albumin (rabbit anti-DNP-BSA) was added.
React with anti-hapteneggG or antiserum.

Anti-hapten IgG and the like are preferably used in the form of a diluted saline solution containing human apmin or goat serum.

After washing with saline, the reacted filter is treated with various peroxidases such as horseradish-vep oxidase-labeled goat anti-Tf IgG antibodies, luciferase-enzyme-labeled anti-IgG antibodies, ethenonucleotides, aminohexane adenosine nucleotides, etc. Fluorescently labeled anti-1
The presence or absence of a specific base sequence in the sibodinucleotide can be determined by photoexciting or adding a substrate to the reacted filter with the gG antibody and detecting the resulting photoresponse. Examples of the substrate include hydrogen peroxide, and examples of the coloring agent include ginseng orthodia.

By the method of detecting a specific base sequence in a polynucleotide using denatured DNA of the present invention, hepatitis B virus (H
It can detect genes such as BV), adult leukemia virus (ATLAV), and large leukocyte antigen (HI, A), and is useful for diagnosing hepatitis B, adult leukemia, etc., and determining HLA type.

Specifically, the animal targeted for diagnosis (mice).

Using serum from dogs, humans, etc., the method described in Example 6 of the present specification and known methods (for example, Proceedings of the National Academy of Sciences USA Vol. 79, 7) are used.
pp. 522-7526.

(1982).

The meanings of the symbols in this specification are as follows.

A: Deoxyadenylic acid residue C: Deoxyadenylic acid residue R
NST: Thymidic acid residue Ph: Feniμ acid residue Bz: Deoxyguaniμ acid residue Bz: Benzoyl The present invention will be specifically explained below with reference to Examples, but the present invention is not limited thereto.

In the examples, the protecting group of the protected nucleotide was dimethoxytrithiμ for the 5' position, and p-chlorophenyltoβ-cyanoe for the phosphoric acid, protected T460q and 1-methylimidazo which deprotected the 5' position. -/l'120
The solution was dissolved in 10 μl of pyridine and 400 μl of diphenylaminophosphoric acid chloride was added.

After 6 hours and 20 hours, 40oq of the above phosphorylating agent and 120μ of 1-methylimidazo-/l'! After 4 hours, 5 ml of IM potassium acetate was added and stirred for 1 minute. Chloroform 20*t was added to the reaction solution for extraction, and the chloroform layer was washed with 0.5M potassium dihydrogen phosphate and then with water, and concentrated to dryness. The residue was 0H0 using silikage/L/30g.
Purified with 13MeOH (97:3) to obtain the target product 370
I got q. R'f = 0.11 (Kieserge/' 60
F'-254, Merlisha, chloroform-methanol/L
/ 19:14/V) Example 2 Coagulation of 1 hour in the presence of 70q of mesitylene μhoniμnitrotriacey # (MSNT) in dry pyridine Iyxl. Add a small amount of water to the reaction solution and concentrate to dryness.

The residue was dissolved in Atenton, water was added until it was boiling, and the residue was adsorbed with 'J flam of Chiricrogrep RP-8 (Merurisha) 309, and acetone-triethyl acetate was added with 7mine (0,
01M) Wash with 3:2 V/V mixture Zoom/, and then concentrate the fraction eluted with the above 7:3 V/V mixture 100 to dryness. The residue was dissolved in a small amount of chloroform and dropped into cyclohexane, and the resulting precipitate was centrifuged to obtain 84 d of white powder.

(1) (Synthesis of phNHJo-TC:TTATGT: 8
Condensation was carried out with NT100q, and powdered target product 60IIv was obtained in the same manner as in method (1).

(In) (phN■)2 '1-o-TCTTATG
Synthesis of TAAGACCTOBz (1, 057 to subtotal 11
Te'p Mara: GACCTOBz 401M;l and MS
Condensate using NTloo, and obtain the target product 60 according to a conventional method.
#7 was isolated.

(1v) Removal of protecting group from fully protected pentadecamer: Dissolve 60q of fully protected pentadecamer in 1.5 ml of #acid-triethymuamine mixture (2:IV/V).

Add 0.15 ml of Isoamide Nitrite 7 and stir at 35°C for 7 hours. After concentrating the reaction solution to dryness, pyridine was added and concentrated again to completely remove isoamyl nitrite.

5 sl of concentrated ammonia water was added to the residue, and the mixture was left sealed at 25°C for 20 hours, and then heated at 60°C for 4 hours. Aqueous ammonia was distilled off, and the residue was dissolved in 0.01M triethylamine bicarbonate and washed twice with ether. Next, Sephadex G-5
The fraction eluted first at
3 AX -10, φ0, 4×30 cva, Na
Linear gradient elution with H2PO4 buffer (pH 6,3), from 0.15 M (containing 15% ethanol-μ) to 0.3 M
(containing 30% ethanol) in 10 minutes], the main fraction eluting most slowly was collected, and 5EP-PA
350. Desalted with K (Waters) and showing a single peak in reverse phase high performance liquid chromatography (Nucleosyl-01B). D.

I got it. The ice crystals are dephosphorylated at the 5' position with bacterial alkaline phosphatase, and then radiolabeled at the 5' position with ATP labeled with 32P at the 1st position and polynucleotide kinase, and then converted into the desired base thread sequence using the Maxam-Gilbert method. Confirmed that they match.

Example 3 850 μg of pentadecamer obtained in Example 20v) was added to 3 parts of water.
0μ! Dissolved in. A 300 μm solution of 2,4-dinitrophenylethylenediamine 13q in N,N-dimethylfopamide was added thereto, and triphenylphosphine 391EI and 2. 'J-dipyridyldisμphyto 33 da was added. Thereafter, at the same temperature, triphenylphosphine and 2,2'-dipyridiμdisp-phyto were added in the same amount as the first time every hour; and two more times, and 2 g of water was added to the reaction solution.
/ was added, and the mixture was washed twice with ethyl acetate/L/2 ml. The aqueous layer was concentrated to dryness, and a small amount of the residue was collected. OIM heavy acid was dissolved in triethyl amine, Sephadex G-50, and then high performance liquid chromatography (particyl/l/5AX-
10) as a 200 μg yellow powder,
2.4-dinitrophenymuNHCH2CH2NH-P
O2-TCTTATGTAAGACCT (DNA
probe) was obtained.

Kakata City Example 4 pBR32 containing all genes of hepatitis B virus Adw
Plasmid pBR322-EcoR engineering/I (B
Y933 (Refer to Japanese Unexamined Patent Application Publication No. 1988-194897) 1
When μ9 was cut with EcoRI, subjected to agarose electrophoresis, and fluorescently stained with ethidium bromide, two bands were detected. D) JA immobilized on 7μ loin was denatured by heating at 80°C for 3 hours, and the high molecular weight band (including the Wimel genome) was translocated onto nitrocellulose.
The sample was hybridized with 2 μl of the DNA probe obtained in Example 3 in 200 μl of buffer. Heptululose was soaked in saline (10 mM Lis-HCl buffer with 9% NaC1, pH 7.4) containing 3% human albumin, washed with saline, and treated with rabbit anti-DNP-BSA serum (10 times diluted). It was soaked for 2 hours in a diluted saline solution containing 3% human 1μ pummin and 10% goat serum. Goat anti-heron IgCtc washed 5 times with saline and labeled with horseradish peroxidase.
The body (diluted 200 times) was immersed in saline solution 1 containing 3% human albumin and 10% goat serum for 2 hours. 5 with salt water again
Wash twice, 0.0025% orthodianisidine, 0.01
% hydrogen peroxide (10mM) in hydrochloric acid buffer, p
When immersed in H14) for 30 minutes, washed with water, and dried, only the band containing the virus genome became reddish brown, and the band derived from pBR322 was not colored at all.

Example 5 Plasmid pBR32 obtained by integrating hepatitis B virus DNA into plasmid pBR322 and cloning
2-EcoRI/HBV933 (previously) 2.3# was digested with the restriction enzyme EcoR, separated and purified by agaronuge/L/electrophoresis, and hepatitis virus DNA (470μ
g) was obtained. Of this, 390 μf was converted into restriction enzyme 5au3A.
After decomposition, the mixture was purified to obtain a mixture (220 μg) containing DNA fragments with an average size of 200 base pairs. this DNA
Dissolve 50μ of this in a mixture of 2,4-dinitrophenylethylenediamine (13,5q) l:KN,N-dimethylhopamide (300pJ) and water (30μ!), and add triphenylphosphine (31g) under water cooling. and 2
, 2'-dipyrimidiμ disulfide (33ffN) was added. The latter two reagents were added twice more every hour, and 1 hour after the last addition, water (also t) and ethyl acetate/'(
4Fl+/) was added for extraction. The aqueous layer was reduced 7 times, and this solution was transferred to a Sephadecnu G-250 column (0.6X 2
5c+n). Kahum was dissolved in Tris-HCl buffer containing 0.15 molar salt (20 mmol/I/concentration,
pH 8,0), and the earliest eluting peak was collected. A DNA mixture (40 gg) modified with 2,4-dinitrophenyl was obtained by ethanol/L'S.

Plasmid PBR322 in Nitrose μLose Phytter
Diluted aqueous solutions of -EcoRI/HIIV933 at various concentrations were spotted, heated and dried for 1 to 3 hours, and then subjected to pretreatment for hybridization using a conventional method. The aforementioned dinitrophenylethylenediamine-haptenated LcDNA (1 μg) was used as a probe and hybridized with the DNA immobilized on the filter in a 400 μE solution at +oM for 16 hours.

Thereafter, the same enzyme immunoassay steps as in Example 4 were performed.

As a result, 1 nanogram of pBR322-
Clear color development was observed even in the EcoRI/HBV933 spot.

Example 6 Hepatitis B surface antigen carrier serum (300 μl) Gold 2
% Dode V/X/Sodium Sulfate, Salmon Semen DNA (4
0μ'i/It) and proteinase x (2i/It)
ml) containing 150mM meal in/10mM ED
'rA/lQmM) in lithic hydrochloric acid (250/jJ) and after incubation for 4 hours at 37°C, an equal volume of the above buffer is washed with saturated phenol and then with chloroform/isoamyl alcohol (24:1). DN using 1/10 volume of 3M sodium acetate and 2 volumes of ethanol-μ.
Precipitate A and dry. This was denatured with 0.3 M sodium hydroxide (10 μm) for 10 minutes at room temperature.
2M ammonium acetate A (10gg)"t"1MTIL
, spotted onto a nitrocellulose filter. After being subjected to conventional hybridization pretreatment,
When treated in the same manner as in Example 5 using haptenized DNA (1 μ9.400 μJ), the spots exhibit a characteristic reddish-brown color.

Claims (2)

[Claims] (1) A polydeoxynucleotide in which a hapten is bound directly to the phosphate moiety at the da terminal or via a Gantt. (2) A polydeoxynucleotide formed by binding a hapten directly to the 5'-terminal phosphate moiety or via a Gantt is hybridized with a polynucleotide in a cell or a sample immobilized on a support, and the hybrid is A method for detecting a specific base sequence in a polynucleotide, which comprises introducing a fluorescent or enzyme label using an immunological method and detecting a photoresponse caused by photoexcitation or addition of a substrate.