JPH0551280B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial application field) Deoxyribonucleic acid (DNA) with a specific structure
Alternatively, the measurement of ribonucleic acid (RNA) is important in the field of biochemistry; for example, by measuring DNA in human serum, it is possible to test for viral infection or discover genetic diseases.
The present invention provides a method for easily and accurately measuring such specific DNA and RNA. (Problems to be Solved by the Prior Art and the Invention) Conventionally, as a method for measuring DNA and RNA, single-stranded DNA (S-
DNA) or single-stranded RNA (S-RNA) is bound to a solid phase, and S-DNA or S-RNA labeled with a radioisotope is applied to the solid phase to
- A method has been used in which a hybrid is formed with DNA or S-RNA, unreacted labeled S-DNA or S-RNA is removed, and radiation on a solid phase is measured. This method requires many steps such as fixation and washing during measurement, and in particular, it takes a long time to fix the sample, which poses problems in terms of both operational labor and time. In addition, 20% of the single-stranded polynucleotide that hybridizes with the single-stranded polynucleotide to be measured is
A method is also known in which a label such as an enzyme, biotin, or hapten is bound to every 150 bases, and this label is used for measurement. However, these methods have a problem in that increasing the amount of labeled material reduces specificity for hybrid DNA, while decreasing the amount of labeled material reduces measurement sensitivity. In addition, a technique has recently been developed in which a DNA probe is cut short and a label is attached to its 5' and 3' ends (Japanese Patent Application Laid-open No. 1983-40099), but this method also requires that the DNA probe is short. Therefore, specificity is low;
In addition, there was a problem that the sensitivity was not sufficient because the amount of labeled substance was small. (Means for Solving the Problems) The present invention provides a method for measuring polynucleotides that solves these problems. 5′ end of nucleotide or
By selectively binding a specific polymer compound to the 3′ end, the specificity of forming a hybrid is not impaired, and
The present invention was also made based on the discovery that this hybrid can be measured with high sensitivity by binding a large number of labels to this polymer compound. That is, the present invention provides a single-stranded polynucleotide capable of forming a double-stranded polynucleotide with a single-stranded polynucleotide to be measured, which is composed of a polypeptide, a polysaccharide, a polyphosphoric acid, a polyethylene glycol, and a polyvinyl alcohol. A polynucleotide selected from the group consisting of: a polymer compound having a label selectively bound to its 5' end or 3' end is brought into contact with a single-stranded polynucleotide to be measured. This invention relates to a method for measuring polynucleotides. The measurement target is a single-stranded polynucleotide (hereinafter referred to as the measurement target polynucleotide). Polynucleotides to be measured include DNA and RNA. If the polynucleotide contained in the sample is double-stranded, it is necessary to make it single-stranded by an alkali treatment such as addition of a sodium hydroxide solution or heat treatment. The type of sample does not matter, but examples include human serum, urine, and tissue extracts.
If there is a possibility that polynucleotides may be bound to proteins, such as in human serum, it is preferable to separate the proteins by treating the sample with protease or the like. The single-stranded polynucleotide (hereinafter referred to as labeled polynucleotide) that is brought into contact with this polynucleotide to be measured is a polymeric compound having a labeled substance.
It is bound to the 5' end or 3' end and can form a double-stranded polynucleotide with the single-stranded polynucleotide to be measured. This single-stranded polynucleotide can be synthesized by the currently known solid-phase polynucleotide synthesis method or the γ-DNA method using genetic engineering using a plasmid. It is also possible to extract DNA using general biochemical methods and denature it with alkali to make it into a single strand. In addition, those already commercially available can also be used. It is preferable to use genetic engineering techniques because single-stranded polynucleotides can be easily obtained in large quantities. For example, single-stranded DNA can be easily obtained by introducing the desired DNA into mp7 phage and culturing it into φ×174 cells. The single-stranded DNA obtained does not become double-stranded by itself, so it can be stored at normal neutral temperatures and at room temperature. Therefore, there is no need for a process to convert the polynucleotide into a single strand after synthesis, and extra effort can be omitted. The polymer compound may be one that is water-soluble and does not non-specifically react with the polynucleotide to be measured. The molecular weight is preferably 1000 or more. Examples of polymeric compounds include polypeptides such as gelatin, hemocyanin, and ferritin, soluble dextran, carboxymethylated dextran, aminated dextran, polysaccharides such as amylose, polyphosphoric acid, polyethylene glycol, and polyvinyl alcohol. . For example, an amino group, a carboxyl group, a thiol group, a hydroxyl group, a reactive chlorine group, etc. may be introduced into these in advance in order to facilitate the introduction of a label. Of the ends of a single-stranded polynucleotide, it is generally easier to bind a polymer compound to the 3' end, and this polynucleotide is less affected. For example, a single-stranded polynucleotide is added to S-acetylhexylaminoadenosine diphosphate, and RNA is attached to the 3' end.
When T4 ligase is applied, an S-acetyl group is introduced at the 3' end. When this is treated with an aqueous sodium hydroxide solution, the acetyl group is removed and becomes an SH group. If water-soluble gelatin into which a maleimide group has been introduced is applied to this, single-stranded polynucleotides can be formed.
Gelatin can be introduced at the 3′ end. RNA
By using T4 ligase, an amino group or a carboxyl group can be easily introduced at the 3' end, so it is also possible to use these to bond a polymer compound. Methods for bonding macromolecular compounds using these functional groups are described, for example, in “Method in Immunology and
“Immunochemistry” (CA Williams et al,
The methods described in books such as "Enzyme Immunoassay" (Ishikawa et al., Igaku Shoin, 1978) can be selected and used as appropriate. For example, when bonding between amino groups, the diisocyanate method, glutaraldehyde method, difluorobenzene method, benzoquinone method, etc. can be used, and when bonding between amino groups and carboxyl groups, In addition to the method of converting a carboxyl group into a succinimide ester, a carbodiimide method, a Woodward reagent method, etc. can be used. There is also a periodate oxidation method (Nakane method) that crosslinks amino groups and sugar chains. When using a thiol group, for example, the carboxyl group on the other side is esterified with succinimide, this is reacted with cysteine to introduce a thiol group, and the two are bonded using a thiol group-reactive divalent cross-linking reagent. can do. Methods that utilize phenyl groups include diazotization and alkylation. In addition to the method described above, methods for binding to the 3' end include, for example, solid-phase synthesis of a single-stranded polynucleotide using a nucleotide to which a polymer compound has been previously bound to the 3' end by the method described above. Finally, there is also a method of separating the polymer compound from the solid phase part. On the other hand, when binding to the 5' end, for example, in a double-stranded DNA state, modified DNA to which amino groups, carboxyl groups, etc. have been bound in advance by the action of DNA T4 ligase is linked to the 5' end, and then If the DNA is separated into single-stranded DNA, amino groups, carboxyl groups, etc. can be introduced at the 5' end. Therefore, a polymer compound may be bonded using this functional group by the method described above. It goes without saying that the polymer compound may be introduced at both the 3' end and the 5' end. The type of sign is not particularly limited,
Enzymes, prosthetic groups, biotin, avidin, haptens, fluorophores, chemiluminescent substances, radioisotopes, etc. can be widely used. It is desirable that these substances can be easily measured and easily bonded to a polymer compound. Examples of enzymes include glucose-6-phosphate dehydrogenase, hexokinase, α-amylase, malate dehydrogenase, alkaline phosphatase, peroxidase, β-galactosidase, creatine kinase, ribonuclease, penicillinase, and the like. The enzyme is preferably thermostable in order to increase specificity. Examples of prosthetic groups include FAD,
Examples include NADH, NADPH ₂ , aminopyrophosphate, pyridoxal phosphate, ADP, and ATP. Biotin may be a derivative capable of binding avidin. Avidin can bind to biotin,
It may be streptavidin or a derivative thereof. Haptens include, for example, dinitrophenol, dinitroaniline, and the like. Fluorescent substances include fluorescein, rhodamine, dansyl chloride, fluorescamine, coumarin, acridine, benzoxadiazole, triarylmethane, and pyrenes. Chemiluminescent substances include luminol, isoluminol, acridinium, hydroperoxide, porphyrin, intren-3-yl hydroperoxide, 2,4,5-triphenylimidazole,
Such as luciferin-luciferase. ³² P, ³ H, ³⁵ S, ¹⁴ C, ¹²⁵ I, etc. may be used as radioactive isotopes. Among these, enzymes, prosthetic groups, biotin and its derivatives, avidin,
Streptavidin and derivatives thereof are preferred in terms of ease of use, sensitivity, etc. The method for binding the label to the polymer compound may be appropriately selected from among the methods described above for binding the polymer compound to the single-stranded polynucleotide. It is desirable that the amount of the label bound to the polymer compound be large. Such a labeled polynucleotide is brought into contact with a polynucleotide to be measured to form a double-stranded polynucleotide. As for the contacting method, first, a nitrocellulose filter is immersed in a solution containing the polynucleotide to be measured to adsorb the polynucleotide to be measured, and then thoroughly washed. Next, this nitrocellulose filter is immersed in a solution containing the labeled polynucleotide and allowed to react. The reaction conditions are preferably such that the polynucleotide to be measured adsorbed on the nitrocellulose filter sufficiently reacts with the labeled polynucleotide to form a hybrid. These conditions depend on the concentration of the labeled polynucleotide, etc., but are usually 20-75°C.
It takes about 0.5 to 48 hours at a pH of about 5 to 9. After the reaction is complete, wash this filter thoroughly.
Measure the label adsorbed on the filter. The measurement method may be carried out according to each known method depending on the type of label. (Function) In the method of the present invention, the labeled polynucleotide specifically forms a hybrid with the polynucleotide to be measured. Then, by measuring the amount of the labeled polynucleotide that has formed this hybrid, the amount of the polynucleotide to be measured can be determined. (Effects of the Invention) In the method of the present invention, the specificity of forming a hybrid with a polynucleotide to be measured is not impaired since no label is bound to any part other than the end of the labeled polynucleotide. Furthermore, since the label is bound to the polymer compound, the amount of the label can be increased, and as a result, the measurement sensitivity can be increased. The method of the invention also has the advantage of being simple to operate. (Example) Example 1 (1) Preparation of HBV-DNA probe 500 ml of pooled serum from chronic hepatitis B patients was
Centrifuge at 9000 rpm for 15 minutes and store the resulting supernatant at 4°C.
The HBV particles were collected as a pellet by ultracentrifugation at 100,000 xg for 5 hours. This pellet was mixed with 0.1M NaCl,
Dissolve in 10 ml of 0.01 M Tris-HCl buffer (PH7.5) containing 1 mM EDTA, 0.1% 2-mercaptoethanol, and 0.1% BSA, save 5 ml of this virus solution, and incubate the remaining 5 ml again at 100,000 × g for 5 ml. A pellet was obtained by ultracentrifugation for an hour. This pellet was treated with 200 µ of a 10 mM Tris-HCl, 0.1M NaClPH7.5 solution containing 0.5% NP-40 to activate DNA polymerase. This solution contains 1mM dATP, 1mM dTTP, 2.5μM dGTP,
50μ of 0.08M MgCl ₂ 0.2M Tris buffer (PH7.5) containing 2.5μM dCTP was added and heated for 3 hours. This solution was layered on a centrifuge tube containing a 30% sucrose solution, and centrifuged for 3 hours at 50,000 rpm using an SW65 rotor (manufactured by Beckman) to obtain a pellet. This pellet was treated with pronase, and the resulting solution was extracted twice with phenol. Prepare the extract using a 5-20% sucrose gradient.
The 15S DNA fraction was collected by centrifugation at 50,000 rpm for 3 hours and pooled. From this fraction, 15S DNA is precipitated with ethanol and dried to obtain the desired
HBV-DNA was obtained. (2) Preparation of DNA probe by Nikk translation Add the sample obtained in the previous section to 100μ of 50mM Tris-HCl buffer (PH7.5) containing 5mM MgCl ₂ , 10mM 2-mercaptoethanol, 5μM dTTP, 5μM dGTP, 5μM dCTP and 5μM dATP. HBV-DNA1μ
g, 100 pg of DNase I and 100 pg of DNA polymerase I were added and incubated at 15°C for 90 minutes. This solution was extracted with phenol,
HBV-DNA was purified using a Sephadex G-50 column.
I got it. (3) Binding of a polymer compound and a label The double-stranded HBV-DNA thus obtained is
Separate into single strands with 0.5N sodium hydroxide solution
This solution was neutralized as DNA. To this was immediately added aminohexyladenosine diphosphate, followed by 10 U of RNA ligase. After 1 hour of reaction, the reaction solution was gel-filtered to obtain aminated HBV-single-stranded DNA. Next, 1 mg of CHM gelatin having a molecular weight of 50,000 was added thereto, and further 1 mg of water-soluble carbodiimide was added thereto, and the mixture was maintained at pH 5.0 at 37° C. for 1 hour. This reaction solution was gel-filtered through a Sephacryl S-300 column,
CHM-formed gelatin-bound HBV-single-stranded DNA was obtained. This CHM-formed gelatin-bound HBV-single-stranded DNA
10mg of SH peroxidase obtained by conventional method
was added, and the mixture was stirred overnight at pH 7.0 and 4°C. The reaction product was gel-filtered with Sepharose 4B to remove unreacted peroxidase, and the desired labeled single-stranded DNA was obtained. (4) Measurement of polynucleotide: 0.5N in 100μ of HB viral hepatitis patient serum
100μ of NaOH was added and stirred at room temperature for 10 minutes.
Next, add 100μ of 0.5N HCl and add 200μg/
Add 200 μl of protein kinase K solution
The reaction was carried out at 70°C for 1 hour. Then, 200μ of a saturated phenol-chloroform aqueous solution (1:1) was added, and 200μ of the separated aqueous layer was placed in a 96-well microplate lined with nitrocellulose at the bottom. The aqueous layer of each well was filtered by suction, incubated at 85° C. for 1 hour, and washed once with PBS. Add to this a solution containing 1 μg of labeled single-stranded DNA obtained in the previous section.
200μ was added and heated at 40°C for 18 hours. This filter was mixed with 0.1M acetic acid - 10μM EDTA - 1M NaCl.
After washing 4 times with PH7.0, add 0.5mM4− to this filter.
100μ of a substrate solution PH7.0 containing methoxynaphthol and 2mMH ₂ O ₂ was added and reacted for 30 minutes. The colored spots on each filter were measured using a refractometer, and the relationship between serum dilution rate and relative reaction intensity was determined. The results are shown in FIG. The amount of HBV-DNA in various serums was measured by the above method, and the results were compared with the values measured by the conventional radioisotope labeling solid phase method, and the results are shown in the table below.

[Table] Example 2 1 mg of double-stranded HBV-DNA prepared in Example 1
(1 ml) and 5 mg (2 ml) of single-stranded M13-phage DNA containing HBV-DNA were added with 8 ml of formamide, and the mixture was boiled for 5 minutes. Next, add 2 ml of buffer (0.07 mol/Tris-
2 mol of HCl/NaCl, 15mM EDTAPH7.5) was added, and the mixture was heated at 50°C for 4 hours and further heated at 60°C for 1 hour. This reaction solution was gel-filtered with Bio-Gel A50m,
Hybridized DNA and unreacted DNA were separated. The first peak eluted near the void fraction was fractionated, and powder was added to it to make NaCl 0.1M and dissolved. Further, 100% ethanol was added at twice the ratio (2 ml) per 1 ml of the solution, and the mixture was left at -70°C for 2 hours. Next, at 17000×g
Centrifuge for 10 minutes and precipitate with 50ml 0.1N ethanol.
Dissolved in NaOH, 0.25mM EDTA, 0.001% phenol. Immediately apply this to Bio-GelA50
The target single-stranded HBV-DNA was obtained by gel filtration. 6-S acetylmercaptohexyladenosine diphosphate (10mM) and 10U of RNA ligase were added to this single-stranded DNA, and 10mM Tris-
The mixture was reacted with HCl, 1mM EDTA, and 0.1M NaClPH7.3 at 37°C for 2 hours. This was gel-filtered with Biogel A60, and a modified single-stranded DNA fraction was collected. PEG−
After concentrating to 2 ml at 15,000, the pH was maintained at 1.0 with 0.1N NaOH to completely remove acetyl groups. Add 10 mg of SPD-modified luminol-conjugated dextran at pH 7.0,
It was left at 4°C overnight. Next, this was gel-filtered through Biogel A60 to obtain the desired luminol-conjugated labeled HBV-single-stranded DNA. 0.5N in 100μ of HB viral hepatitis patient serum
100μ of NaOH was added and stirred at room temperature for 10 minutes.
Next, add 100μ of 0.5N HCl and add 200μg/
Add 200 μl of protein kinase K solution
The reaction was carried out at 70°C for 1 hour. Then, 200μ of a saturated phenol-chloroform aqueous solution (1:1) was added, and 200μ of the separated aqueous layer was placed in a 96-well microplate lined with nitrocellulose at the bottom. The aqueous layer of each well was filtered by suction, incubated at 85° C. for 1 hour, and washed once with PBS. Add to this a solution containing 100 mg of labeled single-stranded DNA obtained in the previous section.
200μ was added and heated at 65°C for 18 hours. This filter was washed with 0.1M acetic acid - 10μM EDTA - 1M NaCl.
After washing 4 times at PH7.0, cut out this filter.
_{H2O2 2mM} , POD10U, in a luminophotometer
Transfer _{Na2CO30.1MPH9.5} to an arc tube containing _200μ ,
The luminescence intensity was measured. The results obtained are shown in FIG.

[Brief explanation of the drawing]

The drawings all show the results of measuring the serum of patients with HB viral hepatitis using the method of the present invention; Fig. 1 shows the relationship between the serum dilution rate and the reflection intensity of the colored spot in one example, and Fig. 2 shows the relationship between the serum dilution rate and the reflection intensity of the colored spot in one example. shows the relationship between serum dilution rate and chemiluminescence intensity in other Examples.

Claims (1)

[Claims] 1. A single-stranded polynucleotide capable of forming a double-stranded polynucleotide with the single-stranded polynucleotide to be measured, selected from the group consisting of polypeptide, polysaccharide, polyphosphoric acid, polyethylene glycol, and polyvinyl alcohol. and the polymeric compound having the label is located at its 5′ end or
A polynucleotide selectively attached to the 3′ end,
A method for measuring polynucleotides, which comprises bringing them into contact with a single-stranded polynucleotide to be measured.