JPS60256059A - Assay of polynucleotide - Google Patents

Abstract

PURPOSE:To enable convenient and accurate assay of specified deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by using labelled polynucleotide. CONSTITUTION:Single-stranded polynucleotide to be in contact with a specimen polynucleotide has a marker-containing high-molecular compound bonded to the 5'- or 3'-terminal thereof and combines with a specimen single-stranded polynucleotide to form a double-stranded polynucleotide. The above single stranded polynucleotide can be synthesized by the solid phase polynucleotide synthesis method or gamma-DNA method according to genetic engineering technology using plasmid. The above-mentioned high-molecular compound may be any one which is water-soluble and does not enter into non-specific reaction with the specimen polynucleotide, pref. one with MW of 1000 or more. The labelled polynucleotide is brought into contact with the specimen polynucleotide to form a double- stranded polynucleotide. After the reaction, the marker adsorbed on a filter is assayed by conventional analytical methods corresponding to the kind thereof.

Description

[Detailed Description of the Invention] (Industrial Application Field) Deoxynucleotide nucleic acid (DNA) or DNA having a specific structure? Measuring nucleic acids (RNA) is important in the field of biochemistry; for example, by measuring DNA in human serum, it is possible to test for viral infections or discover genetic diseases. The present invention furthermore provides beat NA and R.
The present invention provides a method for easily and accurately measuring a specific NA.

(Prior art and problems to be solved by the invention) Conventionally, methods for measuring DNA and RNA include single-stranded DNA (S-DNA) or single-stranded RNA (S-DNA) obtained by denaturing a sample. S-RNA) is bound to a solid phase, and S-DN labeled with a radioisotope is attached to this solid phase.
A or S-RNA is reacted with 5-DNA or S-RNA on the solid phase to form a hybrid, then unreacted labeled S-DNA and S-RNA are removed, and radiation on the solid phase is measured. A method was being used to do so. This method requires numerous steps such as immobilization and washing during measurement, and in particular, it takes a long time to immobilize the sample, which poses problems in both operational labor and time. Single-stranded polynucleotide: 1 to 20 bases of single-stranded I-rinucleotide that hybridizes with single-stranded polynucleotide
A method is also known in which a label such as an enzyme, biotin, or hapten is bound to every 50 bases, and the measurement is performed using this label. However, these methods have the problem that when the amount of labeled substance is increased, the specificity for hybridized DNA is decreased, and on the other hand, when the amount of labeled substance is decreased, the measurement sensitivity is decreased.

In addition, a technique has recently been developed in which the DNA f rope is cut short and a label is attached to the 5' and 3' ends (Japanese Patent Application Laid-Open No. 1983-40099), but this method also requires that the DNA f-lobe is short. There were problems in that the specificity was low and the sensitivity was insufficient due to the small amount of labeled substance.

(Means for Solving the Problems) The present invention provides a method for measuring polynucleotides that solves these problems.
Even if a polymer compound is attached to the 5' or 3' end of a single-stranded polynucleotide that hybridizes with a polynucleotide, the specificity of hybrid formation is not impaired, and the polymer compound can still be attached to a large number of labels. It was discovered that this hybrid can be measured with high sensitivity by combining them.

That is, in the present invention, a single-stranded polynucleotide to be measured is attached to the 5' end or 3' end of a single-stranded polynucleotide,
The present invention relates to a method for measuring a polynucleotide, which comprises contacting the polynucleotide with a single-stranded polynucleotide which is bound to the 'end and can form a double-stranded polynucleotide with the single-stranded polynucleotide to be measured.

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; for example, it may be human serum or a urine tissue extract. 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 slotease or the like.

A single-stranded polynucleotide (hereinafter referred to as a labeled polynucleotide) that is brought into contact with this polynucleotide to be measured.
A polymer compound having a label is bound to the 5' or 3' end and can form a double-stranded polynucleotide with the single S 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. Alternatively, 5 DNA can be extracted using general biochemical techniques and denatured with an alkali to make it 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, introduce the desired DNA into mp7 phage and insert it into φ×
When introduced into 174 and cultured, single-stranded DNA is easily formed.
can be obtained. Since the single-stranded DNA obtained does not become double-stranded by itself, 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 anything as long as it is water-soluble, 6D, and does not non-specifically react with the polynucleotide to be measured. The molecular weight is preferably 1000 or more.

Examples of polymer compounds include gelatin, hemocyanin,
Ferritin, etc., borizeputide, soluble dextran, Cal? Examples include polysaccharides such as methylated dextran, aminated dextran, amylose, phosphoric acid, polyethylene glycol, polyvinyl alcohol, and polynucleotides. For example, an amine group, cardaxyl group, thiol group, hydroxyl group, reactive phosphor group, etc. may be introduced into these in advance in order to facilitate the introduction of a label.

- Of the heavy chain 4 nucleotide ends, generally 3
It is generally easier to attach a polymer compound at the 'end, and there is less influence on the polynucleotide.

As a method for linking to the 3' end, for example, a single-stranded polynucleotide is added to S-acetylhexolaminoadenosine diphosphate, and RNA T4 ligase is applied thereto to introduce S~ruacetyl at the 3' end. When a sodium hydroxide aqueous solution is applied to this, the acetyl group is removed and becomes an SH group.

If we use water-soluble gelatin with maleimide groups introduced into this, it will be a hit. Gelatin can be introduced at the 3' end of the renucleotide. By using RNA T4 ligase, amino groups or carboxyl groups can be easily introduced at the 3' end, and high molecular compounds can also be bound using these. Methods for bonding polymer compounds using these functional groups include, for example, °'
Method in Immunology and
Immunochemistry” (C,A.

Williams et al. + 1976 r
It is possible to use an appropriate method selected from among the methods described at the bottom, such as Academic Press N, Y, ) and "enzyme immunoassay" (Ishikawa et al., Igaku Shoin, 1978). For example, when bonding between amino groups, the soifocyanate method, glutaraldehyde method, difluorobenzene method, benzoquinone method, etc. can be used, and when bonding between amine groups and carboxyl groups, In addition to the method of converting a carboxyl group into a succinimigester, a carpinoimide method, a Woodward reagent method, etc. can be used. There is also a periodic acid oxidation method (Nakane method) in which amine groups and sugar chains are crosslinked. 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 both 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, the 3' end can be bound to the 3' end by solid-phase synthesis of a single-stranded polynucleotide using a nucleotide to which a polymer compound has been bound in advance by the method described above. There is also a method in which the polymer compound is finally separated from the solid phase portion.

On the other hand, when binding to the 5' end, for example, double-stranded DNA
DNA T4 in A state! Modified DJ'JA, which has been bound with amine groups, carboxyl groups, etc. in advance by applying J gauze, is bound to the 5/end, and then the single-strand DNA is
By separating it into the 5' end, an amine group, carboxyl group, etc. can be introduced into the 5' end. Therefore, a polymer compound may be bonded by the above-described method using this functional group.

It goes without saying that the polymer compound may be introduced at both the 3' end and the 5' end.

The type of label is not particularly limited, and includes enzymes, gross stick glue biotin, avidin, bald ten,
Fluorescent substances, chemiluminescent substances, radioactive isotopes, 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 kinase1. Examples include nuclease, nuclease, and nuclease. The enzyme is preferably thermostable in order to increase specificity.

Examples of gross stick groups include FADlNAD
Examples include H, NADPH2, aminopyrophosphate, pyridoxal phosphate, ADP, and ATP.

Biotin may be a derivative capable of binding avidin.

Azetsuno is capable of binding to biotin (l), and streptavidin or a derivative thereof may also be used.

Examples of halogens include dinitrophenol and dinitroaniline.

Fluorescent substances include fluorescein, rhodamine, danzyl chloride, fluoresphamine, coumarin, acrizone, benzoxanoazole, triarylmethane, and pyrenes.

Chemiluminescent substances include luminol, inluminol, acridinium, hydroxide, porphyrin, intren-3-yl hydroperoxide, 2.4.5-triphenylimidazole, luciferin-luciferase, and the like.

Radioactive isotopes include 32P13H135S, '4C11
25I etc. may be used.

Among these, enzymes, glostic groups, biotin and derivatives thereof, avinone, streptaviso/and derivatives thereof are particularly 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-strand polynucleotide.

It is desirable that the amount of the label bound to the polymer compound be large.

Such labeled I polynucleotide is brought into contact with the polynucleotide to be measured to form a double-stranded polynucleotide. First, what is the measurement target? A nitrocellulose filter is immersed in a solution containing polynucleotides to adsorb the target I polynucleotides, and the filter is thoroughly washed.

Next, this nitrocellulose filter is immersed in a solution containing a labeled polynucleotide to react.

The reaction conditions are preferably such that the target polynucleotide adsorbed on the nitrocellulose filter sufficiently reacts with the labeled I polynucleotide to form a hybrid. This condition depends on the concentration of the labeled polynucleotide, etc., but is usually 20 to 75°C, pH 5 to 9, and 05 to 48 hours.

After the reaction is completed, the filter is thoroughly washed and the label adsorbed on the filter is measured. 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 target I polynucleotide. Therefore, by measuring the amount of the labeled polynucleotide that has formed this hybrid, the amount of the target f1) nucleotide to be measured can be determined.

(Effects of the Invention) In the method of the present invention, the specificity of forming a hybrid with the polynucleotide to be measured is not impaired since no label is bound to any part other than the end of the JIJ nucleotide. 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 fo-b 500 m
Pooled serum from patients with chronic hepatitis B was collected at 900 Orpm.
Centrifuge for 15 minutes at
The HBV particles were collected as a pellet by ultracentrifugation at 5' for 5 hours. This R-let was mixed with 0.1M NaCl, 1mM FJ
DTA, 0.1 2-mercaptoethanol and O4
0.01 containing 1 t BSA. OIM) Lis-HCl buffer (pH
7,5) Dissolve 5 of this virus solution in
ml was saved, and the remaining 5 ml was ultracentrifuged again at 100,000X for 5 hours to obtain pellets.

This solution was mixed with 05% NP-40 (-0mM).
Lith-hydrochloric acid, 0.1 M NaClp87.5 solution 20
0 μt to activate DNA polymerase. Add 1 mM dATP z 1 mM a'r' to this solution.
rp z 2.5 μM dGTP.

0.08 MMgC, a20 with 25 μM dCTP
．． 50 μt of 2M) Squirrel buffer (pH 7.5) was added and heated for 3 hours.

This solution was layered on a centrifuge tube containing 30% N-Eucrose solution, and centrifuged at 50,000 rpm for 3 hours using a 5W650-tar (manufactured by Beckman) to obtain pellets. This pellet was treated with zolonase, and the resulting solution was extracted twice with phenol. The extract was centrifuged at 50,000 rpm for 3 hours in a 5-20% sucrose granoent, and the 15S DNA fraction was collected and pooled.

From this fraction, 158 DNA was precipitated using ethanol and dried to obtain the desired HBV-DNA.

(2) Nick Trans Lane Yong KJ: Le DNA
Preparation of 7' lobe 5mM MgC42, 10mM 2-mercaptoethanol, 5μM dTTP, 5μM dGTP, 5μM dC
Add 100 μt of 50rrLM Tris-HC4 buffer (PH 7,5) containing TP and 5 μHdATP (obtained in the previous section) (13V-DNA1 μ&, DNase,
f 100 pl/and DNA polymerase 1100p
F was added and incubated at 15°C for 90 minutes. This solution was extracted with phenol and 5ephadex G-
HBV-DNA was obtained by purification using 50 columns.

(3) Binding of polymer compound and label The double-stranded HBV-DNA thus obtained was
Separate into single stranded DNA with sodium hydroxide solution,
This solution was neutralized. Immediately add amine hexyl acetate/diphosphate to this, then RNA
Added U. After reacting for 1 hour, the reaction solution was filtered through gel to obtain aminated T (BV-single-stranded DNA).

Next, CHM-formed gelatin 1 to 1 with a molecular weight of so, o o o was added to this, and 1 ml of water-soluble rudenoidimide was added.
was added and maintained at H5.0 at 37°C for 1 hour. This reaction solution was filtered through a heptaphacryl S-300 column, and the C.
HBV-single stranded DNA bound to HMized gelatin was obtained.

To this CHM-ized gelatin-bound HBV-single-stranded DNA was added SH-conjugated oxidase 10In9 obtained by a conventional method, and the mixture was stirred overnight at pH 7, 0, and 4°C. The reaction product was filtered through Sepharose 4B to remove unreacted peroxidase, and the target labeled single-stranded DNA was obtained.

(4) Measurement of polynucleotide: 0.5NNa in 100μ of HB viral hepatitis patient serum
Q)l 10 Qμl was added and stirred at room temperature for 10 minutes.

Next, add 0.5 NHCl]OOμj and add 200μ of the solution to 200μ97me of grotein kinase.
was added and reacted at 70°C for 1 hour. Then, saturated phenol-chloroform aqueous solution (1: ]) 20
0 μE was added and 200 μl of the separated aqueous layer was placed in a 96-well microplate lined with nitrocellulose. The aqueous layer of each well was filtered with suction, incubated at 85° C. for 1 hour, and washed once with PBS.

To this was added 200 μβ of the solution containing 1 μy of the labeled single-stranded DNA obtained in the previous section, and the mixture was heated at 40° C. for 18 hours.

This filter was washed with 0.1M acetic acid-10μM EDTA.
After washing four times with -1M NaCL pH 7,0, the filter was treated with 0.5rrM4-methoxynaphthol and 2
100 μl of substrate solution PH7,o containing mM H2O2
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 is shown in Figure 1.

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 radioiso)-f labeling solid phase method, and the results are shown in the table below.

A] OOpg 96 p&B 25 41 C6871 D 365 41 1 FJnd nd F 5 1 0 4. 90 Example 2 1 ml of double-stranded HBV-DNA prepared in Example 1
(1ml) and single-stranded M13-derived HBV-DNA.
Phage DNA5 ■ (2ml) K formamide 8m
1 was added and the mixture was boiled for 5 minutes. This was then added with 2 ml of buffer (0.07 mol/l) Lis-HC.
12 mol/l NaCA, 15 mM EDTA
pH 7.5) and incubated at 50°C for 4 hours and then at 60°C.
It was heated at ℃ for 1 hour.

This reaction solution was filtered through Blo-Ge1 A30FFI to separate hybridized DNA and unreacted DNA.

? The first peak eluted near the id fraction was fractionated, and powder was added to it to dissolve NaCl to 0.1-M. Then, add 100% ethanol at twice the ratio (2 ml) to 1 ml of solution, and -70
It was left at ℃ for 2 hours. Then ], 7000X, 9 to 10
Centrifuge for 50 min and remove the ethanol precipitate in 50 ml of 0.1N
NaOH, 0.25mM EDTA, 0.001
% phenol red. Blot this immediately
- Passed off Gulu with Ge1A50, the main target HBV-D
To this single-stranded DNA, we added 6-S acetylmercaptohexylazo, nosyndiphosphate) (] OmM ) and R
Add IOU of NA ligase, 10 mM Tris-HC
l.

1mM EDTA, 0.1,M NaCl pH
7.3 and reacted at 37°C for 2 hours. This was subjected to gel filtration using Biodal A60, and a modified single-stranded DNA fraction was collected. After concentrating to 2rnl with PEG-15000, 0. I
The pH was maintained at 11.0 with N NaOH to completely remove the butetyl groups. 5PD-luminol-conjugated dextran 1
0■ was added at pH 7.0 and left overnight at 4°C.

Next, this was gel-filtered using Bioglu A60 to obtain the desired luminol-conjugated labeled J (BV-single-stranded DNA).

0.5NNn in 100μl of HB viral hepatitis patient serum
100 μl of OH was added and stirred at room temperature for 10 minutes.

Next, add 1001 tl of 0.5 N HCl and add 20 μg/#I6 of protein kinase to 20 μg/#I6 of the solution.
0μ1lFr was added and reacted at 70°C for 1 hour. Then, a saturated phenol-chloroform aqueous solution (1:1) 2
00 μl was added, and 200 μl 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.

To this was added 200 μl of the solution containing the labeled single-strand DNA 100n9 obtained in the previous section, and the mixture was heated at 65° C. for 18 hours. This filter was mixed with 0.1M acetic acid-10μM EDTA.
After washing 4 times with -I M NaCl pH 7.0, the filter was cut and washed with 2 ml of H2O in a luminobotometer.
M, POD 10 U, NazCO3o, I M
The mixture was transferred to an arc tube containing 200 μl of pH 9.5, and the luminescence intensity was measured.

The results obtained are shown in FIG.

[Brief explanation of drawings]

All of the drawings show the results of measuring serum from patients with HB viral hepatitis using the method of the present invention. Figure 1 shows the relationship between serum dilution rate and reflection intensity of colored spots in one example, FIG. 2 shows the relationship between serum dilution rate and chemiluminescence intensity in other examples. Patent applicant Fujirebio Co., Ltd. Representative Patent Attorney 1) Masahiro Naka

Claims (1)

[Claims] A single-stranded polynucleotide to be measured is double-stranded with a single-stranded polynucleotide to be measured, and a polymer compound having a label is bound to the 5' end or 3' end, and the single-stranded polynucleotide to be measured is double-stranded, j? I.J.
A method for measuring polynucleotide r characterized by contacting with a single-stranded polynucleotide capable of forming nucleotides