JPH0552199B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial application field) Deoxyribonucleic acid (DNA) with a specific structure
The measurement of RNA and 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 hybrids are formed with DNA or S-RNA, unreacted labeled S-DNA or S-RNA is removed, and radiation on the 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. The present inventors conducted various studies to develop a method free of such problems, and found that a pre-labeled single-stranded polynucleotide was bound to a solid phase, and a sample was denatured on this solid phase. He devised a method of reacting single-stranded polynucleotides to form a hybrid and cleaving the hybridized polynucleotides with restriction enzymes, and has already filed a patent application for this method (Japanese Patent Application No. 199702-1981). However, this method still requires separation of the solid phase, and this separation operation is complicated, especially in clinical analyzes where a large amount of specimens are processed at once. (Means for Solving the Problems) The present invention solves these problems by applying an enzyme to the single-stranded polynucleotide hybridized with the single-stranded polynucleotide to be measured and the action of this enzyme. This method utilizes the fact that the continuous reaction between these two types of enzymes changes depending on the cleavage of the polynucleotide. That is, the present invention provides enzyme 1 and enzyme 2 whose substrate is a substance produced by the action of enzyme 1 on a single-stranded polynucleotide that can form a double-stranded polynucleotide with the single-stranded polynucleotide to be measured.
is bound directly or indirectly, or to a labeled polynucleotide to which an affinity substance to which Enzyme 1 and Enzyme 2 can bind, respectively; is contacted with a single-stranded polynucleotide to be measured; forming a double-stranded polynucleotide; cleaving the double-stranded polynucleotide with a double-stranded polynucleotide restriction enzyme; in the above case,
This is a method for measuring polynucleotides, which comprises binding enzyme 1 and enzyme 2 to the affinity substance at any of the steps described above; and then measuring the activity of enzyme 2. 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 treat the sample with protease or the like to separate the proteins. The single-stranded polynucleotide (hereinafter referred to as a labeled polynucleotide) that is brought into contact with this polynucleotide to be measured is directly or indirectly contacted with enzyme 1 and enzyme 2 whose substrate is a substance produced by the action of enzyme 1. It is bound to the single-stranded polynucleotide, or an affinity substance to which Enzyme 1 and Enzyme 2 can bind, respectively, is bound. Therefore,
This labeled polynucleotide is a probe for the polynucleotide to be measured. The single-stranded polynucleotide may be used by denaturing a double-stranded polynucleotide containing the polynucleotide to be measured by alkali treatment, heat treatment, or the like. In this case, it is necessary to remove the complementary single-stranded polynucleotide as much as possible, and therefore purification is preferably performed using electrophoresis, ion exchange chromatography, affinity chromatography, or the like. In addition, recently measured polynucleotides,
Methods for directly synthesizing single-stranded polynucleotides have been developed, such as the nick translation method in which various nucleotides are linked one after another in the presence of DNase I and DNA polymerase I, and the cDNA production method using Escherichia coli. It is also possible to synthesize by Furthermore, only the detection probe may be annealed in a solution, treated with a double-stranded polynucleotide restriction enzyme, and then subjected to gel filtration to remove contaminating complementary polynucleotides. Enzyme 1 and enzyme 2 may be in a relationship such that enzyme 2 uses a substance produced by the action of enzyme 1 as a substrate.
Examples of such enzyme combinations are shown below.

[Table] Fumarase - Malate dehydrogenase These are just examples, and an appropriate combination can be selected and used from the various enzymes listed in the Enzyme Handbook or Biochemistry Handbook. There are various types of enzymes whose optimal pH for action is neutral, acidic or alkaline, and there are also those that are heat resistant, so choose the appropriate one depending on the measurement conditions. Usually two types of enzymes are used, but three or more types of enzymes that are involved in continuous reaction can also be used if necessary. Enzyme 1 and Enzyme 2 can be directly or indirectly bound to a single-stranded polynucleotide using an affinity substance that can easily bind Enzyme 1 and Enzyme 2 in an aqueous solution. In the case of direct binding, for example, it may be combined with antibodies against each of Enzyme 1 and Enzyme 2, or a hapten may be used instead of the antibody.
As an affinity substance that can bind indirectly, for example, biotin bound to a single-stranded polynucleotide,
This corresponds to biotin-avidin in which enzyme 1 and enzyme 2 are bound to avidin, respectively. Alternatively, in another embodiment, an affinity substance to which Enzyme 1 and Enzyme 2 can each bind, such as biotin, bound to a single-stranded polynucleotide is used as a labeled polynucleotide, and this is contacted with the polynucleotide to be measured. Enzyme 1 and Enzyme 2 are added to
The enzyme 1 is reacted with the avidin bound to each, and the enzyme 1
and Enzyme 2 may be linked to the polynucleotide. Enzyme 1 and Enzyme 2 may be directly or indirectly bound to a single-stranded polynucleotide serving as a labeled polynucleotide, for example, in accordance with the known biotin-avidin method. The appropriate gap for binding Enzyme 1 and Enzyme 2 to the polynucleotide is about 10 to 1000 Å, and it is preferable to adjust the amounts added or reaction conditions so that the enzymes 1 and 2 are bonded at such a gap. Such a labeled polynucleotide is brought into contact with a polynucleotide to be measured to form a double-stranded polynucleotide. The contact time is usually such that the polynucleotide to be measured sufficiently reacts with the labeled polynucleotide to form a hybrid, and for example, about 0.5 to 40 hours is appropriate. The temperature is preferably about 20 to 70°C, and the pH is about 5 to 9. After forming a double-stranded polynucleotide, it is treated with a double-stranded polynucleotide restriction enzyme (hereinafter simply referred to as a restriction enzyme). This restriction enzyme preferably acts specifically on double-stranded polynucleotides, and preferably recognizes polynucleotide chains that are not too long. Not only one restriction enzyme but also two or more restriction enzymes may be used in combination. The restriction enzyme is normally applied after the reaction between the polynucleotide to be measured and the labeled polynucleotide has finished, but in some cases it may be added to the reaction system at the same time as or before the polynucleotide to be measured. After allowing the restriction enzyme to act, the activity of enzyme 2 bound to the polynucleotide is measured. This reaction may be measured by a known activity measuring method, such as by colorimetric determination or fluorescence, luminescence, etc., using these conjugated enzymes and chemical substances. (Function) The labeled polynucleotide forms a hybrid with the polynucleotide to be measured. By forming a hybrid, restriction enzymes act to cleave this double-stranded polynucleotide. As a result, the distance between enzyme 1 and enzyme 2 bound to the labeled polynucleotide increases, making it difficult for continuous reactions to occur. Therefore, the reaction rate by enzyme 2 decreases. (Effects of the Invention) Unlike conventional methods, the method of the present invention does not require the complicated operation of immobilizing the polynucleotide to be measured on a solid phase, and compared to the method of the prior application, solid-liquid separation and accompanying It is further simplified in that no cleaning effect is required. (Example) Preparation of HBV-DNA probe 500ml of pooled serum from chronic hepatitis B patients was
Centrifuge at 9000 rpm for 15 minutes and store the resulting supernatant at 4°C.
Hepatitis B virus (HBV) particles were collected as a pellet by ultracentrifugation at 100,000 xg for 5 hours. This pellet was mixed with 0.01M NaCl, 1mM EDTA, 0.1% 2-mercaptoethanol and 0.1% BSA.
Dissolve in 10 ml of Tris-HCl buffer (PH7.5), save 5 ml of this virus solution, and save the remaining 5 ml.
Ultracentrifugation was performed again at 100,000 xg for 5 hours to obtain a pellet. 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,
0.08M containing 2.5μM32PdGTP, 2.5μM32PdCTP
50μ of MgCl ₂ 0.2M Tris buffer (PH7.5) was added and heated for 3 hours. This solution was layered in a centrifuge tube containing 30% sucrose solution, and SW65
A pellet was obtained by centrifugation at 50,000 rpm for 3 hours using a rotor (manufactured by Beckman). This pellet was treated with pronase, 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 gradient to collect 15S32PDNA fractions and pool them.
From this fraction, 15S32PDNA was precipitated using ethanol and dried to obtain the desired HBV-DNA. Preparation of single-stranded HBV-DNA Next, 5mM MgCl ₂ , 10mM 2-mercaptoethanol, 5μM dTTP, 5μM dGTP, 5μM
dCTP, 5μM dATP, 10μM aminohexyl
Contains dATP and 10μM aminohexyl dCTP
Also, 1μg of HBV-DNA, 100pg of DNase I and 100μg of 50mM Tris-HCl buffer (PH7.5) were added.
100 pg of DNA polymerase I was added and incubated at 15°C for 90 minutes. This solution was also extracted with phenol and purified using a Sephadex G-50 column to obtain modified HBV-DNA. This double-stranded HBV-
8 ml of formamide was added to 1 mg (1 ml) of DNA and 5 mg (2 ml) of single-stranded M13-phage DNA introduced with HBV-DNA, and the mixture was boiled for 5 minutes.
Next, 2 ml of buffer solution (0.07 mol/Tris-HCl 2 mol/NaCl, 15 mM EDTAPH 7.5) was added thereto, and the mixture was heated at 50°C for 4 hours and then 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. Then, 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 remove the ethanol precipitate with 50 ml of 0.1N
Dissolved in NaOH, 0.25mM EDTA, 0.001% phenol. Immediately apply this using Bio-Gel A50
The target single-stranded HBV-DNA was obtained by gel filtration. Preparation of biotinylated single-stranded HBV-DNA 100 μg of the single-stranded HBV-DNA prepared in step 1 was reacted with 1 μg of biotin succinimide ester in a carbonate buffer at pH 8.0 at 37° C. for 2 hours. This was gel-filtered with Sephadex G-25 to remove the void fraction.
It was left at 60°C overnight. Add d-DNA restriction enzyme solution (Hae, Hae, Hap, Bgl) to this solution.
, Ava 1U/ml each, 10mM Tris-HCl,
7mM _MgCl2 , 7mM2-mercaptoethanol,
1 ml of PH7.5) was added, and the mixture was reacted at 37°C for 1 hour.
This reaction product was gel-filtered with Sephadex G-100, and the void fractions were pooled and subjected to protein removal treatment. Preparation of SH-modified avidin A DMF solution containing 200 μg of SPDP in 1 ml of a solution of 2 mg of avidin dissolved in 0.1 M phosphate buffer pH 7.5.
100μ was added and allowed to react at room temperature for 30 minutes. This was placed in a Sephadex G-25 column equilibrated with 0.1M phosphate buffer containing 1mM EDTA, pH 7.0, and developed with the same solution for gel filtration. The void fractions were pooled, 200 μg of 2-mercaptoethylamine was added thereto, and the mixture was stirred at 37° C. for 3 hours. Subsequently, gel filtration was performed in the same manner using the above-mentioned Sephadex G-25 column to obtain 2 mg of SH-modified avidin. CHM hexokinase 0.1M phosphate buffer containing 1mg hexakinase
DMF solution containing 200μg of cyclohexyl-4-maleimidomethylcyclohexane-1-carboxylic acid succinimide ester (CHMS) in PH7.01ml
100μ was added and left at room temperature for 1 hour. This was gel-filtered using the same Sephadex G-25 column as in the previous section, and the void fractions were pooled. Preparation of avidin-conjugated hexokinase Add 1 mg of CHM-conjugated hexokinase prepared in step 1 to 1 mg of SH-linked avidin prepared in step 1, and incubate at 37°C for 90 minutes.
Allowed to react for minutes. Next, this was gel-filtered on an ACA34 column and a 1:1 mixture of avidin and hexokinase was added.
The bound product was fractionated. Preparation of avidin-conjugated glucose-6-phosphate dehydrogenase Solution 1 containing 1 mg of CHM-conjugated glucose-6-phosphate dehydrogenase prepared in the same manner as in
1 ml of a solution containing 500 μg of SH-modified avidin was added to each ml, and the mixture was reacted at 37° C. for 90 minutes. reactant
A 1:1 combination of avidin and glucose-6-phosphate dehydrogenase was fractionated by gel filtration using an ACA34 column. DNA measurement 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 0.5NHCl and add 200μg/ml.
200μ of proteinase K solution was added and reacted at 70°C for 1 hour. To this was added 200μ of the biotinylated single-stranded HBV-DNA prepared in Section 2, and the mixture was left at 37°C overnight. d-DNA restriction enzyme solution (Bgl,
Ava, Hae, Hae, Hap,
Hinc 1U/ml each, 10mM Tris-HCl, 7mM
1.0 ml of _MgCl2 , 70mM NaCl, 7mM 2-mercaptoethanol, PH7.5) was added and reacted at 37°C for 1 hour. After the reaction, add 100 μg of a solution containing 1.0 μg each of avidin-conjugated hexokinase and avidin-conjugated glucose-6-phosphate dehydrogenase,
Additionally, substrate solution (0.1M glycylglycine, 20mM
MgCl ₂ Glucose 50mg/ml, 0.5mM ATP,
Add 3 ml of 0.5mM NADP ⁺ , PH8.0), NADPH
The rate increase was assayed by measuring the absorbance at 340 nm. The figure shows the results obtained, with the vertical axis representing the absorbance and the horizontal axis representing the dilution of the serum.

[Brief explanation of the drawing]

The drawing shows the relationship between serum dilution and absorbance measured by the method of the present invention.

Claims (1)

[Claims] 1 Enzyme 1 and enzyme 2 whose substrate is a substance produced by the action of enzyme 1 are directly or indirectly attached to a single-stranded polynucleotide that can form a double-stranded polynucleotide with the single-stranded polynucleotide to be measured. A single-stranded polynucleotide to be measured is brought into contact with a labeled polynucleotide to which it is bound, or an affinity substance to which Enzyme 1 and Enzyme 2 can bind, respectively; to form; the double-stranded polynucleotide is cleaved by the action of a double-stranded polynucleotide restriction enzyme; in the above case, enzyme 1 and enzyme 2 are respectively bound to the affinity substance in any of the above steps; ; A method for measuring polynucleotides, which comprises subsequently measuring the activity of enzyme 2.