JPH0515439B2 - - Google Patents

Description

[Detailed description of the invention] [Object of the invention] (Industrial application field) Deoxyribonucleic acid (DNA) having 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.

(Prior art and problems to be solved by the invention) Conventionally, as a method for measuring this DNA or 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.

On the other hand, recently DNA probes have been cut short and fluorescent substances that transfer energy are bonded to the 5' and 3' ends of the DNA probes, energy transfer is caused by hybridization, and the resulting fluorescence is measured and polynucleotides are analyzed. A technique for measuring the amount of water was developed (Japanese Patent Application Laid-Open No. 1983-40099). However, this method had problems such as a decrease in specificity because the DNA probe was cut short, and insufficient sensitivity due to the low amount of fluorescence.

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.

[Structure of the invention]

The present invention solves these problems by binding two types of luminescent substances to a single-stranded polynucleotide that hybridizes with the single-stranded polynucleotide to be measured, and the energy transfer between them is caused by the energy transfer of this polynucleotide. It takes advantage of the fact that it changes depending on the cutting.

(Means for Solving the Problems) The present invention is characterized in that the luminescent substance 1 binds directly or indirectly to a single-stranded polynucleotide that can form a double-stranded polynucleotide with a single-stranded polynucleotide to be measured. , and a labeled polynucleotide bound to a luminescent substance 2 that emits light when excited by the luminescent substance 1; contacts a single-stranded polynucleotide to be measured to form a double-stranded polynucleotide;
A double-stranded polynucleotide restriction enzyme is allowed to act on the double-stranded polynucleotide to cleave the double-stranded polynucleotide, and then luminescent substance 1 or luminescent substance 2
The present invention relates to a polynucleotide measuring method characterized by measuring the luminescence of a polynucleotide.

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.

A single-stranded polynucleotide (hereinafter referred to as a labeled polynucleotide) to be brought into contact with this polynucleotide to be measured is a luminescent substance to which luminescent substance 1 is directly or indirectly bound and which emits light when excited by luminescent substance 1. 2 can bind to each other and form a double-stranded polynucleotide with the polynucleotide to be measured. Therefore, this labeled polynucleotide is a probe for the polynucleotide to be measured. This labeled polynucleotide may be obtained by denaturing a double-stranded polynucleotide containing the polynucleotide to be measured by alkali treatment, heat treatment, etc., or the polynucleotide to be measured,
It may also be obtained by sequentially linking various nucleotides in the presence of DNase and DNA polymerase.

The luminescent substance 1 may be a fluorescent substance or a phosphorescent substance that emits light by absorbing external light, or may be a chemiluminescent substance or a bioluminescent substance that emits light by a chemical reaction such as an oxidation reaction. .

The luminescent substance 2 is excited by the luminescent substance 1 to emit light, and is therefore a fluorescent substance or a phosphorescent substance.

The fluorescent substances that can be used for the luminescent substance 1 and the luminescent substance 2 are those that normally emit fluorescence when excited at a wavelength of about 320 nm to 600 nm. Examples of such fluorophores include fluorescein, rhodamine, dansyl chloride, fluorescamine,
Examples include coumarin, acridine, NADPH, NADH, benzoxadiazole, triarylmethane, pyrenes, and derivatives or activated forms thereof.

Examples of chemiluminescent substances that can be used as the luminescent substance 1 include luminol, isoluminol, acridinium, hydroperoxide, porphyrin, intren-3-yl hydroperoxide,
Mention may be made of 2,4,5-triphenylimidazole and derivatives thereof. An example of a bioluminescent substance is luciferin, which emits light by luciferase.

When a chemiluminescent substance or a bioluminescent substance is used as the luminescent substance 1, a substance that causes these luminescent substances to emit light can be bound to the labeling substance directly or indirectly. For example, when luciferin is used as the luminescent substance 1, luciferase or its substrate can be bound to it, and the luciferin can be made to emit light by the action of these.

In the method of the present invention, since the luminescent substance 2 is excited by the luminescent substance 1, the combination of the two must be selected such that such a relationship is established. An example of such a combination is fluorescein and rhodamine. Fluorescein is excited by light with a wavelength of 430 to 530 nm and emits fluorescence with a wavelength of 500 to 560 nm. On the other hand, rhodamine is excited by light with a wavelength of 530 to 580 nm and emits fluorescence with a wavelength of 560 to 620 nm. Therefore, when a mixture of the two is irradiated with light of 430 to 530 nm, in addition to the fluorescent light of 500 to 560 nm, it emits light of 560 to 560 nm.
Fluorescence at 620 nm also appears.

The method of binding the luminescent substance 1 to the labeled polynucleotide directly or indirectly via biotin-avizen, and the method of binding the luminescent substance 2 to the labeled polynucleotide may be according to known methods for preparing labeled DNA, such as Alternatively, a modified nucleotide may be obtained by introducing a luminescent substance into a mononucleotide, and an unmodified mononucleotide may be mixed with the mononucleotide to form a polynucleotide by a nick translation method or a chemical polynucleotide synthesis method. In addition, a polynucleotide may be prepared by a nick translation method, a cDNA preparation method using Escherichia coli, or the like, and a luminescent substance may be activated and its active group may be reacted with the polynucleotide. Methods for binding luminescent substances include direct binding to polynucleotides, as well as binding of biotin-avidin to polynucleotides, avidin binding to the luminescent substance, and binding using this biotin-avidin reaction. You can also take indirect methods such as:

The method of the present invention is characterized in that energy transfer occurs from luminescent substance 1 to luminescent substance 2, and in order to cause this energy transfer to occur efficiently, luminescent substance 1 and luminescent substance 2 in the polynucleotide are combined. interval to 50
It is preferable that the two be bonded so that the length is less than .ANG., ie, within 24 to 26 bases.

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 can sufficiently react 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 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 luminescence of Luminescent Substance 1 or Luminescent Substance 2 is measured. The intensity may be measured using an ordinary fluorophotometer.

(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, luminescent substance 1 and luminescent substance 2 bound to the labeled polynucleotide are separated and move around in the solution separately, so that no energy transfer occurs. Therefore, the luminescent intensity of the luminescent material 1 no longer decreases, and on the other hand, the luminescent material 2 no longer emits light.

〔Effect of the invention〕

The method of the present invention utilizes energy transfer from luminescent substance 1 to luminescent substance 2, and since the emission wavelengths of luminescent substance 1 and luminescent substance 2 are different, the luminescence of both can be separately quantified. As a result, unlike the method of the prior application, separation of the solid phase and liquid phase and the accompanying washing operation are not necessary.

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 is even simpler than the method of the previous application in that it does not require a solid-liquid separation operation. ing. The reagents and instruments used in the method of the present invention can be easily made into a kit, and by using this kit, polynucleotides such as DNA can be measured practically and easily.

[Example] (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.
Hepatitis B virus (HBV) was collected as a pellet by ultracentrifugation at 100,000 xg for 5 hours. This pellet was dissolved in 10 ml of 0.01 M Tris-HCl buffer (PH7.5) containing 0.1 M NaCl, 1 mM EDTA, 0.1% 2-mercaptoethanol, and 0.1% BSA, 5 ml of this virus solution was saved, and the remaining 5 ml was 100000×
A pellet was obtained by ultracentrifugation again at g for 5 hours.

This pellet was treated with 200μ of 10mM Tris-HCl, 0.1M NaClPH7.5 solution containing 0.5% NP-40,
Activated DNA polymerase. in this solution
1mM dATP, 1mM dTTP, 2.5μM ^32PdGTP ,
50μ of 0.08M MgCl ₂ 0.2M Tris buffer (PH7.5) containing 2.5μM ³² PdCTP 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 is treated with pronase,
The resulting solution was extracted twice with phenol.
Prepare the extract using a 5-20% sucrose gradient.
The mixture was centrifuged at 50,000 rpm for 3 hours to collect 15S ³² PDNA fractions and pool them. 15S ³² from this fraction
PDNA was precipitated using ethanol and dried to obtain the desired HBV-DNA.

(2) Modified type by Nik translation method
Preparation of HBV-DNA Next, _5mMgCl2 , 10mM2-mercaptoethanol, 5μMdTTP, 5μMdGTP, 5μMdCTP,
5μM dATP, 10μM aminohexyl dATP and
HBV obtained in (1) was added to 100μ of 50mM Tris-HCl buffer (PH7.5) containing 10μM aminohexyl dCTP.
1 μg of DNA, 100 pg of DNase, and 100 pg of DNA polymerase were added and incubated at 15° C. for 90 minutes. This solution was extracted with phenol,
Purify with Sephadex G-50 column to obtain modified HBV-
I got DNA.

This double-stranded HBV-DNA 1mg (1ml) and HBV-
Single-stranded M13-phage DNA introduced with DNA 5mg
(2 ml) was added with 8 ml of formamide and the mixture was boiled for 5 minutes. Next, add 2 ml of buffer (0.07 mol/l, Tris-HCl 2 mol/l)
Add NaCl, 1.5mM EDTAPH7.5) and incubate at 50℃ for 4 hours.
The mixture was further heated at 60° C. for 1 hour.

This reaction solution was gel-filtered with Bio-GelA 50m to separate hybridized DNA and unreacted DNA. 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 remove 50 ml of ethanol precipitate.
Dissolved with 0.1N NaOH, 0.25mM EDTA, and 0.001% phenol. Bio-
Gel-filter with GelA50 and target single-stranded HBV-
I got DNA.

(3) Introducing the luminescent substance into 100μ of a DMF solution containing 1.0μg each of rhodamine isothiocyanate and fluorescein isothiocyanate.
2ml of 0.1M carbonate buffer PH10.0 containing 100μg of DNA
were mixed and reacted at 4°C for 18 hours. This was gel-filtered with Sephadex G-50, and single-stranded HBV-labeled HBV containing fluorescein and rhodamine was added.
100 μg of DNA was obtained.

(4) Measurement of human serum HBV-DNA: 100μ of serum from patients with HB viral hepatitis
100μ of 0.5NNaOH was added and stirred at room temperature for 10 minutes. Next, add 100μ of 0.5NHcl and further 200μg/
Add 200μ of proteinase K solution and heat at 70°C.
The reaction was carried out for 1 hour. 100 µ of a solution containing 10 ng of single-stranded labeled HBV-DNA bound to the luminescent substances fluorescein and rhodamine prepared in section (3) was added, and the mixture was left at 37°C overnight. Add d-DNA restriction enzyme solution (Bgl, Ava, Hae, Hae, Hap) to this.
, Hinc 1U/ml each, 10mM Tris-Hcl,
1.0 ml of _7mMgcl2 , 70mMNaCl, 7mM2-mercaptoethanol, PH7.5) was added and reacted at 37°C for 1 hour. Irradiate the reactant with 470 nm light,
The fluorescence intensity at 600 nm was measured.

The figure shows the results obtained, with the vertical axis representing the relative fluorescence intensity and the horizontal axis representing the dilution of the serum.

Next, HBV-
Indicates the amount of DNA. Present invention method Conventional method A 100pg 110pg B ND ND C 210〃 200〃 D 890〃 860〃 E 60〃 70〃

[Brief explanation of the drawing]

The drawing shows an example of the relationship between the relative fluorescence intensity of the luminescent substance 2 and the serum dilution rate measured by the method of the present invention.

Claims (1)

[Claims] 1. Luminescent substance 1 is directly or indirectly bound to a single-stranded polynucleotide that can form a double-stranded polynucleotide with the single-stranded polynucleotide to be measured, and A labeled polynucleotide bound to a luminescent substance 2 that is excited and emits light; contacts a single-stranded polynucleotide to be measured to form a double-stranded polynucleotide; A method for measuring a polynucleotide, which comprises cleaving the double-stranded polynucleotide using a polynucleotide restriction enzyme; and then measuring the luminescence of luminescent substance 1 or luminescent substance 2. 2. The measuring method according to claim 1, wherein the luminescent substance 2 is a fluorescent substance.