JPH07243971A - Measuring method for dna or rna by fluorescence polarization method - Google Patents

Abstract

PURPOSE:To provide a method for quickly measuring DNA or RNA by utilizing the fluorescence polarization method. CONSTITUTION:A fluorescence marker reagent is added to a sample and mixed, and the fluorescence polarization degree is measured via the signal generated by the fluorescence marker after the hybridization reaction between the DNA or RNA in the sample and the fluorescence marker reagent in this measuring method for DNA or RNA. Inorganic acid salt such as sodium chloride or organic acid salt such as sodium citrate is added before or after the fluorescence marker reagent is mixed during the hybridization reaction between the DNA or RNA in the sample and the fluorescence marker reagent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for rapid measurement in the measurement of DNA or RNA in a sample by using a fluorescence polarization method.

[0002]

2. Description of the Related Art In recent years, DNA and RN which are the main bodies of genes
The necessity of measuring A is increasing, and a measurement method using a radioactive label or an enzyme label has been studied and partially put into practical use, using a conventional immunoassay method as a model. In most cases, the conventional DNA and RNA measurement methods represented by these are constructed as heterogeneous measurement systems. That is, after mixing and reacting a sample containing a measurement substance (DNA or RNA) and a reagent used for measurement (measurement reagent), the unreacted measurement substance or measurement reagent is separated from those already reacted, and then labeled. This is a method of measuring the signal that is generated. The above separation operation is usually called B / F separation. Examples of such B / F separation methods include a method using a DNA reagent immobilized on a reaction container or a film, a method using magnetic fine particles, a method using electrophoresis, etc., but in any case, it is complicated. Alternatively, it requires a long time operation.

A fluorescence polarization method applicable to a homogeneous measurement system is known as a measurement method which does not require such B / F separation. This method has been conventionally known as a simple and rapid method for measuring a drug or the like in a sample.
Alternatively, it is considered to be applicable as a method for measuring RNA (JP-A-5-123196, etc.).

In order to measure DNA or RNA by the method, a fluorescent substance is labeled on DNA or RNA containing a base sequence complementary to the base sequence of the DNA or RNA to be detected, and this is used as a reagent. Here, the above-mentioned reagent is called a fluorescent labeling reagent (labeling probe). Usually, single-stranded DNA or RNA is used as the reagent.

An example of the process of measuring DNA or RNA by the fluorescence polarization method is shown below. First, a fluorescent labeling reagent is added to the sample to be measured. When DNA or RNA containing the target nucleotide sequence is present in the sample, the reagent has a site having the target nucleotide sequence at a certain reaction time,
Sequences complementary to each other associate and bind. This reaction is called hybridization. DNA in the sample here
Alternatively, it is assumed that RNA has been pretreated into a single-stranded state by treatment with temperature or chemicals. Upon hybridization, when the fluorescent-labeled reagent binds to the target DNA or RNA, the apparent molecular weight of the reagent becomes higher than that before binding. Generally, the larger the molecular weight, the slower the molecular motion in a solution. Therefore, when the fluorescence polarization degree before and after the reaction is monitored, the value after the binding by hybridization becomes larger than that before the binding. This is because hybridization with the target DNA or RNA increases the apparent molecular weight of the fluorescent labeling reagent. Given a fixed amount of fluorescent labeling reagent, the extent of this change corresponds to the amount of target DNA or RNA. Therefore, by changing the fluorescence polarization degree before and after the reaction, the target DNA or R
The amount of NA can be measured.

Generally, the degree of polarization of fluorescence is determined by setting a polarization element on both the excitation side and the fluorescence side and rotating the polarization element on the fluorescence side to measure fluorescence having polarization planes parallel and perpendicular to the polarization plane of the excitation light. It is possible to complete one measurement in a short time within 1 minute.

[0007]

Since the rate of hybridization of single-stranded DNA or RNA with them is generally slow, a long reaction time is required after adding the fluorescent labeling reagent to the sample. However, the fluorescence polarization method is characterized in that it can be measured in a short time as already described, so when a long reaction time is required, the time required for measurement is inevitably long, so this characteristic is effectively exhibited. There was a problem that was not done. That is, the fluorescence polarization degree can be measured in a short time, but there is a problem that the waiting time of the reaction before the measurement is long. The object of the present invention is to solve the problems of the above-mentioned conventional measuring methods, and
It is to provide a method for measuring NA or RNA in a short time.

[0008]

That is, according to the present invention, a fluorescent labeling reagent is added to a sample and mixed, and DNA or R in the sample is mixed.
In a method for measuring DNA or RNA by performing a hybridization reaction between NA and a fluorescent labeling reagent and then measuring the fluorescence polarization degree due to a signal generated due to the fluorescent labeling, A method for measuring DNA or RNA by a fluorescence polarization method, which comprises adding an inorganic acid salt or an organic acid salt before or after mixing of a fluorescent labeling reagent when performing a hybridization reaction with a fluorescent labeling reagent. .

The following specific examples are available as methods for measuring DNA or RNA. A. (1) Fluorescently labeled single-stranded DNA or RNA probe is mixed with (2) sample, and the change in fluorescence polarization degree before double-strand formation and fluorescence polarization degree after the double-strand formation is measured. The method for measuring the base sequence complementary to the above-mentioned single-stranded DNA or RNA probe, which is present in the DNA or RNA in the sample.

B. (1) Fluorescently labeled single-stranded DNA or RNA probe and (2) DNA or RNA in a sample, (3) immobilization of DNA or RNA containing a base sequence complementary to the single-stranded DNA or RNA Double-stranded DNA or DNA-RNA is formed by competing with the immobilizing reagent bound to the carrier, and the change in fluorescence polarization degree before the double-strand formation and fluorescence polarization degree after the double-strand formation is changed. A method for measuring a base sequence corresponding to the above-mentioned single-stranded DNA or RNA probe, which is present in DNA or RNA in a sample by measuring.

C. (1) DNA or RNA in the sample and (2) DNA or RNA having a base sequence homologous to the DNA or RNA in the sample are bound to the immobilization carrier, and the immobilization reagent is (3) DNA in the sample or Double-stranded DNA or RN by competing with a fluorescently labeled DNA or RNA probe having a base sequence complementary to RNA
A-DNA is formed, and fluorescence polarization degree before double-strand formation and 2
The change in the fluorescence polarization degree after formation of the main chain was measured, and D in the sample was measured.
A method of measuring a nucleotide sequence existing in NA or RNA and corresponding to the DNA or RNA probe in a complementary manner.

In the present invention, when the hybridization reaction between the DNA or RNA in the sample and the fluorescent labeling reagent is carried out, an inorganic acid salt or an organic acid salt is added before or after the mixing of the fluorescent labeling reagent to carry out the hybridization. , 0.01 to 5 mol / l or more, preferably 0.05 to
It is carried out in a solution containing an inorganic or organic acid salt at a concentration of 3 mol / l. If it is less than 0.01 mol / l, it takes about 30 minutes or more for the hybridization to reach saturation, which is not suitable for practical use. Inorganic or organic acid salt is 5
It is difficult to dissolve it in excess of mol / l.

As the inorganic acid salt in the present invention, hydrochloric acid,
Examples thereof include alkali metal salts, alkaline earth metal salts, and ammonium salts of carbonic acid or phosphoric acid. Examples thereof include chlorides such as sodium chloride, potassium chloride, magnesium chloride, and zinc chloride, or sodium carbonate, calcium carbonate, sodium phosphate, and the like. To be

As the organic acid salt in the present invention, acetic acid,
Examples thereof include an alkali metal salt of citric acid, benzoic acid or phenol, an alkaline earth metal salt or an ammonium salt.

The above salt has a salt concentration of 0.01 to 5 mol in the solution.
Add so that it becomes l / l. For that purpose, the salt may be added in advance to either or both of the measurement reagent and the measurement sample. Further, the measurement reagent or the measurement sample may be diluted with a salt-containing buffer solution or the like before use. Alternatively, a solution containing a salt or the like may be added after mixing the measurement reagent and the measurement sample. Generally, the dissociation constants of salts are very large, so when these salts are added to the solution, most of them are cations,
It exists in a state of being dissociated into anions.

Examples of the fluorescent label used in the present invention include fluorescein, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate. As a method for binding a fluorescent substance to DNA or RNA, there is, for example, a covalent bond such as a thiocarbamide bond. For example, DNA (24 bases) is synthesized by the phosphoramidite method and labeled with a fluorescent label such as fluorescein.

The fluorescent labeling reagent used in the present invention includes
If the number of bases is about 20 to 30 bases, a specific gene can be specifically detected (for example, Eur. J. Clin.
Microbiol. Infect. Dis., 10 (1991) 1048-1055).

DNA or RN in the sample in the present invention
Examples of A include bacteria or DNA or RNA such as virus in measurement samples such as serum, urine, and various culture solutions,
Alternatively, there are tissue cells and their free DNA or RNA.

As the buffer solution for carrying out the hybridization reaction between the DNA or RNA in the sample and the fluorescent labeling reagent, there are Tris buffer solution, phosphate buffer solution, citrate buffer solution and the like. The buffer solution may contain sodium azide, EDTA or the like in addition to the inorganic acid salt or the organic acid salt.

Examples of the carrier used in the present invention include beads of synthetic resin such as polystyrene and nylon, latex particles, glass beads and metal particles such as Au and Ag. A high molecular substance such as protein can also be used. The molecular weight of the solid carrier is based on the principle of the fluorescence polarization method, and the molecular weight of the complementary DNA or RNA is the fluorescent label D.
It is chosen to be sufficiently large relative to the molecular weight of NA or RNA. The molecular weight of the immobilized carrier is fluorescently labeled DNA.
Alternatively, it is preferably 5 times or more than the molecular weight of RNA.

As a method for binding DNA or RNA to the immobilized carrier, there are an adsorption method, a covalent binding method, a method utilizing a specific binding between avidin and biotin, and the like.

The principle of fluorescence polarization measurement will be briefly described below. The light emitted from the light source is filtered by the filter to the excitation wavelength of the fluorescent substance contained in the reagent and is polarized by the polarizing plate. The polarized light of this excitation wavelength is projected on the cell containing the measurement substance (sample) to excite the fluorescent substance in the sample. The excited fluorescent substance emits fluorescence having a wavelength corresponding to the substance, and at this time, the fluorescence causes polarization dispersion in response to the intensity of Brownian motion. The fluorescence passes through a filter that transmits the wavelength, transmits through a polarizing plate, and is converted into an electric signal by a photodetector. By rotating the polarizing plate, a polarized light component Ia in the same direction as the excitation polarized light and a polarized light component Ib perpendicular to this are obtained with respect to the fluorescence of the sample. Using these values, the fluorescence polarization degree P of the measurement substance shown below is determined.

[0023]

[Equation 1] Ia indicates polarized light in the same direction as the excited polarized light. Ib is the above I
The polarization component perpendicular to a is shown.

In this case, the more intense the Brownian motion of the fluorescent substance or the substance that binds the fluorescent substance, the larger the polarization component Ib in the direction perpendicular to the excitation polarization becomes, as compared with the parallel polarization component Ia, that is, P is Get smaller.

In the present invention, a solution containing fluorescently labeled (fluorescently labeled complementary) DNA or RNA is placed in the sample cell, a solution containing the DNA or RNA to be measured is added, and then immobilized complementary (immobilized) DNA or RNA fragment is added. Is added. However, the order of adding these three types of solutions is not limited. However, in carrying out the hybridization reaction between the DNA or RNA in the sample and the fluorescent labeling reagent, an inorganic acid salt or an organic acid salt is added before or after mixing the fluorescent labeling reagent. The concentrations of the fluorescence-labeled DNA or RNA and the immobilized complementary DNA or RNA to be added are appropriately selected according to the measurement concentration range of the measurement target DNA or RNA.

In the present invention, fluorescently labeled DNA or RNA
Binds to the immobilized enzyme DNA or RNA by a complementary binding reaction while competing with the DNA or RNA to be measured in the presence of an inorganic salt or an organic salt. When the fluorescent-labeled DNA or RNA binds to the immobilized complementary DNA or RNA, an apparently large molecular weight change occurs, and therefore the value of the fluorescence polarization degree P described above is obtained corresponding to the bound amount.
Immobilized DNA or R corresponding to the concentration of DNA to be measured
The amount of fluorescently labeled DNA or RNA that binds NA is determined. Therefore, if the polarization degree P is obtained, the concentration of the measurement target DNA or RNA is obtained.

[0027]

EXAMPLES The present invention will be described below with reference to examples. Reference Example 1 The following DNA (24 bases) was synthesized by the phosphoramidite method and labeled with fluorescein to give a measurement reagent. This is described in the above document (Eur. J. Clin. Microbiol. Infect.
Dis., 10 (1991) 1048-1055), the DNA constituting the gene for the heat-labile enterotoxin of toxogenic Escherichia coli.
Is a part of the base sequence of. In addition, a complementary DN
A (24 bases) was similarly synthesized. For control experiments, random sequence DNA (30 bases) was similarly synthesized. Random sequences were determined using a random number table. The nucleotide sequences of these DNAs are shown in order (for convenience, these are referred to as DNA1, DNA2, and DNA3 in this order). Note that
A, C, G and T represent adenine, cytosine, guanine and thymine, respectively. F indicates fluorescein used as a label. The numbers indicate the symbols of the DNA ends.

5'-F AAC AGG GAA TAC AGA GAC CGG TAT -3 '(DNA1) 3'-TTG TCC CTT ATG TCT CTG GCC ATA -5' (DNA2) 3'-GGA TTA CGT ATC TAC GCG ATG ACG GCC ACC-5 '(DNA3)

Example 1 DNA2 was added to a solution containing DNA1, and the fluorescence polarization degree was measured at 1 minute or 2 minute intervals immediately after the addition. It measured using the photomultiplier tube as a photodetector. The result is shown in FIG. Here, DNA2 is used as a model of the target DNA. The solution was maintained at 36 ° C. DNA1,
The concentrations of the DNA2 and DNA3 in the mixed solution are 9 × 10 ⁻⁹ , 7 × 10 ⁻⁸ , and 6 × 10 ⁻⁸ mol / l, respectively. Tris (pH 8.0) was used as the dilution buffer. From FIG. 1, it can be seen that since DNA1 and DNA2 are complementary to each other, they associate by hybridization and the degree of fluorescence polarization gradually increases. However, the reaction did not reach saturation even after about 4 hours. That is, under the condition that the solution does not contain an inorganic acid salt or an organic acid salt, the above D
The rate of hybridization of NA is very low.
Here, FIG. 1 shows the result of mixing DNA1 and DNA3 and measuring in the same manner. Since DNA1 and DNA3 are not complementary to each other, no association reaction occurs and the fluorescence polarization degree does not increase.

On the other hand, DNA1 and DNA2 or DNA3 were preliminarily supplemented with Trimol containing 0.8 mol / l sodium chloride.
It was dissolved in s buffer and the same experiment as in the case of FIG. 1 was performed.
The experimental conditions were all the same as in the case of FIG. 1 except that the solution contained the salt at the concentration. The result is shown in FIG. DN
It can be seen that the hybridization with A1 or DNA2 proceeds very quickly, reaching saturation in less than approximately 10 minutes. That is, it can be seen that the rate of hybridization greatly changes due to the presence of salt ions in the solution.

Similarly, an experimental example in the case where the solution contains 0.05 mol / l sodium chloride is shown in FIG. The experimental conditions are all the same as in the case of FIG. 2 except for the salt concentration in the solution. As in the case of FIG. 2, it can be seen that the hybridization progresses very quickly and reaches saturation as compared with the case where the solution does not contain sodium chloride (the case of FIG. 1).

The base sequence of the heat-labile enterotoxin gene used in the examples is only used as a model and does not limit the assay method of the present invention. In addition, although the measurement of DNA is shown as an example in the examples, it is basically the same even if each of the used DNAs is replaced with RNA.

[0033]

INDUSTRIAL APPLICABILITY In the present invention, 0.01 to 5 mol / in solution
By including l of the inorganic or organic acid salt, the hybridization proceeds sufficiently quickly to reach saturation. Under these conditions, hybridization reaches saturation in less than 10-20 minutes. Therefore, if the hybridized fluorescent labeling reagent is monitored by the fluorescence polarization method, it is possible to complete the measurement within 20 to 30 minutes including the reaction time of hybridization. According to the measuring method of the present invention, it is possible to measure DNA or RNA in a short time, and it is possible to shorten the work such as gene diagnosis, virus and bacteria detection, and reduce the cost.

[0034]

[Sequence listing] SEQ ID NO: 1 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other characteristics: Toxigenic E. coli Part of the nucleotide sequence of DNA that constitutes the heat-labile enterotoxin gene of AACAGGGAAT ACAGAGACCG GTAT 24

SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other characteristics: Toxinogenic E. coli Part of the nucleotide sequence of DNA constituting the heat-labile enterotoxin gene Sequence ATACCGGTCT CTGTATTCCC TGTT 24

SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCACCGGCAG TAGCGCATCT ATGCATTAGG 30

[Brief description of drawings]

FIG. 1 shows the results of hybridizing DNA1 and DNA2 or DNA3 under the condition that no inorganic salt is present.

FIG. 2 shows the results of hybridizing DNA1 and DNA2 or DNA3 in a Tris buffer containing sodium chloride.

FIG. 3 shows the results of hybridizing DNA1 and DNA2 or DNA3 in a Tris buffer containing sodium chloride.

Claims (7)

[Claims] 1. A fluorescent labeling reagent is added to and mixed with a sample, and a hybridization reaction between the DNA or RNA in the sample and the fluorescent labeling reagent is performed. Then, the fluorescence polarization degree due to a signal generated due to the fluorescent labeling is measured. D by measuring
In a method for measuring NA or RNA, D in a sample
When performing a hybridization reaction between NA or RNA and a fluorescent labeling reagent, an inorganic acid salt or an organic acid salt is added before or after mixing of the fluorescent labeling reagent. Measuring method. 2. The hybridization of DNA or RNA in a sample with a fluorescent labeling reagent is performed in an amount of 0.01 to 5 mol.
The method for measuring DNA or RNA by the fluorescence polarization method according to claim 1, which is carried out in a solution containing an inorganic acid salt or an organic acid salt at a concentration of / l. 3. The hybridization of DNA or RNA in a sample with a fluorescent labeling reagent is performed in an amount of 0.05 to 3 mol.
The method for measuring DNA or RNA by the fluorescence polarization method according to claim 1, which is carried out in a solution containing an inorganic acid salt or an organic acid salt at a concentration of / l. 4. The method for measuring DNA or RNA by the fluorescence polarization method according to claim 1, wherein the inorganic acid salt is an alkali metal salt, an alkaline earth metal or an ammonium salt of hydrochloric acid, carbonic acid or phosphoric acid. 5. The method for measuring DNA or RNA by the fluorescence polarization method according to claim 1, wherein the inorganic acid salt is sodium chloride. 6. The measurement of DNA or RNA by the fluorescence polarization method according to claim 1, wherein the organic acid salt is an alkali metal, alkaline earth metal or ammonium salt of acetic acid, citric acid, benzoic acid or phenol. Method. 7. A DN in which a fluorescent labeling reagent is bound with a fluorescent label.
It is A or RNA, The measuring method of DNA or RNA by the fluorescence polarization method of Claim 1 characterized by the above-mentioned.