JPH06261795A - Process for detecting polynucleotides - Google Patents

Abstract

PURPOSE:To provide a quantitative process for detecting polynucleotides in high sensitivity and high accuracy. CONSTITUTION:Five kinds of fluorescent probes 2 to 6 to which two molecules of fluorescent compounds are bonded each, are allowed to react with the target nucleotide chain 1 under the hybridization conditions, the reaction mixture is added to the inlet 21 for the electrophoretic carrier to perform the electrophoresis, thereby the unreacting labeled probes of smaller molecular sizes are removed. The complex of the fluorescent probe with the target polynucleotide chain 10 is a large size of molecule and remains near the inlet to be concentrated. The inlet part is heated to dissolve the fluorescent probe from the target polynucleotide and the probe crosses the a certain zone illuminated with a laser beam in the electrophoretic column to detect the fluorescence. The target polynucleotide of 100 copy order can be detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The object of the present invention is to detect the presence or absence of an exogenous polynucleotide such as a virus, rickettsia or bacterium which causes a disease, or a polynucleotide carrying specific genetic information in an organism and its mutation. To a diagnostic method to do.

[0002]

2. Description of the Related Art A conventional polynucleotide detection method in which a labeled polynucleotide in which a labeled substance is bound to a target polynucleotide (DNA or RNA) sample is associated is described in Proceedings of Natural Academy Science USA, Volume 80 (1983). ) Second
Pages 78 to 282 (Proc. Natl. Aca
d. Sci. USA (1983) 80, 278-2.
82). In this method, first, a sample of polynucleotide fragments whose molecular weights have been separated by electrophoresis is transferred and immobilized on a nitrocellulose membrane, and then this membrane is immersed in a solution containing a polynucleotide probe labeled with a radioisotope to carry out a hybridization reaction. To do. After the reaction, unreacted labeled probe is washed from the membrane by washing. Then, the presence or absence of the target polynucleotide is determined by detecting the amount of residual labeled probe bound to the membrane by autoradiography.

Further, a method for detecting an exogenous target polynucleotide such as a bacterium or a virus in a sample such as blood or excrement is disclosed in JP-A-58-31998. In this method, the polynucleotide in the purified sample is denatured by heating or the like to form a single strand, which is then immobilized on a nitrocellulose membrane. Next, the membrane is washed after reacting with a polynucleotide probe labeled with a radioisotope having a nucleotide sequence complementary to the bacterial or viral polynucleotide to be inspected. If the sample contains bacterial or viral polynucleotides, the labeled polynucleotide probe associates with it and remains on the membrane. The presence or absence of the target polynucleotide can be determined by detecting this by autoradiography.

A method of multiplying a specific portion of a target polynucleotide with a set of DNA primers and a DNA polymerase is described in JP-A-61-274697. In this method, the sense strand and antisense strand of the target double-stranded DNA are 200 to 1000 each other.
A set of DNA primers complementary to sites separated by about bases, 4 kinds of deoxynucleotide triphosphates, and thermostable DN
A target polynucleotide is added to a reaction solution containing A polymerase, and the temperature of the reaction solution is repeatedly raised and lowered to denature the polynucleotide, anneal the target polynucleotide with a DNA primer, and repeat the complementary strand synthesis reaction to repeat the reaction of the target polynucleotide. Proliferate a specific site. According to this method, the polynucleotide fragment at a specific site can be amplified about 100,000 times in about 4 hours. Amplification product detection is
According to the above-mentioned first or second method, or when the amount of the product is sufficiently large, ethidium bromide staining is performed after electrophoresis, and the fluorescence emitted by this dye is detected.

[0005]

In the above-mentioned first and second conventional techniques, the hybridization reaction between the target polynucleotide and the labeled probe was carried out on the membrane carrier. Also, the labeling of the labeled probe required a radioisotope. For this reason, the reaction time is long because the target polynucleotide is immobilized on the membrane carrier and the hybridization reaction is performed on the surface of the solid body. In addition, autoradiography requires a long time of several days for analysis. In addition, this method may require a long time for detection and may lack sensitivity. This is because only a tracer amount of radioisotope can be labeled, so the specific radioactivity per probe cannot be high, and the background radiation that depends on nonspecific adsorption of the probe to the nitrocellulose membrane is Responsible. In addition, it is difficult to quantitatively measure by the conventional method, and most of the time, only semi-quantitative measurement is performed based on the determination of the presence or absence of the target polynucleotide or the degree of blackening of the film in the autoradiogram. Furthermore, since autoradiography is used, it is difficult to automate, and the operator is exposed to radiation, which makes it difficult to test a large amount of samples in the clinical field.

[0006] In the third method described above, although the specific site of the target polynucleotide can be propagated, the detection means often use the first and second autoradiography,
There are the same problems as above. Further, the method using ethidium bromide staining can shorten the time, but is not suitable for automation. The breeding method also needs to raise and lower the temperature precisely. The amount of amplification varies depending on the reaction conditions and the state of the sample, and since amplification is non-linear, quantitative measurement is difficult.

An object of the present invention is, firstly, to provide a highly sensitive and accurate quantitative method for detecting a polynucleotide, which can carry out a hybridization reaction and detection in a short time. Another object of the present invention is to realize non-radioactive isotopeization of the polynucleotide probe.

[0008]

In order to realize the non-radioactive isotopeization of the labeled probe among the above objects, in the present invention, a fluorescent substance is used as the labeled substance. In the present invention,
Although electrophoresis and fluorescence detection are used in combination, the detection limit of the fluorescent substance is 10 ⁻²⁰ to 10 ⁻²¹ mol / band. The minimum copy number to be detected by gene diagnosis is said to be 100 copies for the time being, and it is necessary to increase the current fluorescence detection ability by about 10 times. Therefore, in detecting the target polynucleotide with high sensitivity in a short time, a point to be particularly noted in the present invention was how many fluorophores are introduced into the target polynucleotide. Therefore, using a plurality of labeled probes that can associate with different sites of the polynucleotide to be measured,
A method was devised in which this was simultaneously associated with the polynucleotide chain to be measured, and a substance in which a plurality of fluorophores were bound to each labeled probe was used. Furthermore, removal of unreacted fluorescently labeled probe is an important point for highly sensitive detection. In the present invention, after associating a plurality of labeled probes with a polynucleotide chain to be measured, the unreacted labeled probe is removed using a carrier that removes the labeled probe-bound polynucleotide chain to be measured and the labeled probe at a molecular size. And a step of dissociating a plurality of probes bound to the polynucleotide chain to be measured under denaturing conditions, and the dissociated labeled probe is detected. Specifically, the sample after the hybridization reaction is added to an electrophoretic carrier such as polyacrylamide and electrophoresed to remove the unreacted fluorescent labeled probe. Since the fluorescent-labeled probe hybridized with the target polynucleotide has a large molecular weight, it stays in the vicinity of the sample addition portion of the electrophoretic carrier. Next, when the temperature of the electrophoretic carrier is set to the denaturation temperature of the complex of the target polynucleotide and the fluorescent labeled probe or higher, the fluorescent labeled probe bound to the target polynucleotide is dissociated and migrated. This is accomplished by fluorescently detecting the fluorescently labeled probe bound to the target polynucleotide. Fluorescence detection is performed using a light source and a detector that illuminates a certain portion of the electrophoretic carrier. In the present invention, since electrophoresis is used for detection, it is necessary for higher sensitivity that a plurality of labeled probes have the same migration speed and are detected as one band. Since the electrophoresis used in the present invention is for separating and removing the unreacted fluorescently labeled probe, the separation ability may be low. Therefore, the difference in the base length of each labeled probe is 10% or less, and preferably a uniform band can be obtained by making the base length uniform. In addition, the length of the labeled probe is preferably 12 bases or more from the viewpoint of hybridization efficiency. Further, from the viewpoint of ease of electrophoretic separation and chemical synthesis, 100 bases or less is desirable.

The concept of the present invention will be described with reference to the conceptual diagram of FIG. Multiple fluorescently labeled probes 2-6 having the same base length
And the target polynucleotide strand 1 are reacted under hybridization conditions. At this point of time, what is present in the reaction solution is the fluorescent-labeled probe-target polynucleotide chain complex 10 and the unreacted fluorescent-labeled probe 11. 2 phosphors
It is possible to introduce 10 molecules of fluorophore into 1 molecule of the target polynucleotide by using 5 kinds of probes which are bound to each other. Since the fluorescence intensity obtained by this is improved by about one digit, a target polynucleotide of 100 copy order can be detected by a combination of electrophoresis and fluorescence detection. The fluorescence detection method will be described more specifically. When the reaction solution after hybridization is added to the sample addition section 21 of the electrophoretic carrier and electrophoresed, the unreacted fluorescent labeled probe 11 having a small molecular size is electrophoresed and removed. Since the fluorescent-labeled probe-target polynucleotide chain complex 10 has a large molecular size, it remains concentrated in the vicinity of the sample addition portion and is simultaneously concentrated. When the sample addition part is heated, the fluorescent labeled probe 20 bound to the target polynucleotide chain is dissociated and electrophoresed. When the dissociated fluorescent-labeled probe crosses the laser light that irradiates a certain part of the electrophoretic carrier, it emits fluorescence and is detected by a detector.

[0010]

When a plurality of labeled probes capable of complementary binding to the target polynucleotide are reacted with the target polynucleotide under hybridization conditions, a hybrid of the target polynucleotide and the labeled probe is formed. The unreacted labeled probe can be separated and removed by electrophoresis as described above. The hybrid of the target polynucleotide and the labeled probe is concentrated in the sample addition portion during electrophoresis. High sensitivity can be achieved by hybridizing a plurality of fluorescently labeled probes to one target polynucleotide. When the hybrid of the separated target polynucleotide and the labeled probe is heated above the denaturation temperature, the target polynucleotide and the labeled probe dissociate, so the labeled probe bound to the target polynucleotide is separated from the unreacted labeled probe and detected. It is possible.

As the fluorescent substance for labeling the polynucleotide probe, rhodamine, fluorescein, 4-nitrobenzo-2-oxa-1,3-diazole, phthalocyanine and the like and derivatives thereof, and polymers containing these fluorescent substances are used. be able to. Due to the problem of scattered light from the electrophoretic carrier, a long-wavelength phosphor is preferable, and a combination of sulforhodamine 101 and He / Ne laser is preferable due to the limitation of laser light for excitation.

[0012]

【Example】

Example 1 In this example, M13 was used as a target polynucleotide sample.
mp8 single-stranded DNA was used. The probe complementary to this target DNA is a single-stranded polynucleotide consisting of 24 bases of each of SEQ ID NOs: 1, 2, 3, 4 and 5. In each probe, sulforhodamine 101 is bound to the 5'end and the 12th T via an amino group. These probes are respectively M13 mp8 200-2.
23, 510-533, 6272-6295, 6312.
It binds complementarily to the base portions of ~ 6335 and 6561 to 6584. A method for preparing this fluorescently labeled probe will be described.

Polynucleotides of 24 bases having the structures of SEQ ID NOs: 1 to 3 were prepared by the phosphoamidite method (MacBride,
Elle. J. , Calthers, M. Etch. , Tetrahedron Letters (Mc Bride, LJ a
nd Caruthers, M .; H. , Tetrahhe
dron Letters, vol. 24, 245-3
48 (1983)). At this time, in the final stage of the synthesis, N-monomethoxytritylaminohex-6-oxy-β-cyanoethyl-N, N-diisopropylaminophosphoamidite is reacted to introduce an amino group at the 5 ′ end. As the thymine residue of No. 12, an aminated thymidine represented by the formula 1 in which an amino group is introduced at the 5-position of uridine is protected with a 4,4'-dimethoxytrityl group at the 5'hydroxyl group. Aminated thymidine of this structure is difree bee. Others, Proceeding National Academy Science USS (Ge
offrey B. et al, Proc. Nat
l. Acad. Sci. USA 82,968-9
72 (1985)). The free amino group is trifluoroacetylated and the 3'hydroxyl group is activated with chloro-N, N-diisopropylaminomethoxyphosphine for use. The crude polynucleotide synthesized by the above method is dissolved in 30% aqueous ammonia. After neutralizing with acetic acid under ice cooling, 2.5 times volume of ethanol is added to recover the synthetic polynucleotide. Precipitate with 1M NaCl
And dissolve again in 2.5 times volume of ethanol to precipitate.
By repeating this operation twice more, low molecular weight reaction contaminants such as ammonia and unreacted nucleotides are removed. Next, the synthetic polynucleotide is dissolved in 0.5 M carbonate buffer (pH 9), and 250 times mol of sulforhodamine 101 acid chloride is added. Sulforhodamine 1
The 01 acid chloride is used by dissolving it in acetonitrile in advance, but it is important that acetonitrile is about 20% during the reaction. When the acetonitrile concentration is too high, the polynucleotide is likely to precipitate, and when it is too low, sulforhodamine 101 acid chloride is likely to precipitate. After reacting for 16 hours at room temperature in the dark, neutralized with hydrochloric acid, 2.
Add 5 volumes of ethanol and collect the precipitate. The crude sulforhodamine 101-labeled probe thus prepared is purified by 19% polyacrylamide gel electrophoresis containing 7M urea. The labeled probe thus prepared is electrophoretically pure with a single band.

Next, the target polynucleotide sample M
13 shows the measurement procedure of 13 mp8. 1 μl (microliter) of various concentrations of the target M13 mp8 single-stranded DNA solution is added to the reaction vessel 201 shown in FIG. 2 containing 1 μl (microliter) of the prepared 5 types of fluorescently labeled probes (1 fmol each). After heating at 95 ° C for 30 seconds, 6
Incubate at 5 ° C for 5 minutes. Furthermore, the temperature is kept at 45 ° C. for 30 minutes to complete the hybridization reaction. Next, the reaction solution is added to the electrophoretic carrier 203. The electrophoresis carrier has a structure in which a quartz tube having an inner diameter of 0.2 mm is filled with 12% polyacrylamide gel, and a He / Ne laser is irradiated at a position 4 cm from the sample addition portion at the tip. A photomultiplier tube for detecting fluorescence is arranged near the laser irradiation section. Here, the surface of the reaction container 201 is made of gold or platinum to serve also as an electrode for electrophoresis. By integrating the container and the electrode, there is an advantage that even a trace amount of solution can be directly added to the electrophoretic carrier without loss. When the electrophoretic carrier 203 is brought into direct contact with the reaction solution in the reaction container as shown in FIG. 2 and an electric field is applied between the electrode on the container surface and the other end of the electrophoretic carrier, the target polynucleotide and the labeled probe in the reaction solution. The complex and the unreacted labeled probe are incorporated into the electrophoretic carrier. After a certain period of time, the reaction vessel side of the electrophoretic carrier was treated with 89 mM Tris-89 mM boric acid,
Replace with an electrode tank containing an electrode solution consisting of 2.5 mM EDTA and having a pH of 8.3, and continue to apply an electric field at 45 ° C. Here, in order to prevent the temperature rise of the electrophoretic carrier, electrophoresis was performed at 40 v / cm. Unreacted labeled probe is removed in about 30 minutes. At this point, the complex of the target polynucleotide and the labeled probe is trapped in the vicinity of the sample addition part of the electrophoretic carrier. Next, the vicinity of the sample addition portion of the electrophoretic carrier is heated to 70 ° C. Voltage is 200v / cm for 30 seconds
Then, the electrophoresis is continued at 40 v / cm. The results of the time-dependent change in the fluorescence intensity detected by the detector are shown in FIG.
The electrophoretic separation band 302 derived from the labeled probe dissociated from the target polynucleotide was detected at an elution time that was clearly different from the electrophoretic separation band 301 of the unreacted labeled probe. In FIG. 3, as a control, the results obtained by reacting only one labeled probe of SEQ ID NO: 1 with only the 5 ′ end fluorescently labeled with the target polynucleotide are also shown. The fluorescence intensity 302 derived from the target polynucleotide obtained when 5 types of labeled probes were used was about 8 times the fluorescence intensity 303 when one type of labeled probe was used. In this example, since 5 types of fluorescent-labeled probes each having 2 molecules of the fluorescent substance bound thereto are used, a maximum of 10 molecules of the fluorescent substance are introduced into the target polynucleotide. Originally, a fluorescence intensity 10 times higher than that of the conventional method should be obtained, but it is considered that concentration quenching of about 20% occurs because the distance between the fluorescent molecules is short.

When the present invention is used, a strong signal intensity can be obtained, so that there is an advantage that the target polynucleotide can be detected with higher accuracy. Further, since the hybridization reaction is carried out in a solution, the reaction efficiency is good, and it is conventionally several hours to 1 hour.
There is an advantage that the hybridization reaction which takes a long time can be performed in a short time of about 35 minutes. Further, the present invention has the following advantages. Since single-stranded DNA such as M13 mp8 used in this example has an extremely slow migration speed, it is usually difficult to analyze by electrophoresis using polyacrylamide. However, in the present invention, M13 mp8 single chain DN
Since the labeled probe hybridized to A is released and analyzed, there is an advantage that even a polynucleotide having a slow migration rate such as M13 mp8 can be analyzed using the labeled probe. Furthermore, this method is suitable for high-sensitivity detection because it has the advantage that the sample can be concentrated in the sample addition part of the electrophoretic carrier.

Example 2 In this example, after a plurality of fluorescent-labeled probes were hybridized with a target polynucleotide, the 3'end of the fluorescent-labeled probe was further fluorescent-labeled, so that a larger amount of fluorophore was targeted to the target polynucleotide. The method for quantitatively detecting a target polynucleotide with high sensitivity will be described. Also in the present embodiment, as in the first embodiment, M13 m
p8 single stranded DNA was used as a target polynucleotide sample.

The probe complementary to the target DNA is a single-stranded polynucleotide consisting of 24 bases of each of SEQ ID NOS: 1 to 5, and the amino acid groups are introduced at the 5'end and the 12th T as in Example 1. And sulforhodamine 101 is bound. According to the method of Example 1, the above three types of fluorescently labeled probes (1 fmol each) are hybridized with M13 mp8 at various concentrations. Next, the phosphor-bonded 4
Seed dideoxynucleotide triphosphate and Taq DN
A polymerase is 20 mM Tris-HCl, 50 mM
The reaction is carried out under a solution condition of NaCl, 2 mM MnCl ₂ , and 15 mM isocitrate at pH 8.5 to introduce a base to which a fluorophore is bound to the 3 ′ end of each fluorescent-labeled probe hybridized to the target polynucleotide.
Here, the four types of dideoxynucleotide triphosphate fluorophores to which the fluorophore is bound are the same as sulforhodamine 101 used for the fluorescently labeled probe in Nucleic Acid Research 20 (10) 2471-2483 ( Nucleic Acids Research, V
ol. 20, No. 10 2471-2483) described above and is bound to a dideoxynucleotide triphosphate having a 3-amino-1propyl-1yl linker. In this state, up to 15 molecules of fluorophore are introduced per molecule of target DNA. As in Example 1, the reaction solution is added to an electrophoretic carrier and subjected to electrophoretic analysis. After the unreacted fluorescence derived from the four kinds of dideoxynucleotide triphosphates bound with the fluorescently labeled probe or the fluorescent substance is no longer detected, the vicinity of the sample addition portion is heated to 70 ° C. After raising the voltage to 200 v / cm for 30 seconds, the electrophoresis is continued at 40 v / cm. The result of plotting the fluorescence intensity detected by the detector against the amount of the target polynucleotide M13 mp8 in the sample solution is shown in FIG. As is clear from FIG. 4, about 0.3 zmol (0.0003 amol) of M1
3 mp8 could be detected.

In this example, a plurality of types of probes to which a plurality of fluorophores are bound are used, and a fluorophore is introduced at the 3 ′ end of the fluorescent probe hybridized to the target polynucleotide using a polymerase to obtain the target polynucleotide. We have introduced as many phosphors as possible. The signal intensity obtained by this is the conventionally used phosphor 1.
It is about 12 times stronger than one probe with a molecule attached. Therefore, there is an advantage that the measurement can be performed with higher sensitivity and accuracy.

[0019]

EFFECTS OF THE INVENTION The present invention has the advantage that a plurality of fluorophores can be introduced into a target polynucleotide even with a short probe. Further, in the present invention, since the hybridized fluorescently labeled probe is separated and detected by electrophoresis, the nonspecifically extended probe has an advantage that it can be removed because the migration speed is different. Therefore, quantitative detection with high sensitivity and high reliability is possible. The sensitivity obtained by this method almost clears the sensitivity required for genetic diagnosis. In addition, since it can be analyzed in a short time, it is useful for actual gene diagnosis.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic polymer containing other structure Sequence: GCTGAATCTG GTGCTGTAGGC TCAA SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Synthetic polymer containing other structures Sequence: ATAGCGTCGA ATACTGCGGA ATCG SEQ ID NO: 3 Sequence Length: 24 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Synthetic polymer containing other structures Sequence: TCACGACGTT GTAAAACGAC GGCC SEQ ID NO: 4 Sequence length: 24 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic port containing other structures Mer sequences: TGCAAGGCGA TTAAGTTGGG TAAC SEQ ID NO: 5 SEQ Length: type 24 sequence: the number of a nucleic acid strand: single strand Topology: linear sequence type: synthetic polymer sequences containing other structures: CGGATTGACC GTAATGGGAT AGGT

[Brief description of drawings]

FIG. 1 is a diagram showing the concept of the present invention.

FIG. 2 is a conceptual diagram showing a relationship between a reaction container used in the present invention and an electrophoretic carrier.

FIG. 3 is a view showing a result of time-dependent changes in fluorescence intensity detected in Example 1 of the present invention.

FIG. 4 is a diagram showing an example of a calibration curve of the target polynucleotide obtained in Example 2 of the present invention.

[Explanation of symbols]

1 ... Target polynucleotide, 2-6 ... Fluorescently labeled probe, 10 ... Complex of target polynucleotide and fluorescently labeled probe, 11 ... Unreacted fluorescently labeled probe, 20 ... Released from complex of target polynucleotide and fluorescently labeled probe Fluorescently labeled probe, 21 ... Sample addition section, 201 ... Reaction container / negative electrode, 202 ... Cathode lead wire, 203 ... Electrophoretic carrier, 204 ... Anode, 301 ... Electrophoretic separation band of unreacted fluorescent labeled probe, 302 ... Electrophoretic separation band of fluorescent-labeled probe released from complex of target polynucleotide and multiple types of fluorescent-labeled probe, 303 ... Electrophoretic separation of fluorescent-labeled probe released from complex of target polynucleotide and one type of fluorescent-labeled probe band.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Keiichi Nagai, 1-280 Higashi Koikekubo, Kokubunji, Tokyo, Central Research Laboratory, Hitachi, Ltd. (72) Inventor, Katsuji Murakawa 1-280 Higashi Koikeku, Kokubunji, Tokyo Hitachi, Ltd. Central Research Laboratory (72) Inventor Masahiko Sugiura 1-34-5 Koenji Minami, Suginami-ku, Tokyo Inside BM Corporation (72) Inventor Toshikazu Yamaguchi 1-34-5 Koenji Minami, Suginami-ku, Tokyo No. Stock Company BML

Claims (4)

[Claims] 1. A method for detecting a polynucleotide, which comprises a step of hybridizing a labeled probe to a polynucleotide to be measured, which is an object to be measured, and a step of measuring the amount of the associated probe, to associate with different sites of the polynucleotide to be measured. A method for detecting a polynucleotide, which comprises using a plurality of labeled probes capable of being combined with the polynucleotide chain to be measured. 2. A carrier which, after associating the plurality of labeled probes with the polynucleotide chain to be measured, removes the unlabeled labeled probe unreacted with the polynucleotide chain to be measured bound with the labeled probe at a molecular size. Using the step of removing the unreacted labeled probe using, and dissociating the plurality of labeled probes bound to the polynucleotide chain to be measured under denaturing conditions, the dissociated labeled probe is detected The method for detecting a polynucleotide according to claim 1, wherein 3. The polynucleotide base length of the plurality of labeled probes is 12 bases or more and 100 bases or less, and the difference in the base lengths of the respective labeled probes is 10% or less, preferably a uniform base length. The method for detecting a polynucleotide according to claim 1 or 2. 4. The poly according to claim 1, wherein the labeled substance of the labeled probe is a fluorescent substance, and a plurality of fluorescent substances are bound to each labeled probe. Nucleotide detection method.