JPH0723799A - Method for detecting polynucleotide - Google Patents

Abstract

PURPOSE:To provide a method for detecting polynucleotide, capable of carrying out a reaction at a definite temperature and detecting DNA in high sensitivity by reacting a target nucleotide with ribonuclease and a nucleic acid synthetaase under specific conditions using a nucleic acid probe composed of a ribonucleotide and a deoxyribonucleotide. CONSTITUTION:A nucleic acid composed of deoxyribonucleotide parts 11 and 13, a ribonucleotide part 12 and labelled material 14 is hybridized with the sample DNA101. The resultant complex 102 is made to react with a DNA polymerase 107 and a ribonuclease 106 in the presence of deoxyribonucleotide 3 phosphoric acids 103 and 104 and dideoxynucleotide 3 phosphoric acid 105 different in series of base from deoxyribonucleotide 3 phosphoric acid. Thereby, 3' terminal is elongated by definite base length and a ribonucleotide part 109 is cleaved and a labeled deoxyribonucleotide 108 having definite length is amplified.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for detecting DNA and a gene diagnostic method.

[0002]

2. Description of the Related Art Detection of polynucleotides such as DNA is used for diagnosis of infectious diseases caused by genetic diseases and viruses. When the copy number of the virus to be tested is small, it is several tens or less, and the method of multiplying and detecting DNA by the polymerase chain reaction (PCR) method is a natural method (Natur).
e) 350, 91-92 (1991) or JP-A-61-27
It is disclosed in Japanese Patent No. 4697. The PCR method propagates a specific region of DNA, and propagates a DNA in a region sandwiched by two kinds of oligomers that hybridize to the (+) and (-) strands of the target double-stranded DNA.

Double-stranded DNA is denatured at high temperature (-90 ° C) and separated into (+) strand and (-) strand. Then the temperature is lowered (~ 60
C.) and hybridize DNA oligomers to each, and a complementary strand is synthesized with a heat-resistant DNA polymerase derived from Taq. The temperature was raised again and two pairs of (+) chain and (-)
Make chains. Thereafter, the thermal cycle of temperature decrease and temperature increase is repeated to double the number of DNA chains. Actually, it is known that one heat cycle increases the average by about 1.6 times, and if it is repeated 30 times, the copy number can be increased to 10 ⁶ times. The DNA thus obtained is subjected to gel electrophoresis separation, and the presence or absence of the target DNA is diagnosed by examining the length of the amplified DNA.

[0004]

In this method, an expensive thermostable enzyme is required for the growth of a DNA copy, it takes time and time to repeat heating and cooling, and two types of DN are used.
There is a drawback in that an A-growing oligomer (primer) is required. In particular, depending on the subject, there are cases where two primers cannot be selected properly. Further, it is necessary to separate the PCR products by electrophoresis, which is troublesome and labor-intensive. Further, in the conventional technique, a specific site of the target polynucleotide can be amplified, so that the sensitivity is high, but a RI-labeled substance or a fluorescent staining method using ethidium bromide is used for detection. These are manual methods and are not suitable for automation. In addition, there is a problem that the worker is exposed to radiation when using the RI-marked material.

An object of the present invention is to provide a simple and highly sensitive method for detecting a target polynucleotide, that is, DNA. More specifically, an object of the present invention is to provide a means for amplifying a nucleic acid probe hybridized with a target polynucleotide sample by an enzymatic reaction at a constant temperature. Another object is to provide a method for detecting a target polynucleotide suitable for automation. Another object of the present invention is to realize the non-RI labeling of the nucleic acid probe, to free the operator from radiation exposure and to eliminate restrictions on the work place.

[0006]

In order to achieve the above object, the present invention uses the following method.

First, the nucleic acid probe is amplified by an enzyme reaction at a constant temperature by any of the following methods (1) to (3).

(1) A nucleic acid probe is composed of ribonucleotides and deoxyribonucleotides, and a label is attached to at least one position of deoxyribonucleotides. The nucleic acid probe is bound to the target polynucleotide under hybridizing conditions. next,
By reacting an RNA degrading enzyme such as ribonuclease H (Ribonuclease H) with a nucleic acid synthesizing enzyme in the coexistence of at least one kind of deoxyribonucleotide triphosphate and dideoxynucleotide triphosphate having a base species different from deoxyribonucleotide triphosphate. The 3'end is extended by a constant base length to cut the ribonucleotide portion. The original nucleic acid probe is designed so that the base length of the probe product generated by this series of extension and decomposition reactions is about 10 bases or less. Since such a short probe product cannot retain a hydrogen bond with the target polynucleotide, the shortened probe product is released from the target polynucleotide and regenerates the original target polynucleotide. Replayed,
The target polynucleotide binds to the nucleic acid probe again, and a series of extension decomposition reactions occur. This cycle is repeated multiple times during the reaction for a certain period of time.

(2) A nucleic acid probe composed of ribonucleotides and deoxyribonucleotides is bound to a target polynucleotide under hybridizing conditions to form at least one deoxyribonucleotide triphosphate and a base species different from the deoxyribonucleotide triphosphate. The RNA degrading enzyme and the nucleic acid synthesizing enzyme are reacted in the coexistence of the labeled dideoxynucleotide triphosphate. In this reaction, the 3'end is extended by a certain base length to label the 3'end, and the ribonucleotide portion is cleaved to generate a 3'end-labeled deoxyribonucleotide with a certain length. Similar to (1), the original nucleic acid probe was designed so that the base length of the probe product generated by this series of extension decomposition reactions would be about 10 bases or less, and the extension decomposition of the nucleic acid probe-shortened probe The cycle of product release and target polynucleotide regeneration is repeated multiple times.

(3) The nucleic acid probe is composed of ribonucleotides and deoxyribonucleotides, and a label is bound to at least one position of the deoxyribonucleotides. After binding to the target polynucleotide, at least one deoxyribonucleotide triphosphate and a dideoxynucleotide 3 bound to a label with a base species different from the deoxyribonucleotide triphosphate
An RNA degrading enzyme and a nucleic acid synthesizing enzyme are reacted in the presence of phosphoric acid. In this reaction, the base of the 3'end is extended to a certain length to give a 3'end.
The end is labeled, the ribonucleotide portion is cleaved, and a deoxyribonucleotide of a certain length is produced in which the intermediate base portion and the 3'end are labeled. Similar to (1), the original nucleic acid probe was designed so that the base length of the probe product generated by this series of extension decomposition reactions would be about 10 bases or less, and the extension decomposition of the nucleic acid probe-shortened probe The cycle of product release and target polynucleotide regeneration was repeated several times.

Then, the target polynucleotide is detected by the following method (4) or (5).

(4) In the above (1), the 3'-terminal biotinylated deoxyribonucleotide, which has a fixed length and is composed of dideoxynucleotide triphosphorus bound with biotin, is separated using a carrier on which avidin or streptavidin is immobilized. To do.

(5) In the above (2) or (3),
It was decided to use a nucleic acid probe in which biotin is bound to at least one site of the deoxyribonucleotide portion and to separate a biotinylated probe having a constant length using a carrier on which avidin or streptavidin is immobilized.

Further, non-RI labeling of the nucleic acid probe is realized by the following method (6) or (7).

(6) In the above (1), (2) and (3), a fluorescent substance is used as the labeling substance.

(7) In the above (3), the labeled product of the deoxynucleotide moiety of the nucleic acid probe and the labeled product of labeled dideoxynucleotide triphosphate are composed of different fluorescent substances, and the two types of fluorescent substances are energy-transferred. And one of the phosphors is excited to indirectly excite the other phosphor.

[0017]

When the target nucleic acid is a single-stranded nucleic acid such as single-stranded DNA, it can be used as a sample as it is. Further, in the case of double-stranded DNA, it may be denatured by heat or alkali, but the hybridization of target nucleic acids occurs under the hybridization conditions, and the efficiency of hybridization with the nucleic acid probe decreases, so exonuclease III It is better to treat it with a double-strand-specific nuclease as described above to make it single-stranded.

A nucleic acid probe composed of ribonucleotides and deoxyribonucleotides can bind to a target polynucleotide as long as it has a length of 16 bases or more. When a nucleic acid synthase is reacted in the coexistence of deoxyribonucleotide triphosphate and dideoxynucleotide triphosphate, a base can be added to the 3'end using the nucleic acid probe as a primer. If deoxyribonucleotide triphosphate and dideoxynucleotide triphosphate are different types of base species, the extension reaction can be stopped at the first dideoxynucleotide triphosphate binding site.

Since the extension reaction by the nucleic acid synthase does not always stop 100% at the dideoxynucleotide triphosphate binding site, the deoxygenation corresponding to the 3'-side base portion next to the dideoxynucleotide triphosphate binding site is performed. The extension reaction can be reliably stopped without adding nucleotide triphosphate.

When performing an elongation reaction by a nucleic acid synthase
RN that specifically degrades the RNA part of the hybridization-forming part of DNA and RNA like Ribonuclease H
The A-degrading enzyme can be used to degrade the ribonucleotide portion of the nucleic acid probe bound to the target polynucleotide under hybridization conditions. At this time, the deoxyribonucleotide portion, that is, the target polynucleotide, the deoxyribonucleotide portion of the nucleic acid probe, and the extended portion are not decomposed. Therefore, the nucleic acid probe has a shorter length on the 5'end side and a longer length on the 3'end side.

By designing the probe so that the length of extension is 2 to 5 bases and the total length of the decomposed nucleic acid probe is 10 bases or less, such a short probe retains a hydrogen bond with the target polynucleotide. I can't. Therefore, the nucleic acid probe subjected to extensional decomposition in a series of reactions is released from the target polynucleotide and regenerates the original target polynucleotide. Usually, since the nucleic acid probe is present in a large excess, the regenerated target polynucleotide binds to the nucleic acid probe again, and a series of decomposition and extension reactions occur. Since this cycle occurs until the nucleic acid probe is consumed, the decomposed and extended nucleic acid probe is amplified and produced.

In the present invention, a nucleic acid probe to which a fluorophore is bound is used, or a fluorophore-conjugated dideoxyribonucleotide is used to introduce the fluorophore to the 3'end during the extension reaction. The detection can be performed by fluorescence detection. Fluorescein, tetramethylrhodamine, rhodamine X, sulforhodamine 101 or the like can be used as the fluorescent substance. Alternatively, energy transfer between the two fluorophores can be utilized by changing the types of the fluorophore of the nucleic acid probe and the fluorophore introduced at the 3'end, so that one fluorophore is excited and the fluorescence of the other fluorophore is excited. Can be detected. As a combination of such phosphors, a combination of fluorescein and sulforhodamine 101 is effective.

Since the nucleic acid probe bound to the target polynucleotide and decomposed and extended has a base length different from that of the undecomposed probe or the probe decomposed before the extension, they can be easily separated and detected by electrophoresis.

A probe in which biotin is introduced at the 3'end by a decomposition extension reaction using a product in which dideoxynucleotide triphosphorus is bound to biotin can be easily captured by a carrier having streptavidin or the like immobilized thereon. The probe without biotin, that is, the probe with no extension at the 3'end, can be removed by washing, and the amount of probe with decomposition and extension by measuring the fluorescence on the carrier, that is,
The amount of target polynucleotide in the sample can be known.
Similarly, even when using a nucleic acid probe in which biotin is bound to the deoxyribonucleotide portion, in the case where a fluorophore is introduced at the 3'end in the decomposition extension reaction, the fluorescence intensity on the carrier is measured to measure the target in the sample. The amount of polynucleotide can be known.

[0025]

【Example】

(Example 1) The present invention in which a nucleic acid probe is amplified by an enzymatic reaction at a constant temperature will be described. The structure of the DNA probe of the present invention is shown in FIG. Although the sulforhodamine 101 fluorescent substance was used as the labeled substance in the figure, other fluorescent dyes are also possible.

The basic operation of each probe will be described with reference to FIGS. In the first example, as shown in FIG. 2, the nucleic acid probe 1 shown in FIG. 1 (a) having a single-stranded DNA 101 and a base sequence complementary thereto is used as a sample. The nucleic acid probe 1 has a deoxyribonucleotide moiety 11 having a length of 10 bases at the 5 ′ end, a ribonucleotide 12 having a length of 8 bases at the 3 ′ side, and a deoxyribonucleotide moiety 13 having a length of 6 bases at the 5 ′ end has a total length of 24. It is a hybrid oligonucleotide having a base length. A sulforhodamine 101 fluorophore is bound to the 5th to 4th bases of the deoxyribonucleotide moiety 13.

As shown in FIG. 2, when the sample DNA 101 and the nucleic acid probe 1 are mixed at 37 ° C., they hybridize because they have complementary sequences and form a complex such as 102. Here, two kinds of deoxyribonucleotide triphosphates 103 and 104, dideoxyribonucleotide triphosphate 105, Sequenase (manufactured by Toyobo Co., Ltd.) 107, which is a DNA polymerase, and RNase H (manufactured by Takara Shuzo Co., Ltd.) 106, which is an RNA degrading enzyme, are used. Let it coexist.

At the 3'end of the nucleic acid probe 1 bound to the sample DNA 101, deoxyribonucleotide triphosphate is extended by 3 bases by the sequence, and then dideoxyribonucleotide triphosphate binds to stop the reaction. Also, the ribonucleotide portion of the nucleic acid probe 1 bound to the sample DNA 101 is decomposed by RNase H. The probe that has undergone extension and decomposition becomes about 10 bases long. here,
Since the nucleic acid probe that has been decomposed by RNase H before the extension reaction has a length of 6 bases and is released from the sample DNA 101, the subsequent extension reaction does not occur. As described above, the fragment which was decomposed without the elongation reaction was 6 bases long, and the fragment 108 which was decomposed after the elongation reaction was decomposed.
Has a length of 10 bases, so both can be electrophoretically separated.

The fragment 108 having a length of 10 bases due to the elongation reaction and decomposition is released from the sample DNA 101 at 37 ° C. to regenerate the original sample DNA 101. The regenerated sample DNA 101 binds to the nucleic acid probe 1 again,
Repeat a series of elongation and degradation cycles.

Actually, 1 × 10 to ¹⁸ mol of target DNA1
01 and 1 × 10 to ¹³ mol of the nucleic acid probe 1 were reacted with 1 unit of Sequenase and 1 unit of RNase H at 37 ° C. for 30 minutes in the presence of deoxyribonucleotide triphosphates dT and dC and dideoxyribonucleotide triphosphate ddG. . The reaction solution was heated at 95 ° C. for 2 minutes to stop the reaction, and then separated and analyzed by electrophoresis. Hitachi WS for electrophoresis
The laser for pumping the Q-3000 type DAN sequencer is He /
The laser was changed to a Ne laser (633 nm), and a cylindrical lens was attached to the electrophoretic plate to improve the light collection efficiency, and the electrophoretic plate was modified so that it could be detected with high sensitivity.

As a result, the fragment decomposed after the extension reaction could be detected separately from the unreacted probe and the fragment decomposed before the extension reaction. From the ratio of the sum of the intensities of all the detected fluorescence peaks and the intensities of the peaks of the fragments decomposed after the extension reaction, the amount of the fragments decomposed after the extension reaction was calculated to be about 200 times that of the target DNA. A fragment was being generated. From this, it was confirmed that the nucleic acid probe can be amplified by an enzyme reaction at a constant temperature by using this example.

In this embodiment, the nucleic acid probe is shown in FIG.
Although the one having the structure of was used, the nucleic acid probe 4 having a plurality of ribonucleotides 43 as shown in (d) of FIG.
Similar results can be obtained.

Example 2 In this example, the nucleic acid probe 2 shown in FIG. 1 (b) is used. The nucleic acid probe 2 has a deoxyribonucleotide moiety 21 having a length of 10 bases at the 5 ′ end, a ribonucleotide 22 having a length of 8 bases at the 3 ′ side, and a deoxyribonucleotide moiety 23 having a length of 6 bases at the 5 ′ end has a total length of 24. It is a hybrid oligonucleotide having a base length. The fluorophore is not bound.

As shown in FIG. 3, when the sample DNA 101 and the nucleic acid probe 1 are mixed at 37 ° C., they hybridize because they have complementary sequences and form a complex such as 202. Here, two kinds of deoxyribonucleotide triphosphates 103 and 104, fluorescently labeled dideoxyribonucleotide triphosphate 205, Sequenase (manufactured by Toyobo Co., Ltd.) 107 which is a DNA polymerase, and RNase H (manufactured by Takara Shuzo Co., Ltd.) which is an RNA degrading enzyme. 106 coexist. A sulforhodamine 101 fluorescent substance was used for the fluorescent label. Deoxyribonucleotide triphosphates are extended by 3 nucleotides at the 3'end of the nucleic acid probe 2 bound to the sample DNA 101 by the sequence, and then fluorescence labeled dideoxyribonucleotide triphosphate 205 is bound, and the reaction is stopped. In addition, the ribonucleotide portion 22 of the nucleic acid probe 2 bound to the sample DNA 101 is decomposed by RNase H.

The probe that has undergone extension and decomposition has a length of about 10 bases. As in Example 1, the probe 208 that has undergone extension and decomposition is released from the sample DNA 101, and the original sample D is released.
Play NA101. The regenerated sample DNA 101 is bound to the nucleic acid probe 2 again, and a series of elongation and decomposition cycles are repeated.

As in Example 1, 1 × 10 to ¹⁸ mol of the target DNA 101 and 1 × 10 to ¹³ mol of the nucleic acid probe 2 were coexisted with deoxyribonucleotide triphosphates dT and dC and fluorescence labeled dideoxyribonucleotide triphosphate ddG. Below 1 unit of Sequenace and 1 unit of RNase H
The reaction was carried out at 7 ° C for 30 minutes. After the reaction solution is heated at 95 ° C. for 2 minutes to stop the reaction, gel filtration is performed to such an extent that it does not interfere with the subsequent electrophoretic analysis to roughly remove the unreacted fluorescence-labeled dideoxyribonucleotide triphosphate. Subsequently, they were separated and analyzed by electrophoresis.

As a result, as in Example 1, the fragment decomposed after the extension reaction could be detected separately from the unreacted probe and the fragment decomposed before the extension reaction. In this example, detection of a fluorescent substance incorporated at the 3'end in a series of reactions has the effect of increasing the selectivity for the target polynucleotide.

Although the nucleic acid probe 2 was used in this example, biotin 3 was used as in the nucleic acid probe 3 of FIG. 1 (c).
It is also possible to use those in which 4 is linked.

In this case, 3 '
Deoxyribonucleotide 3 phosphate was extended to the end by a length of 3 bases, and then fluorescently labeled dideoxyribonucleotide 3
Binds phosphoric acid. At the same time, the ribonucleotide moiety 32 is decomposed with RNase H to obtain a decomposed extension fragment.

Subsequently, the reaction solution is separated by electrophoresis, and the fraction containing the decomposed and extended fragments eluted from the electrophoresis carrier gel is collected on a microplate on which streptadipine is immobilized.

By this operation, the biotin-bound fragment is bound to the microplate. At this point, unreacted fluorescently labeled dieoxyribonucleotides left due to insufficient separation by electrophoresis are separated and removed. By measuring the fluorescence derived from the fluorescent substance bound to the surface of the microplate, the same result as in Example 1 could be obtained.

Example 3 In this example, a nucleic acid probe 1 fluorescently labeled with sulforhodamine 101 and a fluorescently labeled dideoxyribonucleotide triphosphate bound with sulforhodamine 101 are used to detect a target polynucleotide by energy transfer. Describe.

Similar to Examples 1 and 2, as shown in FIG. 4, the sample DNA 101 and the nucleic acid probe 1 are hybridized to form a complex such as 102. In a series of reactions, the 3'end is extended by a length of 3 bases, and then fluorescence-labeled dideoxyribonucleotide triphosphate binds to stop the reaction. Further, the ribonucleotide portion of the nucleic acid probe is decomposed by RNase H. About 10 probes were extended and degraded
A probe 208 having a base length and subjected to extension and decomposition in which two kinds of fluorophores are bound is obtained. As in Example 1, the probe that has undergone extension and decomposition is released from the sample DNA and regenerates the original sample DNA 101. Regenerated sample DNA
Binds again with the nucleic acid probe and repeats a series of cycles of elongation and degradation.

In the same manner as in Example 1, 1 × 10 to ¹⁸ mol of the target DNA 101 and 1 × 10 to ¹³ mol of the nucleic acid probe 1 were coexisted with deoxyribonucleotide triphosphates dT and dC and fluorescence labeled dideoxyribonucleotide triphosphate ddG. Below 1 unit of Sequenace and 1 unit of RNase H
Was reacted at 37 ° C for 30 minutes. The reaction solution was heated at 95 ° C. for 2 minutes to stop the reaction, and then separated and analyzed by electrophoresis. A Hitachi WSQ-3000 type DAN sequencer was used for the electrophoresis. The laser used was a 488 nm argon laser.
Fluorescence derived from sulforhodamine 101 was detected using a bandpass filter of 605 nm to 640 nm for detection.

As a result, the fragment decomposed after the extension reaction could be detected separately from the unreacted probe and the fragment decomposed before the extension reaction. When the amount of the decomposed and extended fragment amplified was determined from the fluorescence intensity, it was found that the decomposed and extended fragment was amplified about 100 times the target DNA.

In this example, since the fragments into which the two kinds of fluorescent substances are incorporated are detected by using energy transfer, it is possible to suppress the influence of the electrophoretic separation peaks other than the reaction products. Since there is no need for gel filtration after such a reaction, there is an advantage that detection becomes easier.

(Example 4) As shown in FIG. 5, the fluorescent labeled probe 1 was decomposed and extended by using biotin-conjugated dideoxyribonucleotide triphosphate 408 in the same manner as in the other examples, and biotin was added to the 3'end. A digested extension fragment 418 introduced with is obtained. The reaction solution was transferred to a microplate 411 having streptavidin 410 bound thereto, and
Stir for minutes. In this step, the biotin-introduced degradation extension fragment 409 is captured on the surface of the microplate. After thorough washing, the fluorescence derived from the fluorophore bound to the surface of the microplate is measured. Using this method, 1 × 10 to ¹⁸ mol of target DNA could be measured as in the other examples. Approximately 8 for target DNA from detected fluorescence intensity
It was found that a 0-fold degraded extension fragment was obtained by amplification.

[0048]

EFFECTS OF THE INVENTION A nucleic acid probe for detecting a very small amount of target DNA, which is composed of ribonucleotides and deoxyribonucleotides, is used, and deoxyribonucleotide triphosphates and dideoxynucleotides of different base species from deoxyribonucleotide triphosphates are used. If RNA degrading enzyme and nucleic acid synthesizing enzyme are allowed to react in the presence of triphosphate to extend the 3'end by a certain base length and the ribonucleotide portion is cleaved, it is amplified as a degradable extended fragment generated in a series of reactions. it can. This reaction has the advantage that it can be carried out at a constant temperature. Since the target DNA can be amplified as a degradation extension fragment, the target DNA can be detected with high sensitivity.

In addition, by combining the method of introducing a fluorophore into the nucleic acid probe and the terminal extended with dideoxynucleotide triphosphate, two kinds of fluorophores of different types are introduced and detection is performed by energy transfer. By doing so, the fragment that has been decomposed and extended can be detected more easily. In addition, by introducing biotin into the 3 ′ end of the probe using biotinylated dideoxynucleotide triphosphate, a method that can easily remove the unreacted probe and the like can be used to make the method more suitable for apparatus. it can.

[Brief description of drawings]

FIG. 1 is an explanatory view of a nucleic acid probe.

FIG. 2 is an explanatory diagram of an example of the reaction of the present invention.

FIG. 3 is an explanatory view of a second embodiment of the reaction of the present invention.

FIG. 4 is an explanatory view of a third embodiment of the reaction of the present invention.

FIG. 5 is an explanatory view of a fourth embodiment of the reaction of the present invention.

[Explanation of symbols]

11, 13, 21, 23, 31, 33, 41, 42, 4
4 ... Deoxyribonucleotide moiety, 12, 22, 3
2, 43 ... Ribonucleotide moiety, 14 ... Fluorophore, 34
… Biotin.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroko Furuyama 1-280, Higashi Koigokubo, Kokubunji, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd.

Claims (8)

[Claims] 1. A nucleic acid probe having a sequence complementary to a base sequence at a specific site of a target polynucleotide is composed of ribonucleotides and deoxyribonucleotides, and a label is bound to at least one position of the deoxyribonucleotides. RNA in the presence of at least one kind of deoxyribonucleotide triphosphate and dideoxynucleotide triphosphate having a base species different from that of deoxyribonucleotide triphosphate by binding a nucleic acid probe to a target polynucleotide under hybridization conditions. The method comprises reacting a degrading enzyme with a nucleic acid synthase to extend the 3 ′ end by a certain base length, cleaving the ribonucleotide portion, and detecting the target polynucleotide from the amount of labeled deoxyribonucleotide produced having a certain length. Method for detecting polynucleotide . 2. A nucleic acid probe having a sequence complementary to the base sequence at a specific site of a target polynucleotide is composed of ribonucleotides and deoxyribonucleotides, and the nucleic acid probe is hybridized to the target polynucleotide under conditions for hybridization. After binding, at least one kind of deoxyribonucleotide triphosphate and a labeled dideoxynucleotide triphosphate of a base species different from deoxyribonucleotide triphosphate are allowed to react with an RNA degrading enzyme and a nucleic acid synthase in the presence of a constant 3'end. A method for detecting a polynucleotide, which comprises extending the base length, cleaving the ribonucleotide portion, and detecting the target polynucleotide from the production amount of the 3'-end-labeled deoxyribonucleotide having a constant length. 3. The method for detecting a polynucleotide according to claim 1, wherein the dideoxynucleotide triphosphorus is bound to a label. 4. The process according to claim 1, wherein the 3'-terminal biotinylated deoxyribonucleotide, which has a fixed length and is composed of dideoxynucleotide triphosphorus bound with biotin, is separated using a carrier on which avidin or streptavidin is immobilized. A method for detecting a polynucleotide provided with. 5. The method according to claim 2, wherein the nucleic acid probe is composed of ribonucleotides and deoxyribonucleotides and biotin is bound to at least one position of the deoxyribonucleotides to obtain biotinylated deoxyribonucleotides having a constant length. A method for detecting a polynucleotide, which comprises a step of separating using a carrier on which avidin or streptavidin is immobilized. 6. The method according to claim 1, 2, 3, 4 or 5.
A method for detecting a polynucleotide in which the labeled substance is a fluorophore. 7. The polynucleotide according to claim 3, wherein the labeled product of the nucleic acid probe and the labeled product of labeled dideoxynucleotide triphosphate are different fluorophores, and the two types of fluorophores are polynucleotides that cause energy transfer. Detection method. 8. The deoxyribonucleotide moiety at the 3'-end according to claim 1, 2, 3, 4, 5, 6 or 7,
A method for detecting a polynucleotide having a structure of 1 to 7 bases, a structure having a ribonucleotide moiety at at least one position on the 5'end side thereof, and a base length extending to the 3'end side being 3 to 7 bases.