JPH06189797A - Method for detecting nucleic acid - Google Patents

Abstract

(57) [Summary] [Objective] To provide a simple and highly sensitive method for detecting DNA or RNA. [Structure] A fluorescent-labeled RNA probe 2 having a fluorescent label 10 bound to an end having a sequence complementary to the single-stranded target DNA 1 is mixed with a sample, and hybridized with the target DNA 1. Next, Ribonuclease H is added and reacted. Ribonuclease H selectively decomposes double-stranded RNA of DNA-RNA hybrid 3. RNA probe 2 is again added to the target DNA 1 that has returned to the single-stranded form.
Hybridize, and then Ribonuclease H
Is decomposed and released into the amplified RNA probe small fragment 5 in number. This reaction process is repeated automatically. The reaction is stopped, and the reaction solution is electrophoretically separated to decompose and detect a small fragment of the RNA probe. [Effect] The target DNA can be easily detected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting RNA or DNA and a method for gene diagnosis.

[0002]

DNA for diagnosing infectious diseases caused by viruses and the like.
Alternatively, RNA detection has been used. When the copy number of the virus to be tested is small, it is less than tens of copies. PCR (PCR) (Polymerase Chain Reaction, Polymerase Chain Reaction)
Hoya and NBA (NASBA) (Nucleic and sequence-based amplification, Nucleic and sequence-based amplification
(Nature 350, 91-92 (1991)))
A method of multiplying and detecting a gene by the method is used. The PCR method propagates a specific region of DNA, and the NASBA method propagates a specific region of RNA. For example,
In the PCR method, DNA in a region sandwiched between two types of oligomers that hybridize to the (+) and (−) strands of the target double-stranded DNA is grown. Double-stranded DNA is denatured at high temperature (-90 ° C) and separated into (+) strand and (-) strand. Then, the temperature is lowered (up to 60 ° C), and a DNA oligomer is hybridized to each, and heat-resistant DNA such as Taq.
A complementary strand is synthesized by a polymerase. The temperature is raised again to produce two pairs of (+) chain and (-) chain. 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 thermal cycle increases the average number to about 1.6 times, and if it is repeated 30 times, the copy number can be increased to 10 ⁶ times.

[0003]

In this PCR method, D
The need for expensive thermostable enzymes to grow NA copies,
Need for a device that repeats temperature rise and fall 2 types of DNA
There is a problem in that there is a drawback that an oligomer for proliferation (primer) is required. In particular, depending on the subject, it may not be possible to select two primers well. An object of the present invention is to solve the above problems and to provide a simple and highly sensitive method for detecting DNA or RNA in place of the PCR method and the like, and a gene diagnostic method using this detection method.

[0004]

In order to achieve the above object, in the present invention, an RNA oligomer or a DNA-RNA copolymer labeled with a fluorescent substance or the like is used as a probe and hybridized with a target DNA. Then RNA
The presence of target nucleic acid is detected by allowing a degrading enzyme to act, degrading the DNA-RNA double-stranded portion, and detecting the shortened probe. A probe that has a structure in which the binding portion of DNA and RNA repeats a plurality of times, and has a labeling site that is fluorescently labeled in a DNA portion that repeats a plurality of times, or a probe that binds to the microparticles at at least one end of the probe Target D between labeled labeling sites
It is also possible to use a probe having an RNA strand capable of hybridizing with NA.

[0005]

[Function] RNA probe and RNase (R
Injecting ibonuclease H, etc. causes the RNA probe to become D only when the target (complementary pair) DNA sequence is present.
Form an NA-RNA hybrid. Since RNase cleaves the RNA portion of this hybrid, the RNA probe is shortened and is eventually released from the target DNA strand. Then, unreacted RNA probe is present in the sample solution D
Hybridization with NA and the action of RNase also cause a shortening reaction of the RNA probe. This reaction is 3
When a DNA having a complementary sequence to the RNA probe that can be repeatedly generated at 7 ° C. is present in the sample solution, the number of shortened R is much larger than the number of the DNA.
NA probes can be made. By measuring this shortened RNA probe, it is possible to detect the presence of a very small amount of DNA to be inspected. DNA-RNA
When a copolymer is used as a probe, only the RNA part of the probe is decomposed, and a short DN having a fluorescent label bound thereto is used.
Since the A-oligomer has a small binding force and is detached from the target DNA chain, a large number of short DNA oligomers to which a fluorescent label is bound are released and produced, and the presence of the target DNA can be detected with high sensitivity.

When a probe having a structure in which the binding portion of DNA and RNA is repeated a plurality of times and having a fluorescently labeled labeling site in the DNA portion of a plurality of repetitions is used, the RNA portion of the probe is decomposed and the DNA portion becomes the target DNA chain. It becomes a small fragmented oligomer (probe) hybridized with, but when the length of the DNA part is short, the binding force is not so large and it is detached from the target DNA strand. A plurality of short DNA probes having different lengths are released and generated. Keep the length of the DNA portion of the probe to be the same and release DN
When the A probe is separated and measured by electrophoresis, it can be detected at the same migration distance and highly sensitive. In addition, an RNA probe described above as a probe, a probe composed of a DNA-RNA copolymer, a probe including a structure in which a binding portion of DNA and RNA repeats a plurality of times, and a probe having a labeling site fluorescently labeled in a DNA portion that repeats a plurality of Either one of the probes is bound to the microparticles at at least one end of the probe so that it has an RNA chain capable of hybridizing with the target DNA between the binding site of the nucleic acid probe and the microparticles and the fluorescently labeled labeling site, Probe and target DNA
Form a hybrid with. Since RNase cleaves the RNA portion of this hybrid, magnetic beads can be used as the microparticles, and the microparticles can be easily removed using a magnet after the decomposition reaction, and the portion containing the labeled fluorescent substance generated from the decomposed probe is contained in the container. To remain. The amount of the sample DNA can be easily known by measuring the fluorescence of the labeled fluorescent substance in the portion containing the labeled fluorescent substance.

[0007]

EXAMPLES The present invention will be described with reference to examples. In FIGS. 1 and 3, 3 ′ and 5 ′ indicate the 3 ′ end and 5 ′ end of the nucleic acid molecule chain. In the following examples, as fluorescent labels, sulforhodamine 101, emission wavelength 615n
m), FITC, fluorescein isothiocyanate (fluorescence wavelength 525 nm)
Alternatively, TRITC, Tetra methyl rohodamine isothioc
yanate; emission wavelength 580 nm) or the like can be used, and both are covalently bonded to the RNA molecule via an amino linker. First Embodiment The process of the first embodiment of the present invention is shown in FIG. An RNA oligomer having a sequence complementary to the single-stranded target DNA 1 was prepared, and a fluorescent label 10, sulforhodamine 101 (emission wavelength 615) was prepared at the 5'end thereof.
nm) to produce a fluorescently labeled RNA probe 2 (FIG. 1 (1)). The length of this oligomer is preferably about 20 to 50 mers. The reason for this is that when the length of the oligomer becomes shorter, the binding force in hybridization becomes weaker, and when the length of the oligomer becomes longer, a non-specific reaction occurs because the preparation takes time and costs, which is not preferable. is there. Sample and fluorescent labeled RNA probe 2, 0.1pm
The ole is mixed at 37 ° C. and hybridized with the target DNA 1 (FIG. 1 (2)). Then, 1 unit of Ribonuclease H is added and the reaction is carried out for 20 minutes. Ribonuclease H is a double-stranded RN of DNA-RNA hybrid 3
Single-stranded RNA, DNA and double-stranded DN that degrade A
Since A is not degraded, only hybridized RNA is selectively degraded (FIG. 1 (3)). When single-stranded target DNA 1 is present in the sample, RNA probe 2 hybridizes to it and is decomposed by Ribonuclease H to be released as small fragment 4 (FIG. 1 (4)). (When the target is double-stranded DNA, it is separated into two single strands in advance and then hybridized with an RNA probe, or exonuclease is used to decompose from the ends of the double strands to form single strands. RNA probe 2 is hybridized again to the target DNA 1 which has returned to the single-stranded form, and then similarly degraded by Ribonuclease H,
The number becomes the amplified RNA probe small fragment 5 and is released. This reaction process is repeated automatically and the number of repeated small fragments of RNA probe is increased (n). After reacting for 20 minutes, the reaction solution is heated to 90 ° C. to thermally decompose and deactivate the enzyme to stop the reaction. The reaction solution is electrophoretically separated using polyacrylamide gel (6 w / v%). The migration path length is preferably 10 cm to 30 cm, but 15 cm. The lower part of the migration path is 594 nm H
Irradiation with an e-Ne laser is performed, and the fluorescence emitted from the passing RNA fragment is detected. The migration path may be either a capillary or a flat plate. The reaction solution contains undegraded RNA probe and degraded RNA probe, and in most cases, the degradation product is 5 mer or less. Since the migration speed of the RNA fragment differs depending on the length, the undecomposed probe and the decomposed probe are detected by separating the undecomposed RNA probe 22 and the RNA fragment 21 which is decomposed into small fragments, as shown in FIG. The presence of the target DNA 1 can be confirmed by detecting the degraded RNA probe 21. Furthermore, it goes without saying that the presence of the target DNA 1 can be quantitatively determined by a quantification method using a normal calibration curve.

Second Example Although the fluorescence labeled RNA probe was used in the first example, a DNA-RNA copolymer in which a DNA chain and an RNA chain are linked in a chain can be used as the probe.
That is, 4 with a fluorescent label bound at or near the end
Alternatively, a fluorescence-labeled DNA-RNA copolymer probe in which a 5-mer DNA oligomer and a 20-50-mer RNA oligomer are bound is prepared. The base sequence is complementary to the target DNA. The fluorescence-labeled DNA-RNA copolymer probe hybridized with the target DNA is decomposed with Ribomuclease H. This enzyme does not decompose the DNA-DNA double strand, but only the DNA-RNA double strand, so that only the RNA part of the fluorescently labeled DNA-RNA copolymer probe is decomposed and the fluorescently labeled short DNA oligomer. Is 4 ~
Since it is a 5-mer, the binding power is not so large and the sample (target) D
Detach from NA. Thereafter, similar to the first embodiment, a large number of short DNA oligomers to which a fluorescent label is bound are released and produced, and the presence of the target DNA can be detected with high sensitivity. In this example, since a fluorescent label-bonded 4-5-mer DNA oligomer and a 20-50-mer RNA oligomer-bound fluorescent-labeled DNA-RNA copolymer probe are used, the degradation products are fluorescent-labeled DNA. The lengths of the parts are standardized, and when separated and measured by electrophoresis, fluorescently labeled DNA is detected at the same migration distance, which is convenient because it can be detected with high sensitivity. A probe having a structure in which the structure of DNA-RNA has a plurality of repeating structures may be used, which will be described below.

Third Example This is an example of using a probe composed of a fluorescently labeled DNA-RNA block copolymer having the same partial structure and repeating DNA-RNA structure. FIG. 3 shows the fluorescent labeled DNA-RNA block copolymer probe 31 and target D.
The outline of the reaction process with NA1 is shown. Fluorescent labeled DN
The DNA portion 32 of the A-RNA block copolymer probe 31 is fluorescently labeled with the fluorescent substance 10, and the DNA portion 32 is
Is composed of 7-mers, and the phosphor 10 is attached to the central part. R
The NA portion 33 is composed of 5 mers and has a total length of 65 mers (FIG. 3 (1)). Of course, the number and length of the DNA portion 32,
The number and length of the RNA portion 33 may be changed. When the fluorescent-labeled DNA-RNA block copolymer probe 31 is added to the sample, it hybridizes with the complementary portion in the strand of the target DNA 1. Reference numeral 34 represents a hybridized fluorescently labeled DNA-RNA block copolymer probe (FIG. 3 (2)). That is, this hybridoma is DNA
-DNA and a DNA-RNA hybrid part, the RNA part of the probe is decomposed by adding Ribonucoease H, and the DNA part 32 becomes the target DNA.
This results in a small fragmented oligomer (probe) 35 hybridized to 1 (FIG. 3 (3)). In the example of FIG. 3, when the DNA portion 32 is a 7-mer, the binding force is not so large and it is detached from the target DNA 1. Therefore, five fluorescent-labeled DNA-RNA block copolymer probes are used to bind five fluorescent-labeled DNA-RNA block copolymer probes. The DNA probe 36 is released and generated (FIG. 3 (4)). On the other hand, fluorescent labeled DNA
-When the DNA portion 32 in the RNA block copolymer probe 31 is long, the labeled DNA probe generated by the decomposition of the probe 31 does not detach from the target DNA 1 and RNA
Only part 33 is completely disassembled. In the example of FIG. 3, the fluorescence-labeled DNA-RNA block copolymer probe 31 was hybridized to the target DNA 1 and then Ribonuc was used.
The number of short labeled DNA probes 36 increases with time as the process of being degraded again by oease H is repeated.
Since the fluorescently labeled DNA portion 32 is a 7-mer, D
The NA probe has a uniform length, and its copy number is 5 times the copy number of the target DNA1. Repeating the above process n times will yield 5n copies. In this case, a highly sensitive fluorescence detector (<10 ^-21 mol / ban
It is possible to detect hundreds of copies of the sample DNA. Furthermore, the fluorescence-labeled DNA of this example
-Using an RNA block copolymer probe, mutations of 1-2 bases in the DNA chain can be investigated. Fluorescently labeled DNA-RNA block copolymer probe and target DNA
If a non-complementary portion is present when a hybridoma of (1) is prepared, the binding strength of the degraded fragment portion remaining after being digested by Ribonucoease H, including this non-complementary portion, weakens, and is released as a long fragment. Therefore, by investigating this long fragment, information about the mutated portion can be obtained and can be applied to a gene diagnostic method.

Fourth Embodiment This embodiment is an example of using a probe having a microparticle or a macromolecule such as a protein at at least one end. Examples of fine particles include magnetic beads, polystyrene particles, latex particles, etc., and examples of proteins include:
Streptavidin, adibin and the like and polymers thereof can be used. The probe itself may be any type of the fluorescent-labeled RNA probe used in the first example, the probe used in the second example, and the probe used in the third example. Macromolecules such as proteins are bound. When the probe used in the second embodiment and the probe type used in the third embodiment are used, the RNA portion is used as an end and a macromolecule such as a fine particle or a protein is bound to this end. In such a probe, Ribonuclease H separates the part to which the microparticle (or protein) is bound from the part to which the fluorophore is bound. The probe hybridized to the sample DNA is decomposed by Ribonuclease H, and the portion containing the labeled fluorophore is released from the fine particles. When the fine particles are magnetic beads, the fine particles can be removed using a magnet after the decomposition reaction, and the portion containing the labeled fluorescent substance generated from the decomposed probe remains in the container. The sample DN can be easily measured by measuring the fluorescence of the labeled fluorescent substance in the portion containing the labeled fluorescent substance.
A quantity can be known. When the type of probe used in the second example and the type of probe used in the third example are used, the degradation products of Ribonuclease H are uniform in the length of the fluorescently labeled DNA portion, and Highly sensitive detection is possible by separation measurement by electrophoresis. When organic beads or proteins are used as the fine particles, a molecular sieve film or gel is used to separate and measure only the free-label fluorescent substance.

The embodiment of the present invention has been described above.
Furthermore, in order to change the probe fragment length after digestion with Ribonuclease H for each target, DN was used in the above examples.
By designing the probe by changing the length of the A portion for each target, it is easy to detect many kinds of different target DNAs. Also, by changing the type of fluorescent label for each target,
Even if a probe is designed, various kinds of different target DNAs can be easily detected.

[0012]

EFFECTS OF THE INVENTION In the detection of a very small amount of target DNA, it is necessary to detect the nucleic acid probe hybridized to it with high sensitivity. A labeled probe containing RNA was labeled with DNA-
RNA double-stranded (hybridomer) degrading enzyme (Ribonucle
The reaction of degrading only the probe that formed a hybridoma with the target DNA and changing the probe length can be regarded as a kind of catalytic reaction using the target DNA as a catalyst, and is irreversible with the reaction time. The decomposition of the probe proceeds. Therefore, even if only a very small amount of target DNA is present, a large amount of a decomposed product of the probe can be obtained, and highly sensitive detection is possible. The decomposition probe and the non-decomposition probe are distinguished by the number of nucleic acids constituting the probe, but it can be easily performed by using gel electrophoresis. By attaching fine particles or magnetic beads to one end of the probe, separation of decomposed products and undecomposed products can be made easier and simpler. The method according to the present invention is different from the conventional method such as PCR,
This can be performed simply by injecting the probe and the enzyme into the sample, which is highly effective in simplifying the device.

[Brief description of drawings]

FIG. 1 is a diagram showing a reaction process of a first embodiment of the present invention.

FIG. 2 is a view showing an electrophoresis result of a reaction solution in the reaction process of the first example of the present invention.

FIG. 3 is a diagram showing a reaction process of a third embodiment of the present invention.

[Explanation of symbols]

1 ... Single-stranded target DNA, 2 ... Fluorescently labeled RNA probe, 3 ... R hybridized to target DNA
NA probe, 4 ... R fragmented by RNase H and fragmented
NA probe, 5 ... Small fragment of RNA probe amplified by free amplification, 10 ... Fluorescent label, 21 ... Degraded and fragmented RN
A probe, 22 ... Undegraded RNA probe, 31 ... Fluorescently labeled DNA-RNA block copolymer probe, 32 ...
DNA portion, 33 ... RNA portion, 34 ... Fluorescence-labeled DNA-RNA block copolymer probe bound to target DNA, 35 ... Probe in which RNA portion is decomposed into small fragments, 36 ... Released probe.

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

Claims (21)

[Claims] 1. A method of detecting a target nucleic acid by decomposing a nucleic acid probe hybridized with a sample using an RNA degrading enzyme and detecting a decomposition product in the method of detecting a nucleic acid using a nucleic acid probe. 2. The method for detecting a nucleic acid according to claim 1, wherein
A method for detecting a nucleic acid, wherein the RNA degrading enzyme has a property of degrading an RNA portion of a DNA-RNA hybridomer. 3. The method for detecting a nucleic acid according to claim 1,
A method for detecting nucleic acid, wherein the nucleic acid probe has a labeling site containing a fluorophore or a dye. 4. The method for detecting a nucleic acid according to claim 3,
A method for detecting a nucleic acid, wherein the nucleic acid probe comprises RNA. 5. The method for detecting a nucleic acid according to claim 3,
A method for detecting a nucleic acid, wherein the nucleic acid probe is made of a copolymer of DNA and RNA and has the labeling site at the DNA portion. 6. The method for detecting a nucleic acid according to claim 3,
The method for detecting a nucleic acid, wherein the nucleic acid probe has a structure in which a binding portion of DNA and RNA repeats a plurality of times, and has the labeling site in a DNA portion that repeats a plurality of times. 7. The method for detecting a nucleic acid according to any one of claims 4 to 6, wherein the nucleic acid probe is bound to the microparticles at at least one end, and the binding site between the nucleic acid probe and the microparticles is A method for detecting a nucleic acid, wherein the nucleic acid probe has an RNA chain capable of hybridizing with the target DNA between the labeling sites. 8. The method for detecting a nucleic acid according to claim 3,
A method for detecting a nucleic acid, wherein the nucleic acid probe comprises RNA having 20 to 50 bases. 9. The method for detecting a nucleic acid according to claim 3,
The nucleic acid probe contains at least one DNA portion consisting of 4 to 7 bases and RNA consisting of 20 to 50 bases
A method for detecting a nucleic acid, which comprises the above-mentioned copolymer and has the labeling site in the DNA portion. 10. The method for detecting a nucleic acid according to claim 3, wherein the nucleic acid probe contains at least one DNA portion consisting of 4 to 7 bases, and has a structure in which a binding portion of DNA and RNA is repeated a plurality of times and DNA is repeated a plurality of times. A method for detecting a nucleic acid, characterized in that the portion has the labeling site. 11. The method for detecting a nucleic acid according to any one of claims 8 to 10, wherein the nucleic acid probe is bound to the fine particles at at least one end, and the binding site between the nucleic acid probe and the fine particles is A method for detecting a nucleic acid, wherein the nucleic acid probe has an RNA chain capable of hybridizing with the target DNA between the labeling sites. 12. A nucleic acid probe used in the method for detecting a nucleic acid according to claim 1, which comprises a copolymer of DNA and RNA or RNA. 13. A nucleic acid probe used in the method for detecting a nucleic acid according to claim 3, which is composed of a copolymer of DNA and RNA and has the labeling site at the DNA portion. 14. A nucleic acid probe used in the method for detecting a nucleic acid according to claim 3, wherein the binding portion of DNA and RNA includes a structure in which a plurality of repeating portions have a repeating structure, and the repeating DNA portion has the labeling site. Nucleic acid probe. 15. The nucleic acid probe according to any one of claims 12 to 14, wherein the nucleic acid probe is bound to the fine particles at at least one end, and the nucleic acid probe, the binding site of the fine particles, and the label. A nucleic acid probe having an RNA chain capable of hybridizing with the target DNA between the sites. 16. The nucleic acid probe used in the method for detecting a nucleic acid according to claim 3, wherein the nucleic acid probe is 4 to 7.
A nucleic acid probe comprising a copolymer of DNA consisting of bases and RNA consisting of 20 to 50 bases, and having the labeling site in the DNA portion. 17. The nucleic acid probe used in the method for detecting a nucleic acid according to claim 3, wherein the nucleic acid probe is 4 to 7.
A nucleic acid probe comprising a structure in which a DNA-RNA binding portion consisting of a base is repeated a plurality of times, and having the labeling site in the DNA portion repeated a plurality of times. 18. The nucleic acid probe according to claim 16 or 17, wherein the nucleic acid probe is bound to the microparticles at at least one end, and the nucleic acid probe, the binding site between the microparticles and the label. A nucleic acid probe having an RNA chain capable of hybridizing with the target DNA between the sites. 19. The method for detecting a nucleic acid according to claim 1, wherein the decomposition product is detected by electrophoresis. 20. An RNA having a property of degrading an RNA portion of a DNA-RNA hybridomer of a nucleic acid probe hybridized with a sample in a nucleic acid detection method using a nucleic acid probe having a labeling site containing a fluorophore or a dye.
A method for detecting a nucleic acid, wherein a target nucleic acid is detected by degrading a nucleic acid probe hybridized with a sample using a degrading enzyme and detecting a degradation product. 21. A nucleic acid detecting method using a nucleic acid probe having a labeling site containing a fluorophore or a dye, wherein the RNA has a property of degrading the RNA portion of the DNA-RNA hybridomer of the nucleic acid probe hybridized with the sample.
A nucleic acid detection method for detecting a target nucleic acid by degrading a nucleic acid probe hybridized with a sample using a degrading enzyme and detecting a degradation product by electrophoresis.