JPH10290692A - New amplification of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To amplify a nucleic acid by binding a primer to a template RNA in the presence of a deoxynucleoside triphosphate, etc., forming a cDNA in the form of a hybrid with a reverse transcriptase, then separating the resultant cDNA and repeating the operations. SOLUTION: A primer is annealed to a template RNA in a buffer solution containing a deoxynucleoside triphosphate, a dideoxynucleoside triphosphate, a derivative thereof and/or a modification thereof, the template RNA, the primer bindable to a specific site of the template RNA and/or the primer bindable to an unspecific site of the template RNA and a reverse transcriptase to form a cDNA for the template RNA with the reverse transcriptase. The resultant RNA/DNA hybrid is then separated into the RNA and cDNA. The separated RNA is used as the template RNA and the above steps are repeated at least once to thereby amplify the RNA as the cDNA. Thereby, the nucleic acid useful for research on the nucleic acid, clinical tests, food tests, environmental tests, etc., is amplified with a high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for converting specific RNA into cD
The present invention relates to a novel nucleic acid amplification method aimed at amplifying as NA, and more particularly to a method of cycle-synthesizing cDNA from specific RNA. INDUSTRIAL APPLICABILITY The present invention enables use in a wide range of academic and industrial fields, such as nucleic acid research, clinical testing, food testing, and environmental testing, particularly using a highly sensitive and reproducible reverse transcription reaction.

[0002]

2. Description of the Related Art When a specific RNA in a sample is detected, sequenced or isolated, a reverse transcriptase reaction is used.
It is widely used to utilize cDNA synthesized from RNA. A conventional method of synthesizing cDNA from RNA is to add a template RNA, a reverse transcriptase, a buffer, four kinds of deoxynucleoside triphosphates (also referred to as deoxynucleotides) and a primer, and then incubate at a constant temperature. Had been taken.

[0003] In such a cDNA synthesis reaction, M-MLV (mouse leukemia virus) or AM is used as a reverse transcriptase.
V (avian granuloma virus), other viruses, and reverse transcriptases derived from microorganisms are widely used. In addition, thermostable DNA polymerase (for example, Tth DNA polymerase, Taq DNA polymerase, Stoffe)
RNA fragment using the reverse transcriptase activity of
A method of synthesizing a cDNA using a DNA as a template has also been developed (Japanese Patent Application Laid-Open No. 5-505105).

[0004] However, in the conventional cDNA synthesis reaction, 100% of the abundance of the template RNA is not always converted into cDNA. Sambrook, Fritz, Maniatis, Ed., Molecular Cloning
), Cold Spring Harbor Laboratories, 1982, p. 233.
Regarding the rate of reverse transcription to DNA, when AMV-derived reverse transcriptase is used, only about 10 to 30% of template RNA is converted to cDNA. Further, the present inventors have determined that Tth DNA
When polymerase is used, template RNA is 1 to 3%
It was found that only a small amount was converted to cDNA. Therefore, the ability to detect, analyze, or isolate RNA that originally has a low abundance using a normal reverse transcription reaction has a limited sensitivity.

Therefore, as one means for detecting, analyzing or isolating trace RNA, specific RNA is reverse transcribed (reaction is performed at a constant temperature as described above), and the obtained cDNA is subjected to PCR (polymerase).・ Amplification method by chain reaction) has been used. A reaction system in which such a reverse transcription reaction and PCR are coupled is called RT-
PCR (Reversetranscription-Polymerasse Chain Reac
Option).

However, PCR is basically performed using DNA as a template (Japanese Patent Publication No. 4-67957,
4-67960, JP-A-5-308971, JP-A-6-2925
No. 79, etc.) cannot be applied to a reaction for synthesizing cDNA using RNA as a template.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to increase the efficiency and reproducibility of a generally performed reverse transcription reaction. Is to provide a method for increasing the amount of cDNA to be generated by repeatedly performing the above-mentioned steps.

[0008]

Means for Solving the Problems The present inventors have focused on the stability of RNA, and have studied RNA-generated RNA by a reverse transcription reaction.
R isolated from the cDNA hybrid by thermal denaturation
The inventors have found that NA can be used again as a template for a reverse transcription reaction, and have reached the present invention.

That is, the present invention relates to (1) deoxynucleoside triphosphate, dideoxynucleoside triphosphate, a derivative and / or a modified product thereof, (2) a template RNA, and (3) a specific site of the template RNA. Primer and / or template RNA
(A) annealing the primer (3) to the template RNA (2) in a buffer solution containing a primer capable of binding to the unspecified site and (4) reverse transcriptase; ) Forms cDNA on template RNA (2) at a temperature sufficient to express reverse transcriptase activity to obtain an RNA / DNA hybrid. (C) The RN
A / DNA hybrid is separated into RNA and cDNA, and (d) cDNA obtained by repeating the above steps (a), (b) and (c) at least once using the obtained RNA as template RNA (2) This is a novel nucleic acid amplification method characterized by amplifying.

The present invention also relates to a cD amplified by the above method.
A novel nucleic acid amplification method characterized by further amplifying cDNA by performing polymerase chain reaction (PCR) using NA as a template.

[0011] Further, the present invention provides a method in which a detection probe is reacted with cDNA amplified by the above-described method from RNA in a sample, and the detection probe hybridized or non-hybridized detection probe is measured. RNA characterized by detecting RNA
It is a detection method.

The present invention is characterized in that the RNA in the sample is subjected to sequence analysis by analyzing the sequence of the cDNA amplified by the above method from the RNA in the sample.
A sequence analysis method.

[0013] The present invention is characterized in that a cDNA amplified by the above method is inserted from a RNA in a sample into a cloning vector, introduced into a competent cell, and screened to obtain a cDNA. This is a method for isolating RNA.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, the term "deoxynucleoside triphosphate" refers to deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, thymidine triphosphate, deoxyinosine triphosphate, deoxyuridine triphosphate and the like.

In the present invention, dideoxynucleoside triphosphate refers to dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxycytidine triphosphate, deoxythymidine triphosphate and the like.

In the present invention, these derivatives are functionally or structurally similar. For example,
Nucleoside triphosphates such as ATP, CTP, GTP, TTP, UTP, ITP, and G (5 ′) p
Cap structure analogs such as pp (5 ') A,
2'-deoxyguanosine 5'-O- (1-thiotriphosphate) (dGTP-α-S), adenosine 5'-
O- (3-thiotriphosphate) (ATG-γ-S)
Etc. are included. Furthermore, these also include those which are radioactively labeled. For example, adenosine 5 '-[γ- ³²
P] triphosphate and the like. Labeled deoxycytidine 5 '-[α- ³² P] triphosphate is also exemplified.

[0017] These modified products are those in which a part of a nucleic acid is chemically modified. For example, 7-deaza-deoxynucleoside triphosphate, biotinylated deoxynucleoside triphosphate, biotinylated dideoxynucleoside triphosphate and the like are included.

In the present invention, deoxynucleoside triphosphate, dideoxynucleoside triphosphate, a derivative thereof or a modified product thereof may be used alone or in combination.

In the present invention, (2) the template RNA may be any of messenger RNA, ribosomal RNA and transfer RNA, and may be total RNA in which these are mixed. RNA derived from a virus or RNA derived from a phage may be used. Furthermore, RNA synthesized enzymatically or chemically may be used. The concentration of the template RNA is not particularly limited.

In the present invention, (3) a primer capable of binding to a specific site of the template RNA includes a primer completely complementary to the sequence of the specific site of the template RNA,
And primers that are not necessarily completely complementary (substantially complementary) to the specific site sequence. The number of oligonucleotides or similar oligonucleotides in the primer is generally 16-30.

In the present invention, a primer that is not completely complementary (substantially complementary) refers to at least a part of the base sequence other than the 3 'end of the primer (generally, one or more bases, the number of primer bases minus 16 bases). Is appropriate) is not complementary. Also, similar nucleotides are
For example, peptide-nucleic acid (DNA)
Is bound to the backbone of N- (2-aminoethyl) glycine and binds specifically to the DNA sequence).

Further, the primer capable of binding to a specific site of the template RNA includes both those containing an appropriate analog at the end or in the sequence and those not containing the appropriate analog.

Examples of the primer containing an appropriate analog at the end or in the sequence include a primer labeled with a radioisotope, a primer labeled with biotin, and a primer labeled with a fluorescent substance. Further, these primers include not only primers composed of oligonucleotides, but also primers formed by analogs of oligonucleotides.

In the present invention, the primer that binds to an unspecified site of the template RNA refers to a random primer, and it is preferable that the 5 ′ end is phosphorylated. In addition, both those containing the appropriate analog at the terminal or in the sequence and those not containing the analog are included. Furthermore, not only primers composed of oligonucleotides but also primers formed by analogs of oligonucleotides are included.

The oligonucleotide analog (similar nucleotide) includes, for example, peptide nucleic acid.

In the present invention, (4) the reverse transcriptase is generally used from the viewpoint of cDNA synthesis efficiency, for example, M-MLV (mouse leukemia virus) -derived reverse transcriptase and AMV (bird). A reverse transcriptase derived from various other viruses and microorganisms such as a reverse transcriptase derived from granuloma virus) can be widely used. In addition, thermal stability DN
A polymerase (eg, Tth DNA polymerase,
The reverse transcriptase activity of Taq DNA polymerase (Stoffel fragment) can also be used. Furthermore, the simultaneous use of a plurality of reverse transcriptases is also included. Furthermore, as long as the enzyme group does not hinder the cDNA synthesis reaction (= reverse transcription reaction), a reverse transcriptase and another type of enzyme may be used simultaneously.

In the present invention, (5) the buffer solution may be any one having a composition capable of expressing reverse transcriptase activity.
Although not particularly limited, it is preferable that the composition be such that the activity as a reverse transcriptase can be sufficiently exhibited. In the case of M-MLV-derived reverse transcriptase, an example of a preferred composition is 50 mM Tris / HCl (pH 8.3) 75 mM KCl 3 mM MgCl ₂ 10 mM DTT.

It is preferable that the reagents used in the present invention have high purity with no or almost no ribonuclease (RNase) contamination.

In the present invention, the following steps (a) to (c) are performed to amplify cDNA. (A) annealing the primer (3) to the template RNA (2); (b) reacting the template RNA (2) with the template RNA (2) at a temperature sufficient for the reverse transcriptase (4) to exhibit reverse transcriptase activity. The cDNA is formed to obtain an RNA / DNA hybrid, (c) the RNA / DNA hybrid is separated into RNA and cDNA, and (d) the obtained RNA is used again as a template RNA (2). a),
Repeat (b) and (c) at least once.

The conditions suitable for annealing are as follows:
And the temperature at which the primer binds at the desired site. The liquid property can be generally used as long as the composition can sufficiently express the activity of the reverse transcriptase to be used. Further, the temperature may be equal to or lower than the dissociation temperature (Tm) of the primer. Preferably, Tm-5 (° C) is appropriate.

The conditions for obtaining an RNA / DNA hybrid are as follows: in the state where the primer is bound to the template RNA, the reverse transcriptase sufficiently expresses its activity, and
A liquid and temperature at which A can be synthesized. The liquid property can be used as long as the composition can sufficiently express the activity of the reverse transcriptase used. Further, the temperature is a temperature at which the activity of the reverse transcriptase can be sufficiently expressed. Generally, M-MLV
In the case of reverse transcriptase derived from AMV,
7-42 ° C is preferred.

The means for separating the RNA / DNA hybrid is a condition under which the hydrogen bond between the RNA / DNA hybrids is broken, and includes thermal, chemical or enzymatic means. As a thermal means, a typical heating temperature is 80-9.
5 ° C. is appropriate. Chemically, raising the salt concentration in the liquid can be mentioned. Enzymatically, helicase-like enzymes are used.

As one embodiment of the present invention, (1) deoxynucleoside triphosphate, dideoxynucleoside triphosphate, derivatives and / or modified products thereof, (2) template RNA, (3) template RN
A primer capable of binding to a specific site of A and / or a primer capable of binding to an unspecified site of template RNA and (4) reverse transcriptase are mixed in (5) buffer solution,
The primer is annealed to the template RNA at, for example, 37 ° C. for 30 seconds, and then reacted at a temperature condition sufficient for the reverse transcriptase to exhibit the activity, for example, at 37 ° C. for 5 to 60 minutes. Enzyme cDNA to template RNA
A, cDNA synthesized with template RNA strand
The temperature is raised to a temperature sufficient to dissociate the RNA strand from the hybrid molecule consisting of strands, for example, 94 ° C. for 30 seconds,
It is separated into a cDNA strand and an RNA strand, and the temperature is again lowered to a temperature at which the RNA and the primer can be annealed, for example, at 37 ° C. for 30 seconds.
Reaction method that repeats cDNA synthesis in seconds (see Fig. 1)
There is.

In the present invention, the repetition (cycle) frequency of the steps (a) to (c) is suitably several times to several tens. Furthermore, although it depends on the amount of cDNA to be obtained and the purpose, when M-MLV-derived reverse transcriptase or AMV-derived reverse transcriptase is used, several to ten and several cycles are usually sufficient.

When the number of cycles is increased,
Because efficient cDNA synthesis can be performed compared to conventional cDNA synthesis reactions, a limited amount of RNA can be used effectively, and a small amount of RNA can be detected, analyzed, and isolated. It becomes possible.

In another embodiment of the present invention, a novel cDNA characterized by further amplifying the cDNA by performing polymerase chain reaction (PCR) using the cDNA amplified by the above method as a template. This is a nucleic acid amplification method. Additional amplifying means include other amplifying means such as ligase chain.
Reaction (LCR), isothermal transfer replication (TAM),
(NASBA) can also be used.

All that is required to perform the PCR is at least two primers, a thermostable DNA polymerase and a buffer. Here, the two primers are substantially complementary to each of the two strands of the specific cDNA, and the extension product of one pramer serves as a template for the other primer. Examples of the thermostable DNA polymerase include Taq DNA polymerase and Tth.
DNA polymerase, KOD DNA polymerase, P
Fu DNA polymerase, Vent DNA polymerase and the like are exemplified.

As the PCR conditions, (1) 90 to 90
(2) at a temperature not higher than the Tm of the primer for 30 seconds to 2 minutes, (3) 60 to 75 ° C.,
The reaction is performed for 30 seconds to 3 minutes, and the cycle of (1) to (3) is repeated 12 to 50 times. In addition, when the temperature of (3) is substantially equal to or lower than the Tm of the primer,
Step (2) can be omitted. The buffer used for PCR is selected according to the DNA polymerase used. The other components used are also selected depending on the DNA polymerase used.

[0039] Another embodiment of the present invention provides a method for preparing RNA in a sample.
Detecting a RNA in a sample by reacting a detection probe with the cDNA amplified by the above method, and measuring a detection probe hybridized to the cDNA or a non-hybridization detection probe. It is. A detection probe is a specific RNA
An oligonucleotide having a sequence homologous to the sequence (or a complementary sequence), which binds a labeling substance. Labeling substances include radioactive substances, enzymes, fluorescent substances, biotin and the like.

In the present invention, it is possible to detect RNA in a sample without using a detection probe. The simplest detection means is a detection method using ethidium bromide which is a nucleic acid intercalator. Sample R
When the cDNA amplified from NA by the nucleic acid amplification method of the present invention is electrophoresed on an agarose gel and the gel is irradiated with ultraviolet light in the presence of ethidium bromide, an amplified cDNA fragment derived from a specific RNA can be detected as a band.

[0041] Another embodiment of the present invention provides a method for preparing RNA in a sample.
And analyzing the sequence of the cDNA amplified by the above method.
Here, the type of RNA to be sequenced is not limited. Specifically, there are two methods, one of which is a method of using a labeled analog when amplifying as a cDNA from RNA in a sample by the above method. That is, cDN amplified by incorporating the labeled analog
A is a method of directly sequencing A. Here, examples of the labeled analog include a primer labeled with a labeling substance, deoxynucleoside triphosphate, dideoxynucleoside triphosphate, and the like. Labeling substances include radioactive substances, enzymes, fluorescent substances, biotin and the like.

Another means is to convert RNA in a sample into c
This is a method of performing sequence analysis by combining the above method of amplifying as DNA with the Sanger method or the Maxam-Gilbert method. For example, in the case of the Sanger method, the amplified cDNA, a primer capable of binding to a specific site of the cDNA, deoxynucleoside triphosphate, and dideoxynucleoside triphosphate are mixed in a buffer, and first, the primer is annealed to the cDNA. Then, a DNA polymerase is added, and a reaction is performed for a certain period of time under a temperature condition sufficient to express the activity, thereby performing an extension reaction. Here, any of the primer, deoxynucleoside triphosphate and dideoxynucleoside triphosphate needs to be labeled with a labeling substance. Labeling substances include radioactive substances, enzymes, fluorescent substances, biotin and the like.

The thus prepared sample to be sequenced was subjected to a heat denaturation treatment and a quenching treatment, if necessary, by adding a reaction stop solution containing formamide (including EDTA, BPB, XC, etc.). Thereafter, electrophoresis is performed on an acrylamide gel (sequencing gel) containing urea. The electrophoresed gel is subjected to a deurea treatment as necessary, developed on a filter paper and dried, and then contacted with an X-ray film or the like to take an autoradiogram. By sequentially reading the bands on the obtained autoradiogram, the sequence of the target RNA can be analyzed.

DNA sequencing required for sequencing
Kits include Sequencing PRO, Se
quenching High Plus (Toyobo)
and so on.

[0045] Another embodiment of the present invention provides a method for preparing RNA in a sample.
Thus, a method for isolating RNA in a sample, comprising inserting the cDNA amplified by the above method into a cloning vector, introducing the cDNA into competent cells, and screening to isolate the cDNA. Specifically, first, a portion containing the target gene is cut out from the amplified cDNA using an appropriate restriction enzyme, and inserted into a cloning vector (such as pBluescriptII; sold by Stratagene, etc.). Next, by introducing the integrated cloning vector into competent cells, a recombinant bacterium retaining the target gene fragment can be produced.

Further, only a recombinant bacterium having the target gene is selected using an appropriate selection marker.
If this recombinant bacterium is cultured and then prepared as a plasmid, a plasmid containing the target gene fragment can be obtained. Here, a suitable selection marker is, for example, an ampicillin resistance gene and a lacZ gene. As an example, pBluescriptII in which these genes are encoded on a cloning vector, and competent cells E. coli. In the case of using E. coli DH5α, after selecting a transformant in an ampicillin-containing medium, a transformant retaining the target gene can be further selected based on the deficiency of the function of the lacZ gene on the cloning site. The amplification method of the present invention has a good cDNA acquisition rate, and is also effective for cloning trace genes.

Reagents required for cloning include restriction enzymes, DNA ligase, cloning vectors,
For example, pBluescriptII (manufactured by Stratagene), restriction enzymes, competent cells, for example, Competent high E.coli
DH5α (manufactured by Toyobo) and the like.

[0048]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. Example 1 RNA of MS2 phage (abbreviated as MS2 RNA; Boehringer Mannheim 165948) as a template
Using a specific site-binding antisense primer (SEQ ID NO: 1, see FIG. 2) and M-MLV-derived reverse transcriptase (RN
The heat cycle reaction was carried out 1, 2, 5 and 8 times using aseH ⁻ , Toyobo Co., Ltd. RTM-101) with the following reaction solution composition.

Reaction solution composition: 50 mM Tris · Cl (pH 8.3) 75 mM KCl 3 mM MgCl ₂ 10 mM DTT 1 mM 4 kinds of deoxynucleoside triphosphates 100 pmoles Specific site-binding antisense primer (SEQ ID NO: 1) 20 U RNase Inhibitor 4 μg MS2 RNA 200 U M-MLV-derived reverse transcriptase (RNaseH ⁻ ) Total reaction volume: 50 μl

Reaction conditions: 94 ° C., 30 seconds ↓ 37 ° C., 30 seconds ↓ (add 200 UM MLV-derived reverse transcriptase (RNaseH ⁻ )) 37 ° C., 5 minutes

The reverse transcriptase derived from M-MLV was heated at 94 ° C.
Since it was deactivated by heating, it was added every cycle when the temperature was lowered to 37 ° C. As a control, a sample that had been reacted at 37 ° C. for 1 hour was prepared as in the conventional method. Further, M-ML is obtained by heat denaturation (94 ° C., 30 seconds) during the cycle cDNA synthesis reaction.
In order to confirm that V-derived reverse transcriptase was inactivated and did not contribute to the next cycle reaction, the reaction system to which M-MLV-derived reverse transcriptase was added was first heat-treated at 94 ° C. for 30 seconds. After the reaction, a sample in which the reaction was performed at 37 ° C. for 1 hour was also prepared.

When the sample thus obtained was subjected to RNase A treatment (for the purpose of intentionally decomposing the template RNA and observing only the generated cDNA), the RNase A treatment was not carried out. In some cases, electrophoresis was performed using a 1% agarose gel (using a TBE buffer). The result is shown in FIG.

FIG. 3A shows an electrophoresis image of a sample not subjected to the RNase A treatment, and FIG. 3B shows an electrophoresis image of the sample subjected to the RNase A treatment. In each lane, A represents a sample obtained by heat denaturation at 94 ° C. for 30 seconds and then incubated at 37 ° C. for 1 hour, B represents a sample subjected to incubation at 37 ° C. for 1 hour, and lane M represents a size marker (λ / λ). HindIII), C is cycle cDNA
The sample subjected to the synthesis reaction (one cycle) and the sample D subjected to the cycle cDNA synthesis reaction (cycle 2)
E) shows a sample subjected to a cycle cDNA synthesis reaction (5 cycles), and F shows a sample subjected to a cycle cDNA synthesis reaction (8 cycles).

As shown in lane B of FIG.
It was confirmed that the LV-derived reverse transcriptase specifically generated a fragment corresponding to a length of about 3 Kb, which is the full length of the template RNA. Also, as shown in lane A,
It was confirmed that the M-MLV-derived reverse transcriptase activity was completely lost by heat treatment at 94 ° C. for 30 seconds.
Furthermore, it was confirmed that the amount of generated fragments increased as the number of reaction cycles increased in lanes C, D, E, and F. Compared with the generated fragment in lane B, the concentration of the generated fragment in lane F, which was obtained by repeating the cycle eight times, is about 5 times or more, and this technology has a clear improvement effect as compared with the conventional reaction technology. It turns out that this is an effective technology.

Example 2 In order to confirm that the reaction product of Example 1 was a cDNA of a template RNA, a specific site-binding primer pair (SEQ ID NOs: 1 and 2) was used to prepare the following reaction solution and reaction mixture. PCR was attempted under certain conditions.

Reaction solution composition: Cycle cDNA synthesis product (1/50 volume of PCR reaction solution) 120 mM Tris · Cl (pH 8.0) 10 mM KCl 6 mM (NH ₄ ) ₂ SO ₄ 0.1% Triton X-100 0 0.001% BSA 0.2 mM deoxynucleoside triphosphate 20 pmoles MS2 RNA specific site binding antisense primer (SEQ ID NO: 1) 20 pmoles MS2 RNA specific site binding sense primer (SEQ ID NO: 2) 1 mM MgCl ₂ 2.5 U KOD polymerase Total reaction volume : 50 μl

Reaction conditions: 98 ° C., 20 seconds ↓ 60 ° C., 30 seconds (The above cycle was performed 25 times.)

FIG. 4 shows the results of performing a PCR on the cDNA synthesis reaction product and subjecting the obtained sample to agarose electrophoresis. As a result, about 1 Kbp
CDN amplified specifically by the above method
A shows that cD obtained using MS2 RNA as a template
It was confirmed to be NA (see FIG. 4).

In FIG. 4, each lane is as follows. M: Size marker (φX174 / HincII) A: Sample obtained by performing normal cDNA synthesis using M-MLV RTase, followed by PCR B: Cycle cDNA using M-MLV RTase
After performing a synthesis reaction (once), a sample obtained by PCR C: cycle cDNA using M-MLV RTase
A sample obtained by performing a synthesis reaction (8 times) and then PCR. D: A sample obtained by directly performing PCR on MS2 RNA.

[0060]

According to the present invention, by repeatedly using RNA as a template for cDNA synthesis, the amount of cDNA to be obtained can be increased, and the detection, analysis and isolation of trace RNA can be facilitated. In addition, by using it in combination with an auxiliary amplification means such as PCR, it is possible to detect, analyze and isolate RNA that is unprecedentedly sensitive. Furthermore, operability is improved by providing reagents and kits using these techniques.

[0061]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCACGCTATG TAGCGACCAC 20

SEQ ID NO: 2 Sequence length: 19 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCCGTGGATC AGACACGCG 19

[Brief description of the drawings]

FIG. 1 is a diagram showing the principle of the cycle cDNA synthesis method of the present invention.

FIG. 2 is a view showing an annealing site of a template MS2 RNA and a primer pair capable of binding to a specific site of the RNA.

FIG. 3 is a photograph replacing a figure showing the results of agarose gel electrophoresis of the cDNA synthesis product obtained in Example 1.

FIG. 4 is a photograph replacing a figure showing the result of agarose gel electrophoresis of a sample obtained by PCR with respect to the cycle cDNA synthesis product of Example 1.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Fumiyoshi Kawakami 10-24, Toyocho, Tsuruga-shi, Fukui Toyobo Co., Ltd. Inside Tsuruga Bio-Research Laboratory (72) Inventor Yoshihisa Kawamura 10-24, Toyocho, Tsuruga-shi, Fukui Toyobo Co., Ltd., Tsuruga Bio Research Laboratory

Claims (5)

[Claims] 1. A deoxynucleoside triphosphate, a dideoxynucleoside triphosphate, a derivative and / or a modified product thereof, (2) a template RNA, (3) a primer capable of binding to a specific site of the template RNA, and / or Alternatively, in a buffer solution containing (4) a reverse transcriptase and a primer capable of binding to an unspecified site of the template RNA,
(B) annealing the template RNA at a temperature sufficient for reverse transcriptase (4) to exhibit reverse transcriptase activity
A cDNA is formed with respect to (2), and RNA / DNA
Obtaining a hybrid, (c) separating the RNA / DNA hybrid into RNA and cDNA, and (d) performing the above steps (a), (b) and (c) using the obtained RNA as a template RNA (2). A novel nucleic acid amplification method characterized in that amplification is performed as cDNA by repeating at least once. 2. A novel nucleic acid amplification method, wherein cDNA is further amplified by performing polymerase chain reaction (PCR) using the cDNA amplified by the method of claim 1 as a template. Method. 3. A detection probe is reacted with a cDNA amplified by the method according to claim 1 or 2 from RNA in a sample, and a cDNA to which the detection probe is hybridized or a detection probe not to be hybridized is measured. A method for detecting RNA in a sample. 4. The method according to claim 1, wherein the RNA in the sample is obtained from the RNA.
A method for analyzing the sequence of RNA in a sample, comprising analyzing the sequence of the RNA in the sample by analyzing the sequence of the cDNA amplified by the method described in (1). 5. The method according to claim 1, wherein the RNA in the sample is selected from the group consisting of:
A method for isolating RNA in a sample, comprising inserting a cDNA amplified by the method described in 1) into a cloning vector, introducing the cDNA into a competent cell, and screening to obtain the cDNA.