JPH0716094A - Production of single-stranded oligonucleotide having promoter sequence of rna polymerase - Google Patents

Abstract

PURPOSE:To obviate the necessity of troublesome heating treatment by hybridizing a peeling primer and a primer having the promoter sequence of RNA polymerase in a specific mode to the nucleic acid sequence followed by extension reaction using a DNA polymerase with helicase-like activity. CONSTITUTION:(A) A peeling primer and (B) a primer having the promoter sequence of RNA polymerase are hybridized to the nucleic acid sequence so as to situate the primer A toward 3'-side off the primer B. An extension reaction from the primer B is conducted by the action of DNA polymerase having helicase-like activity, and, furthermore, by the extension reaction from the primer A, the objective single-stranded oligonucleotide is separated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a single-stranded oligonucleotide having an RNA polymerase promoter sequence, and a method for amplifying a specific nucleic acid sequence using a single-stranded oligonucleotide having the RNA polymerase promoter sequence. Regarding

[0002]

2. Description of the Related Art Nucleic acid detection by hybridization has come to be widely used as an effective means in diagnosis of genetic diseases, cancers, infectious diseases and the like. However,
If the specific nucleic acid sequence is very small, it is difficult to detect it due to problems such as sensitivity. Therefore, attempts have been made to develop a method for amplifying a specific nucleic acid sequence. As the first attempt, a method for amplifying a target nucleic acid with a DNA polymerase (JP-A-61-274697, hereinafter abbreviated as PCR method) was developed. However, the PCR method has a drawback that it requires a special temperature control device in principle, and that the amplification efficiency can be amplified only twice. Also DN
Amplification method using A ligase (WO89 / 12696, JP-A-2-2934
The following abbreviations such as the LCR method) were also devised, but similar to the PCR method, they require a special temperature control device.

Therefore, there is a need for a method for efficiently amplifying genes without the need for a special temperature control device.
Gene amplification method using A polymerase (JP-A-2-5864)
The following is abbreviated as "NASBA method", such as Japanese Patent Laid-Open Publication No. 4-501057. In the NASBA method, a primer having a transcription initiation sequence and a specific nucleic acid sequence are annealed, an extension reaction is performed with DNA polymerase, and then heat is applied to obtain a single-stranded oligonucleotide having a promoter sequence of RNA polymerase. After that, the gene amplification reaction is performed at a constant temperature by utilizing the property of the promoter sequence (transcription initiation sequence) of RNA polymerase. In addition, the NASBA method enables gene amplification from messenger RNA. In this method, a primer having a transcription initiation sequence is used to specify a transfer RNA.
To RNA and synthesize complementary DNA with RNA-dependent DNA polymerase (reverse transcriptase).
H) treatment decomposes the transcribed RNA to obtain a single-stranded oligonucleotide having a promoter sequence of RNA polymerase. After that, the gene amplification reaction is performed at a constant temperature by utilizing the property of the transcription initiation sequence.

[0004]

As described above, the PCR method has a drawback that it requires a special temperature control device and an expensive thermostable DNA polymerase, and the amplification efficiency can be amplified only twice. The NASBA method was developed to solve this drawback. However, in this NASBA method, in the step of obtaining a single-stranded oligonucleotide having a promoter sequence of RNA polymerase, which is an intermediate step of the reaction, heat treatment (treatment at 95 ° C. for 5 minutes followed by cooling) is unavoidable. It becomes complicated. Further, the NASBA method allows gene amplification from transducing RNA, and the complicated heat treatment can be omitted by using RNA ribonuclease, but is applied only to the amplifying method from transducing RNA and is not suitable for amplification from DNA.

The object of the present invention is to solve the above-mentioned conventional problems, and the object thereof is to complicate the step of obtaining a single-stranded oligonucleotide having a promoter sequence of RNA polymerase. It is to provide a simple manufacturing method that does not require heat treatment.

[0006]

The present inventors have arrived at the present invention as a result of intensive studies to solve these problems. That is, the present invention is a method for producing a single-stranded oligonucleotide having a promoter sequence of RNA polymerase, which comprises the following steps. (A) A primer for peeling and RNA for a specific nucleic acid sequence
A first primer having a polymerase promoter sequence is hybridized. (Here, a first primer having a peeling primer and a promoter sequence of RNA polymerase
A primer can bind to each complementary sequence of a particular nucleic acid sequence,
The positional relationship is such that the complementary sequence of the specific nucleic acid sequence of the peeling primer is present 3'to the complementary sequence of the specific nucleic acid sequence of the first primer having the promoter sequence of RNA polymerase. (B) Then, using a DNA polymerase having helicase-like activity, an extension reaction from the first primer having a promoter sequence of RNA polymerase is performed. (C) Furthermore, with the extension reaction from the peeling primer,
A single-stranded oligonucleotide having an RNA polymerase promoter sequence formed by a first primer having an RNA polymerase promoter sequence and an extension product from the first primer is separated from the specific nucleic acid sequence.

In the present invention, it is preferable that step (b) and step (c) are carried out simultaneously. Further, in the method for producing a single-stranded oligonucleotide having a promoter sequence of RNA polymerase of the present invention, 1 having a promoter sequence of RNA polymerase formed in step (c) is used.
The double-stranded region of the double-stranded oligonucleotide and the specific nucleic acid sequence is unwound by the action of the DNA polymerase having helicase-like activity in association with the extension reaction from the primer for peeling, and the single-stranded region having the promoter sequence of RNA polymerase It is preferred that the oligonucleotide is separated from the specific nucleic acid sequence.

The present invention is also a method for amplifying a specific nucleic acid sequence using a single-stranded oligonucleotide having an RNA polymerase promoter sequence, which comprises the following steps. (A) RN obtained through the above steps (a) to (c)
A second primer is hybridized with a single-stranded oligonucleotide having a promoter sequence of A polymerase. (B) Next, an extension reaction is performed using a DNA polymerase to synthesize double-stranded DNA, and then (C) the double-stranded DNA is reacted with RNA polymerase to synthesize a plurality of single-stranded RNAs. (D) The second primer is hybridized with the single-stranded RNA as a template. (E) Next, an RNA-DNA hybrid is formed using an RNA-dependent DNA polymerase. (F) The RNA portion of the RNA-DNA hybrid is decomposed to obtain single-stranded DNA. (G) The obtained single-stranded DNA is hybridized with a first primer having an RNA polymerase promoter sequence, and an extension reaction is performed to synthesize a double-stranded DNA. (H) Further, steps (C) to (G) are repeated to amplify the specific nucleic acid sequence.

The terms used in the present invention are explained below. “Extension reaction” means that after a primer having a sequence sufficiently complementary to a nucleic acid sequence is hybridized with the nucleic acid sequence, D
NA polymerase and deoxyadenosine 5'-triphosphate (dATP), deoxycytidine 5'-triphosphate (dCTP), deoxyguanosine 5'-triphosphate (dGTP), and deoxythymidine 5'-triphosphate (dTTP) In the presence of a), a deoxynucleotide is covalently bound to the primer to sequentially synthesize a DNA sequence complementary to the nucleic acid sequence. The "peeling primer" is a primer for separating a single-stranded oligonucleotide having an RNA polymerase promoter sequence from a specific nucleic acid sequence. RN existing upstream of the extension reaction direction with the extension reaction from the stripping primer
A single-stranded oligonucleotide having a promoter sequence for A polymerase is separated from a specific nucleic acid sequence by the action of a DNA polymerase having helicase-like activity.

The "RNA polymerase promoter sequence" is a sequence to which RNA polymerase specifically binds. RNA polymerase specifically binds to this promoter sequence and DNA downstream of the promoter sequence.
Synthesize RNA homologous to the sequence. The “first primer having a promoter sequence of RNA polymerase” has a promoter sequence such as T3RNA polymerase, T7RNA polymerase, SP6RNA polymerase at the 5 ′ end and a nucleic acid sequence complementary to a specific nucleic acid sequence at the 3 ′ end side. It is a primer. The "single-stranded oligonucleotide having a promoter sequence of RNA polymerase" is a combination product of a first primer having a promoter sequence of RNA polymerase and an extension product from the first primer by DNA polymerase.

The "DNA polymerase having helicase-like activity" is a polymerase that synthesizes DNA by performing an extension reaction from a primer and unwinds a double-stranded DNA site existing in the extension direction to convert it into a single-stranded DNA. That is. For example, PHI29 DNA polymerase, Klenow fragment, M2 DNA polymerase and the like have helicase-like activity and can be used in the present invention. In addition, Tt which is a thermostable DNA polymerase
h (Thermus thermophilus) DNA polymerase, Ta
q (Thermus aquaticus) DNA polymerase, Ven
t (Thermococcus litoralis) DNA polymerase, B
st (Bacillus stearothermophilus) DNA polymerase and the like also have helicase-like activity and can be used in the present invention.

The specific nucleic acid sequence used in the present invention is a single-stranded chain D
It may be NA or double-stranded DNA. It does not matter whether they are artificially created or naturally occurring. When natural DNA is used, it can be used in the present invention after being extracted from a sample according to a conventional method.
As a specific method for preparing a sample, for example, a strain of Vibrio parahaemolyticus is inoculated into a brain heart infusion medium and cultured overnight. Scrap the grown colonies in an Eppendorf tube, suspend in a diluting buffer, add proteinase and lysate to this, and stir at about 60 ° C.
Incubate for 30 minutes at
After extraction with phenol, chloroform, etc., ethanol precipitation is performed to obtain a nucleic acid.

In the present invention, the promoter sequence of the first primer having the promoter sequence of RNA polymerase is T3 RNA polymerase, T7 RNA polymerase, SP
6 A promoter sequence for RNA polymerase or the like may be used. The sequence length of the annealing site between the first primer and the specific nucleic acid sequence is not particularly limited, and is preferably 10 to 100b.
p, particularly preferably 10 to 50 bp, further preferably 15 to 30 bp
However, the present invention is not limited to this. The method for preparing the primer follows a conventionally known method. As a specific method, for example, it is produced by the phosphoramidide method according to the manual of ABI company. Deprotection of various oligonucleotides is performed with aqueous ammonia. Purification may be performed by FPLC on a reverse phase column.

In the present invention, the stripping primer and RN are used.
The first primer having the promoter sequence of A polymerase can bind to each complementary sequence of the specific nucleic acid sequence, and its positional relationship is such that the complementary sequence of the specific nucleic acid sequence of the peeling primer has the RNA polymerase promoter sequence. It is located 3'to the complementary sequence of the specific nucleic acid sequence. When designing the stripping primer, the sequence length of the annealing site between the stripping primer and the specific nucleic acid sequence is not particularly limited, preferably 10 to 100 bp, particularly preferably 10 to 50 bp.
bp, more preferably 15 to 30 bp, but not limited thereto.

The present invention will be described below with reference to the drawings (FIGS. 1 and 2). The stripping primer a and the first primer b having the promoter sequence of RNA polymerase can bind to the complementary binding sequence of the specific nucleic acid sequence, and the positional relationship is such that the complementary binding sequence a ′ of the specific nucleic acid sequence of the stripping primer a is It must be present 3'to the complementary binding sequence b'of the specific nucleic acid sequence of the first primer b. The distance between the complementary binding sequences of the specific nucleic acid sequences of the first primer b and the stripping primer a (distance between b ′ and a ′) is not particularly limited, and the stripping primer can be formed by a DNA polymerase having helicase-like activity. With the extension reaction from a,
The distance may be set such that the first primer b and the extension product c from the first primer b are separated from the specific nucleic acid sequence.
It is preferably 0 to 2000 bp, particularly preferably 20 to 1000 bp, but is not limited thereto.

The stripping primer a and the first primer b having an RNA polymerase promoter sequence are annealed to the specific nucleic acid sequence according to the following method (step a).
When the specific nucleic acid sequence is double-stranded DNA, the specific nucleic acid sequence, the first primer b and the stripping primer a are mixed, heated at 80 to 105 ° C. for 2 to 5 minutes, and then allowed to stand at room temperature. . When the specific nucleic acid sequence is single-stranded DNA, the specific nucleic acid sequence, the first primer b, and the stripping primer a
You may mix and leave for a while, or 2 at 50 ~ 105 ℃
After heating for ~ 5 minutes, it may be left to stand until it reaches room temperature. After completion of the annealing reaction, a DNA polymerase having helicase-like activity was used in the presence of dNTP (4 types of deoxyribonucleotides of dATP, dCTP, dGTP, and dTTP) to remove the primer a and a first primer having a promoter sequence of RNA polymerase. The extension reaction from b is caused (step b). The first primer b and the extension product c from the first primer b are produced by the action of a DNA polymerase having a helicase-like activity in the double-stranded region formed with a specific nucleic acid sequence according to the extension reaction from the stripping primer a. When unwound, the single-stranded oligonucleotide d having the promoter sequence of RNA polymerase is separated from the specific nucleic acid sequence (step c). Extension reaction from stripping primer a and RN
Extension reaction from the first primer b having the A polymerase promoter sequence (step b), and single-stranded oligonucleotide d having the RNA polymerase promoter sequence
There is no problem even if the reaction (step c) in which is separated from the specific nucleic acid sequence occurs at the same time.

As an example of a specific method for producing a single-stranded oligonucleotide having a promoter sequence of RNA polymerase, the genome of Vibrio parahaemolyticus is suspended in a buffer solution to obtain a deoxyribonucleotide, a T7 transcription initiation sequence, and a heat-resistant hemolysin gene. Primer 1 having a homologous sequence and peeling primer 2 having a sequence homologous to the thermostable hemolysin gene sequence are mixed, heated to about 95 ° C., and then allowed to stand until room temperature. Add DNA polymerase and incubate at about 30 ° C to carry out reaction.

In order to amplify a nucleic acid using a single-stranded oligonucleotide d having a promoter sequence of RNA polymerase produced by the production method of the present invention, first, a second primer complementary to the single-stranded nucleotide d is used. e is annealed (FIG. 2, step A), and double-stranded DNA is formed by a DNA polymerase reaction (step B). The double-stranded DN
A is reacted with RNA polymerase to synthesize single-stranded RNAf (step C). A second primer e complementary to the single-stranded nucleotide can be annealed to the single-stranded RNA (step D), and DN complementary to the single-stranded RNAf can be formed by RNA-dependent DNA polymerase (reverse transcriptase).
A sequence g is synthesized (step E). The DNA sequence g complementary to the single-stranded RNA and the single-stranded RNAf are separated using, for example, ribonuclease (step F), and the first primer b having the DNA sequence g complementary to the single-stranded RNA and the promoter sequence of RNA polymerase Are annealed and double-stranded with DNA polymerase (step G). The above reaction is repeated at least once to carry out the amplification reaction.

The second primer used in the present invention is RN
A primer complementary to a single-stranded nucleotide having a promoter sequence of A polymerase.

In the present invention, in order to detect a specific nucleic acid sequence, it is necessary to subject the sample amplified by the above-mentioned amplification method to electrophoresis, Southern blotting, dot blotting, etc. As one example of a concrete detecting method, A second primer complementary to a single-stranded nucleotide having an RNA polymerase promoter sequence, an RNA-dependent DNA polymerase (reverse transcriptase), an RNA polymerase, and optionally a ribonuclease are added to the reaction solution obtained in the steps a to c. Then, the reaction is carried out at about 42 ° C., and several μl of the reaction solution is dropped on, for example, a nylon membrane, and then it can be detected by hybridizing with a radiolabeled probe or an enzyme labeled probe. As another detection example, the second primer, DTT,
RNA-dependent DNA polymerase (eg AMV reverse transcriptase), RNA polymerase (eg T7 RNA polymerase) and optionally ribonuclease (eg RNaseH)
Is added and reacted at about 42 ° C., the reaction solution is subjected to agarose gel electrophoresis and stained with ethidium bromide, and then fluorescence from ultraviolet rays is detected. In addition to the reaction solution, a molecular weight marker is also electrophoresed at the same time, and the length of the detected nucleotide fragment can be calculated by comparing the relative mobilities.

[0021]

INDUSTRIAL APPLICABILITY In the conventional method, heat treatment (treatment at 95 ° C. for 5 minutes, followed by cooling) is required to obtain a single-stranded oligonucleotide having a promoter sequence of RNA polymerase, and this heating operation is used for automating the reaction. Barrier to
A heating device must be provided on the device itself. However, in the present invention, a DNA polymerase having helicase-like activity is used, and its operation can be simplified,
RN without the need for such complicated operations and devices
A single-stranded oligonucleotide having a promoter sequence for A polymerase can be obtained. The method for producing a single-stranded oligonucleotide of the present invention can be applied to an amplification method in which a nucleic acid having a known base sequence is produced in a larger amount as compared with the amount present in the initial stage. It can also be applied to a method for detecting a specific nucleic acid sequence.

[0022]

EXAMPLES The present invention will be described in detail with reference to examples. Example 1 (Synthesis of various oligonucleotides) Using a DNA synthesizer type 391 manufactured by ABI, a sequence shown in the sequence listing by the phosphoamidite method, that is, a promoter sequence of T7 RNA polymerase (T7 transcription initiation sequence) and a heat-resistant hemolytic poison A first primer having a sequence homologous to the gene (SEQ ID NO: 1), a stripping primer having a sequence homologous to the thermostable hemolysin gene (SEQ ID NO: 2), a single-stranded oligonucleotide having an RNA polymerase promoter sequence A second primer (having a sequence complementary to the sequence of the thermostable hemolytic poison gene) (SEQ ID NO: 3) complementary to was synthesized. The specific method was carried out on a 0.2M scale according to the ABI company manual. Deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C overnight. Purification was carried out by FPLC manufactured by Pharmacia on a reverse phase column.

(Preparation of Genome from Vibrio parahaemolyticus) The strain of Vibrio parahaemolyticus was inoculated into Brain Heart Infusion Agar containing 3% NaCl and cultured at 37 ° C. overnight. Each grown colony
It was scraped into a 1.5 ml Eppendorf tube and suspended in 300 μl of a dilution buffer (0.1 M NaH ₂ PO ₄ , pH 7.0). Further to this Proteinase K (Nacalai Tesque) 0.6
mg, lysate (8M urea, 0.25% sodium dodecyl sulfate,
0.25% sodium lauryl sarcosine, 50 mM EDTA, pH
7.6) 600 μl was added, stirred and incubated at 60 ° C. for 30 minutes. The resulting solution was extracted twice with phenol and once with chloroform and then ethanol precipitated to obtain a nucleic acid.

(Preparation of Probe for Detection) (1) Synthesis of Oligonucleotide for Vibrio parahaemolyticus Having a Linker Arm Using a DNA Synthesizer 392 from ABI, the sequence shown in the sequence listing by the phosphoramidide method, that is, the probe for detection (SEQ ID NO: 4) was synthesized. At this time, uridine having a linker arm at the 5-position, which was chemically synthesized from deoxyuridine, was introduced into the above-mentioned oligonucleotide by the synthetic method described in JP-A-60-500717. This uridine can replace any T in the oligonucleotide, but in this example the 5'end T was replaced.
The synthesized linker oligonucleotide was deprotected with ammonia water at 50 ° C overnight, and then FPLC manufactured by Pharmacia
It was purified by using an anion exchange column.

(2) Labeling of Linker Oligonucleotide with Alkaline Phosphatase Alkaline phosphatase was added to the above linker oligonucleotide via its linker arm (Ncleic Acid).
s Research, vol.14, p.6114, 1986). Linker oligonucleotide 1.5 A ₂₆₀ with 0.2 M NaHCO
_{3 It} was dissolved in 12.5 μl, and 25 μl of 10 mg / ml succinimidyl suberate (DSS) was added thereto and reacted at room temperature for 2 minutes. Seph equilibrated with 1 mM CH ₃ COONa (pH 5.0)
Excess gel filtration with an adex G-25 column (1 cmφ × 30 cm)
The DSS was removed. An alkaline phosphatase-labeled probe was prepared by reacting a linker oligonucleotide in which the terminal amino group was activated with a double molar amount of alkaline phosphatase (dissolved in 100 mM NaHCO ₃ , 3M HCl) at room temperature for 16 hours. Obtained. The obtained labeled probe was purified by Pharmacia FPLC using an anion exchange column. Fractions containing the labeled probe were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

(Synthesis of Single-Stranded Oligonucleotide Having RNA Polymerase Promoter Sequence) 1 ng of Vibrio parahaemolyticus genome was buffered (50 mM Tris-HCl, pH 8.3, 6 mM MgC
was suspended in l _2, 40 mM KCl), was mixed with 1mM deoxyribonucleotides (Pharmacia) and primer 2, after heating for 3 minutes 95 ° C., and allowed to come to room temperature. PHI91 RN
4 units of A polymerase was added, and the reaction was carried out by keeping the temperature at 30 ° C. for 1 hour.

(Detection) Primer 3 (SEQ ID NO: 3), 10 mM DTT, 40 units of RNA-dependent DN were added to the above reaction solution.
A polymerase (AMV reverse transcriptase) (Toyobo) 0.4 unit of ribonuclease (RNaseH) (Pharmacia), 20 units of T7 RNA polymerase (Stratagene) were added, and the temperature was 42 ° C.
And reacted for 3 hours. After dropping 1 μl of the reaction solution onto a nylon membrane, the nucleic acid was immobilized under alkaline conditions. After neutralizing this membrane, it was transferred to a hybridization bag, and the hybridization buffer (5 × SSC, 0.5 × 0.5) containing the above-mentioned alkaline phosphatase-labeled nucleic acid probe (SEQ ID NO: 4) was used.
% Bovine serum albumin, 0.5% polyvinylpyrrolidone,
1% sodium dodecyl sulfate) was added, and hybridization was performed at 50 ° C for 15 minutes. The nylon membrane was taken out from the polybag and washed with Washing liquid 1 (1 × SSC, 1% sodium dodecyl sulfate) at 50 ° C. for 10 minutes with shaking. Further cleaning liquid
2 (1 × SSC) was washed by shaking at room temperature for 10 minutes. Transfer the membrane to a new hybridization bag and add substrate solution (0.1M Tris
-HCl, 0.1 M NaCl, 0.1 M MgCl ₂ , 0.3 mg / ml nitroblue tetrazolium, 0.3 mg / ml bromochloroferryl phosphate pH 7.5) was added, and after sealing, the mixture was incubated at 37 ° C for 30 minutes.

(Results) In the product, a spot of a purple dye generated by alkaline phosphatase when primers 1 and 2 (SEQ ID NOs: 1 and 2) were used simultaneously could be detected.

FIG. 1 shows an overall diagram for producing a single-stranded nucleotide having a promoter sequence of the RNA polymerase of the present invention. FIG. 2 shows a method for amplifying a specific nucleic acid sequence using a single-stranded nucleotide having an RNA polymerase promoter sequence obtained according to the present invention. FIG. 3 shows the first embodiment.
The result of the hybridization of is shown. In the figure, spots 1 to 4 show the results of the execution in the presence and absence of the primers (SEQ ID NOS: 1 and 2). Spot 5 represents a control in the absence of the specific nucleic acid sequence in the presence of the primers (SEQ ID NOS: 1 and 2).

[0030]

[Sequence list]

SEQ ID NO: 1 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Method for determining features : S Other features: It has a T7 transcription initiation sequence and a sequence homologous to the thermostable hemolytic toxin gene. Sequence: AATACGACTC ACTATAGCCCC GGTTCTGATG AGATATTGTT 40

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Features Determined method: S Other characteristics: Having a sequence homologous to the sequence of thermostable hemolytic toxin gene. Sequence: GCTGCATTC AAAACATCTGC 20

SEQ ID NO: 3 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Features Determined method: S Other characteristics: having a sequence complementary to the sequence of thermostable hemolytic toxin gene. Sequence: ATTTTACGAA CACAGCAGAA TGACCG 26

SEQ ID NO: 4 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Determined method: S Other characteristics: having the sequence of thermostable hemolytic toxin gene. Sequence: TCAGGTACTA AATGGTTGAC ATCCT 25

[Brief description of drawings]

FIG. 1 shows steps a to c for producing a single-stranded nucleotide having a promoter sequence of the RNA polymerase of the present invention.

FIG. 2 shows a method for amplifying a specific nucleic acid sequence using a single-stranded nucleotide having an RNA polymerase promoter sequence of the present invention.

FIG. 3 shows the result of hybridization in Example 1.

Claims (4)

[Claims] 1. RNA comprising the following steps:
A method for producing a single-stranded oligonucleotide having a polymerase promoter sequence. (A) A primer for peeling and RNA for a specific nucleic acid sequence
A first primer having a polymerase promoter sequence is hybridized. (Here, a first primer having a peeling primer and a promoter sequence of RNA polymerase
A primer can bind to each complementary sequence of a particular nucleic acid sequence,
The positional relationship is such that the complementary sequence of the specific nucleic acid sequence of the peeling primer is present 3'to the complementary sequence of the specific nucleic acid sequence of the first primer having the promoter sequence of RNA polymerase. (B) Then, using a DNA polymerase having helicase-like activity, an extension reaction from the first primer having a promoter sequence of RNA polymerase is performed. (C) Furthermore, with the extension reaction from the peeling primer,
A single-stranded oligonucleotide having an RNA polymerase promoter sequence formed by a first primer having an RNA polymerase promoter sequence and an extension product from the first primer is separated from the specific nucleic acid sequence. 2. A method for producing a single-stranded oligonucleotide having a promoter sequence of RNA polymerase, wherein step (b) and step (c) in claim 1 are performed simultaneously. 3. A double-stranded region of a single-stranded oligonucleotide having a promoter sequence of RNA polymerase formed in step (c) of claim 1 and a specific nucleic acid sequence,
RNA characterized by being unwound by the action of a DNA polymerase having a helicase-like activity in association with an extension reaction from a stripping primer, and a single-stranded oligonucleotide having a promoter sequence of RNA polymerase is separated from a specific nucleic acid sequence. A method for producing a single-stranded oligonucleotide having a polymerase promoter sequence. 4. RNA comprising the following steps:
A method for amplifying a specific nucleic acid sequence using a single-stranded oligonucleotide having a polymerase promoter sequence. (A) The second primer is hybridized with the single-stranded oligonucleotide having the promoter sequence of RNA polymerase produced in claim 1. (B) Next, an extension reaction is performed using a DNA polymerase to synthesize double-stranded DNA, and then (C) the double-stranded DNA is reacted with RNA polymerase to synthesize a plurality of single-stranded RNAs. (D) The second primer is hybridized with the single-stranded RNA as a template. (E) Next, an RNA-DNA hybrid is formed using an RNA-dependent DNA polymerase. (F) The RNA portion of the RNA-DNA hybrid is decomposed to obtain single-stranded DNA. (G) The obtained single-stranded DNA is hybridized with a first primer having an RNA polymerase promoter sequence, and an extension reaction is performed to synthesize a double-stranded DNA. (H) Further, steps (C) to (G) are repeated to amplify the specific nucleic acid sequence.