JP3275969B2 - Method for amplifying nucleic acid sequence - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for amplifying and detecting a specific nucleic acid sequence present in a sample. The invention particularly relates to a method of producing a given nucleic acid sequence from a given nucleic acid, optionally in greater amounts, as compared to the amount initially present. The nucleic acid may be single-stranded or double-stranded, in a relatively pure state, or as a component of a mixture.

[Prior art]

In recent years, detection of nucleic acid sequences by hybridization has been widely used as an effective means for diagnosis of genetic diseases, cancers, infectious diseases and the like. In the nucleic acid sequence detection method, the target base sequence may be only a small part of the target nucleic acid, and in the detection method using a non-radiolabeled probe or an oligonucleotide probe labeled at the end with a radioisotope, Its detection is difficult due to sensitivity problems and the like. Therefore, many efforts have been made to improve the sensitivity of probe detection systems (eg, W087 / 03622). Also, as a means of improving sensitivity,
A method of amplifying a specific nucleic acid sequence of interest with a DNA polymerase (Japanese Patent Application Laid-Open No. 61-274697, etc .;
May be abbreviated as "). However, in this method, it is necessary to frequently carry out heating and cooling operations for repeating annealing, extension reaction, and denaturation of the primer, which requires special equipment or requires complicated labor. Further, in this method, two oligonucleotides are used as primers.
A polymerase may cause amplification, and the specificity of the primer is strictly required. An amplification method using DNA ligase was also disclosed (WO89 / 12696, JP-A-2-29)
No. 34 gazette). However, in this method, non-specific amplification occurs by a reaction in which DNA ligase ligates blunt ends (blunt end ligation). WO89 / 12696 as a workaround for this
Although three or more sets of probes are used, the number of probes is large and the cost is high. Further, a method combining DNA ligase and DNA polymerase has been disclosed.
Further, in one method (WO90 / 01069), it is necessary to use two sets of four or more oligonucleotides, and there is a problem that the cost is increased. The other method (Japanese Patent Application Laid-Open No. Hei 2-28683) requires three oligonucleotides, two of which require several tens of nucleotides, which increases the cost. In addition, RNA polymerase is used to convert R from DNA.
It is well known that NA is produced, and a method for amplifying a nucleic acid sequence using RNA polymerase is also disclosed.
(WO89 / 01050). However, in this method, it is difficult to perform sufficient amplification only by transcription amplification using RNA polymerase. Therefore, an operation is performed in which reverse transcriptase acts again on the generated RNA to generate DNA. Generally, transcription to a DNA by a reverse transcriptase has a drawback that reading errors are more likely to occur than DNA replication by a DNA polymerase. On the other hand, after a probe is hybridized to a nucleic acid of interest, a method of amplifying only a probe that has hybridized correctly (BIO / TECHNOLOGY, vol. 6, 1197,
1988) is also known. However, this method has a problem that a probe bound by a non-specific reaction is also amplified, resulting in an increase in a blank value.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for easily amplifying a target specific nucleic acid sequence.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve these problems, and as a result, from the 5 'end to the 3' end, the homologous part of the specific nucleic acid sequence is sequentially obtained. The present inventors have found that the above problem can be solved by using an oligonucleotide having at least a base sequence A and a base sequence B complementary to a part of the specific nucleic acid at a position 3 ′ downstream, and completed the present invention. Was. That is, the present invention has the following gist. (1) A method for amplifying at least one specific nucleic acid sequence contained in a sample, the method comprising the following steps: (A): From the 5 ′ end to the 3 ′ end, at least a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to a part of the specific nucleic acid sequence 3 ′ downstream are at least sequentially. The oligonucleotide having the mixture is reacted with the sample, and the specific nucleic acid sequence in the sample and the oligonucleotide are annealed. (B): using the oligonucleotide as a primer,
Perform an extension reaction. (C): leaving at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence of the unreacted oligonucleotide,
Disassemble other parts. (D): denaturing the product extended in operation (b) to generate a single-chain molecule. (E): An elongation product is synthesized using the single-stranded molecule generated in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. (2) In addition to the above operations (a) to (e), the following operation (f) or (f ′) is included. Operation (f): The extension product obtained in operation (e) is denatured to generate a single-stranded molecule, and the synthesis of the extension product is performed at least once using the oligonucleotide obtained in operation (c) as a primer. Repeat. Operation (f ′): The above operations (a) to (e) are repeated at least once on the extension product obtained in operation (e). (3) In addition to the above operations (a) to (e), the following operations (f) and (g) are included. Operation (f): The extension product obtained in operation (e) is denatured to generate a single-stranded molecule, and the synthesis of the extension product is performed at least once using the oligonucleotide obtained in operation (c) as a primer. repeat. Operation (g): The above operations (a) to (e) or operations (a) to (f) are repeated at least once on the amplification product obtained in operation (f).

[0005] The oligonucleotide of the present invention is complementary to a base sequence A homologous to a part of a specific nucleic acid sequence and 3 'downstream of a part of the specific nucleic acid sequence in order from the 5' end to the 3 'end. It is designed to have at least a basic base sequence B. Its structure and length are not particularly limited. Typically,
The length of the base sequence A homologous to a part of the specific nucleic acid and / or the base sequence B complementary to a part of the specific nucleic acid is 6 to 10
A length of 0 nucleotides, preferably 10-30 nucleotides, is used.

[0006] In the oligonucleotide used in the present invention, a spacer may be provided between the base sequence A homologous to a part of the specific nucleic acid and the base sequence B complementary to the 3 'downstream phase thereof, if desired. It is possible. The spacer may generally be from 1 to 100, preferably from 1 to 20, nucleotides. This oligonucleotide can be synthesized by a phosphoramidite method using, for example, a DNA synthesizer Model 391 of ABI (Applied Biosystems Inc.). Alternatively, it may be synthesized by any method such as the phosphoric acid triester method, the H-phosphonate method, and the thiophosphite method. It may also be isolated from biological sources, for example, from restriction endonuclease digests.

Next, the present invention will be described with reference to the drawings. FIG. 1 schematically illustrates the principle of the present invention. In the figures, 1 indicates a specific nucleic acid sequence, and 2 indicates an oligonucleotide. The oligonucleotide has a base sequence A (5 ′ end) homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to a part of the specific nucleic acid sequence.
(3 'end side). A spacer is contained between the base sequence A and the base sequence B. Operation (a): Anneal the oligonucleotide to the specific nucleic acid sequence. When the specific nucleic acid sequence is a double strand, it is denatured by heating, alkali treatment, acid treatment, etc. to make it a single strand. Heat denaturation can be carried out, for example, by treating at 80 to 105 ° C for 1 to 5 minutes. The alkali treatment can be performed, for example, by treating in the presence of 0.2 to 1 N caustic soda for 1 to 30 minutes and neutralizing with an equal amount of hydrochloric acid. The acid treatment can be carried out, for example, in the presence of 0.01 to 1 N hydrochloric acid for 1 to 30 minutes and neutralized with caustic soda before use. Alternatively, enzymatic chain degradation can be performed. Annealing is performed on the oligonucleotide, preferably at a temperature selected to provide maximum annealing selectivity. Generally, the temperature is raised so that the specific nucleic acid sequence specifically binds to the oligonucleotide and non-specific binding due to mismatch is minimized. Usually, annealing is performed at around 30 to 70 ° C., but there is no particular limitation.

[0008] Operation (b): A nucleic acid synthesis reaction is carried out using the above oligonucleotide as a primer and a nucleic acid polymerase. The operation is performed by, for example, dNPT (dATP, dCTP, dGTP, dTTP 4).
Species deoxyribonucleotides) and DNA polymerases, such as E. coli DNA1A polymerase, Klenow fragment, T4DN
A polymerase, T7 DNA polymerase, Thermus aqua
ticus DNA polymerase, Thermus thermophilus DN
The extension is carried out by using an A polymerase or a reverse transcriptase and using a specific nucleic acid as a template to carry out an extension reaction. The above method is described in, for example, Journal of Molecular Biology, 56,
341-361, 1971). Operation (c): The unreacted oligonucleotide that has not reacted in the extension reaction leaves at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence, and degrades the other part. The procedure involved exonuclease III, Bal31
This is performed using a nuclease such as a nuclease or a restriction enzyme. The remaining nucleotide sequence is desirably the same as the nucleotide sequence A having homology to the above-mentioned specific nucleic acid, but it is only required to have a sequence included in the sequence A. Also, if necessary, in order to assist the operation, a method in which a base sequence portion A homologous to a part of the specific nucleic acid sequence of the oligonucleotide is set as a phosphorothioate-type oligonucleotide, or the oligonucleotide has a base sequence A and There is also a method of adding an oligonucleotide having a sequence complementary to B to make it double-stranded. Operation (d): The extension product obtained in operation (b) is denatured to a single chain. In this operation, the denaturation can be carried out by the same method as the above-mentioned denaturation of the specific nucleic acid sequence, but the denaturation by heating is particularly preferable. Operation (e): A nucleic acid synthesis reaction is performed using the single-stranded extension product obtained in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. In the present invention, one nucleic acid can be amplified to two by performing the above operations (a) to (e). In the present invention, in addition to the operations (a) to (e), an operation (f) is further performed.
Or, perform (f ′). Operation (f): The extension product obtained in operation (e) is denatured to generate a single-stranded molecule, and the synthesis of the extension product is repeated using the oligonucleotide obtained in operation (c) as a primer. Operation (f ′): The above operations (a) to (e) are repeated at least once on the extension product obtained in operation (e). By this operation, a specific sequence of the nucleic acid can be easily obtained in a large amount. In the present invention, an operation (g) is performed in addition to the operations (a) to (f). Operation (g): The above operations (a) to (e) or operations (a) to (f) are repeated at least once on the amplified product obtained in operation (f). That is, using the product of the above operation (e) and / or the amplification product obtained by repeating the operation (f) as a sample nucleic acid, the operations (a) to (e) or (a) to (f) are repeated at least once. By doing so, a larger amount of amplified product can be obtained. At this time, an amplified product of the specific nucleic acid sequence having higher specificity can be obtained by replacing the oligonucleotide with an oligonucleotide different from the oligonucleotide used in the first operation (a).

[0009]

According to the nucleic acid amplification method of the present invention, oligonucleotides are sequentially transferred from the 5 'end to the 3' end.
By having at least a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary 3 ′ downstream of a part of the specific nucleic acid base sequence, the base of the oligonucleotide in the specific nucleic acid sequence in the sample is The array portion B anneals, and then an extension reaction is performed. A base sequence A ′ complementary to the base sequence A of the specific nucleic acid is formed. Next, the unreacted oligonucleotide is replaced with a base sequence portion A homologous to a part of the specific nucleic acid.
Is left at least in part, and the other part is decomposed into a primer. This primer was used as the base sequence A 'in the extension product.
And an elongation reaction is repeated to perform an amplification reaction. That is, non-specific reactions are suppressed because only one kind of oligonucleotide is used to be mixed and reacted with the sample. Furthermore, in the operation in which the operations (a) to (e) are repeated, one kind of the oligonucleotide obtained in the operation (c) is used as a primer. Even in the case of hybridization, a significant S / N (Signal / Noise) ratio can be obtained because remarkable amplification of non-specific substances is not performed. Furthermore, the nucleic acid amplification method of the present invention has no amplification of the probe remaining due to mismatching or non-specific hybridization, compared to the conventional amplification method using a probe, and
/ N ratio can be increased.

[0010]

EXAMPLES The effects of the present invention will be further clarified by specifically describing the present invention. Reference Example 1 Synthesis of Various Oligonucleotides Using an ABI DNA synthesizer Model 391, various oligonucleotides having the following sequences were synthesized by the phosphoramidite method. 1) First oligonucleotide: The present oligonucleotide is Vibrio parahaemolyticus TDH (Thermosta).
ble Direct Haemolysin) has a sequence complementary to the nucleotide sequence at positions 483 to 504 of the gene, a spacer and a sequence homologous to the nucleotide sequence at positions 52 to 74 of the Vibrio parahaemolyticus TDH gene (Sequence Table 1). 2) Second oligonucleotide: The present oligonucleotide is the same as the first oligonucleotide 19
It has a complementary sequence from the 50th to the 50th (Sequence Table 2).
In addition, an amino group is bonded to the 3 'end. 3) Third Oligonucleotide: The present oligonucleotide is Vibrio parahaemolyticus TDH (Thermos
oligonucleotide probe (24) complementary to the 103rd to 126th sequence of the “Table Direct Hemolysin” gene.
mer). If necessary, the phosphate group at the 5 'end is labeled with ³² P. The method was 0.2 μm according to the ABI manual.
Performed on the M scale. Deprotection of various oligonucleotides was performed overnight at 55 ° C. with aqueous ammonia. Purification was carried out on a reverse phase column with FPLC manufactured by Pharmacia.

(Example 1) Method for amplifying specific nucleic acid Procedure (a) 40 pg of the first oligonucleotide of Reference Example 1 together with 1 ng and 100 pg of genomic nucleic acid separated and partially purified from cultured cells of TDH-producing Vibrio parahaemolyticus Added to 50 μl reaction. After keeping at 94 ° C. for 5 minutes, 4 units of Tth DNA polymerase (manufactured by Toyobo) was added, and after keeping at 94 ° C. for 1 minute, 60 ° C.
For 2 minutes and annealed. Reaction solution 10 mM Tris-HCl (pH 8.9) 1.5 mM MgCl 2 80 mM KCl 500 μg / ml BSA 0.1% sodium cholate 0.1% Triton X-100 2 mM dATP, dGTP, dCTP, dTTP Operation (b) 1 minute and 30 seconds at 75 ° C. The elongation reaction was performed while keeping the temperature. Operation (c) The temperature was lowered to 37 ° C., 340 units of Exonuclease III were added, and the mixture was kept warm for 5 minutes. In operation (b), a part of the unreacted oligonucleotide was decomposed. Operation (d) After incubation at 94 ° C for 2 minutes, amplification was carried out according to the following cycle. Denaturation 94 ° C, 60 seconds Annealing 60 ° C, 120 seconds Extension reaction 75 ° C, 90 seconds Number of cycles 30 operations (e) Then, electrophoresis was performed on agarose gel to confirm the DNA synthesized by ethidium bromide staining (Fig. 2). As a result, DNA was synthesized around 480mer. This is because the first oligonucleotide is extended and unreacted first
The oligonucleotide was degraded, and a sequence homologous to a part of the specific nucleic acid sequence remained to act as a primer, indicating that a DNA molecule was synthesized using the extension product as a template.

Example 2 Amplification Method of Specific Nucleic Acid Procedure (a) 40 μmol of the oligonucleotide of Reference Example 1, 1 ng and 100 pg of partially purified genomic nucleic acid separated and partially purified from cultured cells of TDH-producing Vibrio parahaemolyticus, were mixed with 50 μl each. Added to the reaction solution. 94
After keeping at 5 ° C. for 5 minutes, 4 units of Tth DNA polymerase (manufactured by Toyobo) was added, kept at 94 ° C. for 1 minute, kept at 60 ° C. for 2 minutes, and annealed. Reaction solution 10 mM Tris-HCl (pH 8.9) 1.5 mM MgCl 2 80 mM KCl 500 μg / ml BSA 0.1% sodium cholate 0.1% Triton X-100 2 mM dATP, dGTP, dCTP, dTTP operation (b) 1 minute 30 seconds at 75 ° C. The elongation reaction was performed while keeping the temperature. Operation (c) The second oligonucleotide was added in an amount of 40 pmol, kept at 75 ° C. for 1 minute, kept at 60 ° C. for 2 minutes, and annealed to the unreacted first oligonucleotide. Operation (d) The temperature was lowered to 37 ° C., 340 units of exonuclease III were added, and the mixture was kept warm for 5 minutes. In operation (b), a part of the unreacted oligonucleotide was decomposed. Operation (e) After incubation at 94 ° C. for 2 minutes, amplification was carried out according to the following cycle. Denaturation 94 ° C, 60 seconds Annealing 60 ° C, 120 seconds Extension reaction 75 ° C, 90 seconds Cycle number 30 times Operation (f) Then, electrophoresis was performed on an agarose gel to confirm the DNA synthesized by ethidium bromide staining (Fig. 2). As a result, DNA was synthesized around 480mer. This is because after the first oligonucleotide is extended, the unreacted first oligonucleotide leaves a base sequence homologous to a part of the specific nucleic acid sequence, and the other part is degraded, and as a result, acts as a primer, This indicates that a DNA molecule was synthesized using the product as a template. (Example 3) The amplification product of the agarose gel obtained in the operation (e) of Example 1 was subjected to Southern blotting using a nylon membrane Hybond-n.
⁺ (Amersherm). 6 × S nylon film
SC, 5x Dane Heart solution, 1 mM EDTA, 10μ
After prehybridization at 60 ° C. for 1 hour in 100 μl of a solution containing 1 l of boiled salmon semen DNA (average 500 bases), the 5′-terminal phosphate of the third oligonucleotide prepared in Reference Example 1 was added to the above solution. A probe labeled with ³² P was added thereto, followed by hybridization at 60 ° C. for 1 hour. 6 at 60 ° C
After thoroughly washing the nylon membrane in × SSC, it was dried. An X-ray film (New ALF RX manufactured by Fuji Photo Kogyo) was adhered and exposed to light at -80 ° C for 24 hours. As a result, the film was exposed at the same place as the DNA observed by ethidium bromide staining. This indicates that the DNA observed by ethidium promide staining was a product obtained by accurately amplifying the target nucleic acid. Also,
This shows that a trace amount of target nucleic acid could be easily detected.

[Sequence list]

SEQ ID NO: 1 Sequence length: 50 Sequence type: Nucleic acid Topology: Single-stranded Sequence type: Other nucleic acid synthetic DNA Sequence characteristics Location: 1..22 Method for determining characteristics: S, etc. Features: Vibrio parahaemolyticus TDH (Thermostable Direct H
aemolysin) has a sequence complementary to the nucleotide sequence from position 483 to position 504 of the gene. Location: 23..27 Other features: Spacer Location: 27..50 Other features: Vibrio parahaemolyticus TDH (Thermostable Direct H)
aemolysin) has a sequence homologous to the nucleotide sequence from the 52nd to the 74th of the gene. Sequence ACTACCACTC TCATATGCTT CTAAAAAGCT GCATTCAAAA CATCTGCTTT 50

SEQ ID NO: 2 Sequence length: 32 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid synthetic DNA Sequence characteristics Location: 1..32 Characteristics Method determined: S Other characteristics: It has a sequence complementary to the nucleotide sequence of positions 19 to 50 of the first oligonucleotide. Sequence AAAGCAGATG TTTTGAATGC AGCTTTTTAG AA 22

SEQ ID NO: 3 Sequence length: 24 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid synthetic DNA Sequence characteristics Location: 1..24 Characteristics Method determined S: Other characteristics: Vibrio parahaemolyticus TDH (Thermostable Direct H)
aemolysin) has a sequence complementary to the 103rd to 126th nucleotide sequence of the gene. Array AAACAATATC TCATCAGAAC CGGG 24

[Brief description of the drawings]

FIG. 1 is a diagram schematically illustrating the principle of the present invention. In the figure, 1 indicates a specific nucleic acid, and 2 indicates an oligonucleotide.

FIG. 2 is a view showing an electrophoresis pattern of DNA synthesized in Example 1. Lane 1 is marker 2,
3 and 4 are 1 ng, 100 pg and 0 nucleic acid samples of Example 2, respectively.
g is supported.

FIG. 3 is a view showing an electrophoresis pattern of DNA synthesized in Example 2. Lane 1 is marker 2,
3 and 4 are 1 ng, 100 pg and 0 nucleic acid samples of Example 2, respectively.
g is supported.

FIG. 4 is a view showing a photosensitive pattern of an X-ray film exposed in Example 3. The exposed part shows the same part as the DNA observed by the ethidium promide staining method of Example 1.

Continuation of front page (56) References JP-A-64-60390 (JP, A) JP-A-5-84079 (JP, A) JP-A-7-143900 (JP, A) JP-A-5-199900 (JP, A) , A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 15/09-15/11 C12Q 1/68 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) PCI (DIALOG) WPI ( DIALOG)

Claims (6)

(57) [Claims] 1. A method for amplifying at least one specific nucleic acid sequence contained in a sample, the method comprising the following steps: Operation (a): From the 5 ′ end to the 3 ′ end, a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to a part 3 ′ downstream of the specific nucleic acid sequence are sequentially formed. The oligonucleotide having at least the oligonucleotide is mixed and reacted with the sample, and the specific nucleic acid sequence in the sample is annealed with the oligonucleotide. Operation (b): An elongation reaction is performed using the annealed oligonucleotide as a primer. Operation (c): In the unreacted oligonucleotide, at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence is left, and the other part is decomposed. Operation (d): The product extended in operation (b) is denatured to generate a single-chain molecule. Operation (e): An elongation product is synthesized using the single-stranded molecule generated in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. 2. A method for amplifying at least one specific nucleic acid sequence contained in a sample, comprising the following steps: Operation (a): From the 5 ′ end to the 3 ′ end, a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to a part 3 ′ downstream of the specific nucleic acid sequence are sequentially formed. The oligonucleotide having at least the oligonucleotide is mixed and reacted with the sample, and the specific nucleic acid sequence in the sample is annealed with the oligonucleotide. Operation (b): An elongation reaction is performed using the annealed oligonucleotide as a primer. Operation (c): In the unreacted oligonucleotide, at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence is left, and the other part is decomposed. Operation (d): The product extended in operation (b) is denatured to generate a single-chain molecule. Operation (e): An elongation product is synthesized using the single-stranded molecule generated in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. And operation (f): denaturing the extension product obtained in operation (e) to generate a single-stranded molecule, and using the oligonucleotide obtained in operation (c) as a primer, synthesizing at least one extension product. Repeat several times. 3. A method for amplifying at least one specific nucleic acid sequence contained in a sample, the method comprising the following operations: Operation (a): From the 5 ′ end to the 3 ′ end, a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to a part 3 ′ downstream of the specific nucleic acid sequence are sequentially formed. The oligonucleotide having at least the oligonucleotide is mixed and reacted with the sample, and the specific nucleic acid sequence in the sample is annealed with the oligonucleotide. Operation (b): An extension reaction is performed using the oligonucleotide as a primer. Operation (c): In the unreacted oligonucleotide, at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence is left, and the other part is decomposed. Operation (d): The product extended in operation (b) is denatured to generate a single-chain molecule. Operation (e): An elongation product is synthesized using the single-stranded molecule generated in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. Operation (f ′): The above operations (a) to (e) are repeated at least once on the extension product obtained in operation (e). 4. A method for amplifying at least one specific nucleic acid sequence contained in a sample, comprising the following steps: Operation (a): From the 5 'end to the 3' end, at least a base sequence A homologous to a part of the specific nucleic acid sequence and a base sequence B complementary to the 3 'downstream of a part of the specific nucleic acid sequence are sequentially formed. The oligonucleotide having the mixture is reacted with the sample, and the specific nucleic acid sequence in the sample and the oligonucleotide are annealed. Operation (b): An extension reaction is performed using the above oligonucleotide as a primer. Operation (c): Of the unreacted oligonucleotide, at least a part of the base sequence portion A homologous to a part of the specific nucleic acid sequence is left, and the other part is decomposed. Operation (d): The product extended in operation (b) is denatured to generate a single-chain molecule. Operation (e): An elongation product is synthesized using the single-stranded molecule generated in operation (d) as a template and the oligonucleotide obtained in operation (c) as a primer. Operation (f): The extension product obtained in operation (e) is denatured to generate a single-stranded molecule, and the synthesis of the extension product is performed at least once using the oligonucleotide obtained in operation (c) as a primer. Repeat. And operation (g): The above operation (a) to (e) or operation (a) to (f) is repeated at least once on the amplification product obtained in operation (f). 5. The method for amplifying a nucleic acid sequence according to claim 3, wherein the oligonucleotide used in the operation (f ′) is different from the oligonucleotide used in the first operation (a). 6. The method for amplifying a nucleic acid sequence according to claim 4, wherein the oligonucleotide used in the operation (g) is different from the oligonucleotide used in the first operation (a).