JPH05199900A - Amplification and detection of nucleic acid sequence and reagent kit therefor - Google Patents

Abstract

PURPOSE:To readily amplify and detect a specified nucleic acid. CONSTITUTION:A sample is reacted with the first oligonucleotide comprehensive to a part of a specified nucleic acid sequence and with the second oligonucleotide having, at least, a sequence comprehensive to another part of the specified nucleic acid sequence from 5' terminal to 3' terminal in order and a base sequence comprehensive to the first oligonucleotide. Thereby, an elongation reaction is carried out by allowing the first annealed oligonucleotide to function as a primer and the second annealed oligonucleotide is subsequently bonded thereto. A single strand obtained by separating the resultant bonded material from the template is used as a template so as to carry out an elongation reaction by allowing the first oligonucleotide function as a primer. The specified nucleic acid is amplified thereby and the amplified nucleic acid is detected. Thereby, non-specific reactions can be inhibited and only the specified sequence can be amplified.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art In recent years, the detection of nucleic acid sequences by hybridization has become widely used as an effective means for diagnosing genetic diseases, cancers, infectious diseases and the like. In the nucleic acid sequence detection method, the target base sequence may be a very small part of the target nucleic acid, and in the detection method using a non-radioactive labeled probe or an oligonucleotide probe labeled with a radioactive isotope, It is difficult to detect it due to sensitivity problems. Therefore, much effort is being made to improve the sensitivity of probe detection systems (eg W087 / 03622). Also, as a means of improving sensitivity,
A method for amplifying a specific nucleic acid sequence of interest with a DNA polymerase (JP-A-61-274697, etc .; hereinafter referred to as "PCR").
May be abbreviated) was disclosed. However, in this method, it is necessary to frequently perform annealing of the primer, extension reaction, and heating and cooling operations for repeating denaturation, which requires a special device or requires complicated labor. Further, in this method, although two oligonucleotides are used as primers, amplification by DNA polymerase may occur even when these are non-specifically annealed, and the specificity of the primer is strictly required. An amplification method using a DNA ligase has also been disclosed (WO089 / 12696, Japanese Patent Laid-Open No. Hei 10 (1999) -26498)
2-2934, etc.). However, in this method, DNA ligase ligates blunt ends (blunt and ligation).
Causes non-specific amplification. WO as a way around these
Although 089/12696 uses three or more pairs of probes, the number of probes is large and the cost is high. More DNA
Methods of using a combination of ligase and DNA polymerase have been published. One method requires the use of two sets or more of four or more oligonucleotides, which causes a problem of high cost (WO90 / 01069), and the other method requires three oligonucleotides, two of which are required. Requires several tens of nucleotides, resulting in high cost (JP-A-2-
No. 268683). In addition, D using RNA polymerase
It is well known that RNA is produced from NA.
A method for amplifying a nucleic acid sequence using a polymerase has also been published (WO089 / 01050). However, in this method, sufficient amplification is difficult only by transcription amplification with RNA polymerase. Therefore, an operation is carried out to cause the generated RNA to act again with the reverse transcriptase to generate DNA. Generally, transcription into DNA by reverse transcriptase is performed by DN
Compared with DNA replication by A polymerase, there is a drawback that read errors are likely to occur. On the other hand, there is also known a method of hybridizing a probe to a target nucleic acid and then amplifying only the correctly hybridized probe (BIO / TECHNOLOGY vol.6, 1197, 1988). However, this method has a problem that the probe bound by the non-specific reaction is also amplified and the blank value is increased.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for conveniently amplifying a specific nucleic acid sequence of interest. Another object of the present invention is to provide a method for detecting a specific nucleic acid sequence in a sample.

[0004]

As a result of intensive studies to solve these problems, the present inventors have found that a first oligonucleotide complementary to a part of at least one specific nucleic acid sequence contained in a sample. And a second oligonucleotide having a base sequence B complementary to the other part of the specific nucleic acid sequence and a base sequence A complementary to the first oligonucleotide in sequence from the 5 ′ end to the 3 ′ end. The present invention has been completed by finding that the above problems can be solved. That is, the present invention provides an oligonucleotide containing at least base sequence A and base sequence B,
An oligo having a base sequence B complementary to a part of a specific nucleic acid sequence and a base sequence A homologous to a 3'downstream part of the specific nucleic acid sequence in order from the 5'end to the 3'end. It is a nucleotide (Fig. 1).

The method for amplifying a specific nucleic acid of the present invention basically includes the following operations (a) to (e). Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized.

The following are examples of embodiments of the present invention. (1) A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following operations: Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized.

(2) A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following steps: Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. And operation (f): the amplification product obtained in operation (e) is separated from the template synthesized in operation (d), and the single chain molecule generated in operation (e) is used as a template, and The oligonucleotide extension product synthesis of the nucleotide extension product is repeated at least once.

(3) A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following steps: Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): Operation (c) The linked products are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. And operation (f ′): the above-mentioned operations (a) to (e) are repeated at least once for the amplification product obtained in operation (e).

(4) A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following steps: Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. Operation (f): The amplification product obtained in operation (e) was separated from the template synthesized in operation (d) and the single-stranded molecule generated in operation (e) was used as a template, and the first oligonucleotide was used as a primer. The synthesis of the nucleotide extension product is repeated at least once. And operation (g): the above-mentioned operations (a) to (e) or operations (a) to (f) are repeated at least once for the amplification product obtained in operation (f).

The reagent kit for amplifying the target nucleic acid sequence of the present invention comprises the above-mentioned first oligonucleotide and / or second oligonucleotide, ligase, nucleic acid polymerase and / or reverse transcriptase, and four kinds of deoxyribonucleotide triphosphates. Contains an acid and a reaction buffer.

The method for detecting a specific nucleic acid sequence of the present invention comprises
Using the above-mentioned first oligonucleotide and / or second oligonucleotide labeled with the first oligonucleotide and / or the second oligonucleotide, the above operations (a), (b), (c), (d) and (e), or operations (A) (b) (c) (d) (e) and / or (f), or after carrying out operations (a) (b) (c) (d) (e) and (f '), The method is characterized in that the target nucleic acid sequence is detected by measuring the label. Furthermore, the method for detecting a specific nucleic acid sequence of the present invention uses the above-mentioned first oligonucleotide and / or second oligonucleotide, and performs the above-mentioned operations (a) (b) (c) (d) and (e),
Or after performing operations (a) (b) (c) (d) (e) and (f) or operations (a) (b) (c) (d) (e) and (f ') A labeled oligonucleotide probe capable of hybridizing to the sequence to be detected and / or a variant thereof is added to the object to detect the target nucleic acid sequence.

The reagent kit for detecting a specific nucleic acid sequence of the present invention comprises the above-mentioned first oligonucleotide and / or
Alternatively, a first oligonucleotide and / or a second oligonucleotide labeled with a second oligonucleotide, a ligase, a nucleic acid polymerase and / or a reverse transcriptase,
Includes four deoxynucleotide triphosphates, a reaction buffer, and a system to detect the label. Furthermore, the reagent kit for detecting a specific nucleic acid sequence of the present invention comprises the above-mentioned first oligonucleotide and / or second oligonucleotide, ligase, nucleic acid polymerase and / or reverse transcriptase,
Includes four deoxynucleotide triphosphates, a reaction buffer, a labeled oligonucleotide probe and a system to detect the label.

In the present invention, the first oligonucleotide and / or the second oligonucleotide used in the operation (f ') is the first oligonucleotide and / or the second oligonucleotide used in the first operation (a). It may be different from the oligonucleotide. Further, the first oligonucleotide and / or the second oligonucleotide used in the operation (g) is different from the first oligonucleotide and / or the second oligonucleotide used in the first operation (a). Good.

The first oligonucleotide of the present invention may have a base sequence complementary to a part of a specific nucleic acid in a sample, and its structure and length are not limited (see 2 in FIG. 2). Generally, its length is from 6 to 100 nucleotides, preferably from 10 to
Those that are 30 nucleotides are used. The second oligonucleotide of the present invention sequentially has a base sequence complementary to another part of the specific nucleic acid and a base sequence complementary to the first oligonucleotide from the 5 ′ end to the 3 ′ end (FIG. 2).
3). The first oligonucleotide so that the portion complementary to the first oligonucleotide which is a part of the specific nucleic acid is present at the 3'end side of the portion complementary to the second oligonucleotide which is another part of the specific nucleic acid. And a second oligonucleotide is selected. The structure and length are not limited as long as the first oligonucleotide is extended so that the 3'end of the second oligonucleotide and the 5'end of the extension product are linked. Generally, the length of a base sequence complementary to another part of a specific nucleic acid is 6 to 100.
Nucleotides, preferably 10-30 nucleotides in length, are used. In the second oligonucleotide of the present invention, a spacer can be optionally provided between the base sequence complementary to the other part of the specific nucleic acid and the base sequence complementary to the first oligonucleotide. The spacer may generally have 1 to 100 nucleotides, preferably 1 to 20 nucleotides. These oligonucleotides are, for example,
It can be synthesized by the phosphoramidite method using a DNA synthesizer type 391 manufactured by ABI (Applied Biosystems Inc.). In addition, any method such as the phosphoric acid triester method, the H-phosphonate method and the thiophosphite method may be used. It may also be isolated from biological sources, such as restriction endonuclease digests. It is preferable to add a phosphate group to the 5'end of the second oligonucleotide. The addition of a phosphate group can be performed by T4 polynucleotide kinase in the presence of ATP, for example.

To understand the present invention, the principle of the present invention is schematically shown in FIG. The present invention will be described below based on this drawing. In the figure, 1 is a specific nucleic acid sequence, 2 is a first oligonucleotide, and 3 is a second oligonucleotide. Operation (a): The specific nucleic acid sequence is annealed with the first oligonucleotide and the second oligonucleotide simultaneously or separately. In the present invention, the second oligonucleotide needs to be designed to anneal to the 3'downstream side of the first oligonucleotide. When the specific nucleic acid sequence is a double strand, it is denatured by heating, alkali treatment, acid treatment or the like to be a single strand. The heat denaturation can be carried out, for example, by treating at 80 to 105 ° C for 1 to 5 minutes. Alkali treatment is, for example, 0.
Treated in the presence of 2 to 1 normal NaOH for 1 to 30 minutes to obtain an equal amount of HCl
Can be used after neutralizing with. Acid treatment is, for example, 0.01-
It can be used by treating it in the presence of 1N HCl for 1 to 30 minutes and neutralizing with NaOH. As another method, chain decomposition can be performed enzymatically. Annealing is performed for each of the first and second oligonucleotides, preferably at a temperature selected to provide maximum annealing selectivity. Generally, the heating is performed so that the specific nucleic acid sequence specifically binds to the first oligonucleotide and the second oligonucleotide, and nonspecific binding due to mismatch is minimized. Usually, it is annealed at around 30 to 70 ° C., but it is not particularly limited. Operation (b): A nucleic acid synthesis reaction is performed using a nucleic acid polymerase with the first oligonucleotide as a primer.
This operation can be performed, for example, with dNTP (dATP, dCTP, dGTP, dTTP).
Species deoxyribonucleotides) and DNA polymerases (eg E. coli DNA1A polymerase, Klenow fragment, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus DNA
Polymerase, Thermus thermophilus DNA polymerase, or reverse transcriptase) is used to perform an extension reaction using a specific nucleic acid as a template. The above method is described in, for example, Journal of Molecular Biology; 56,341-361,1971.
It is described in. Operation (c): When the extension reaction proceeds to the 5'end of the second oligonucleotide annealed in operation (a), it is ligated with the second oligonucleotide. The operation is
T4 DNA ligase, T7 DNA ligase, E. coli DNA
A method using a ligase such as ligase or Thermus thermophilus DNA ligase is preferable. Operation (d): The extension and ligation products obtained in operation (c) are denatured into a single chain. In the operation, the denaturation can be carried out by the same method as the denaturation of the specific nucleic acid sequence, but the denaturation by heating is particularly preferable. Operation (e): A nucleic acid synthesis reaction is carried out using the above single chain, extension, and ligation products as templates and the first oligonucleotide as a primer.

By repeating the production of the single strand in the operation (d) and the nucleic acid synthesis using the first oligonucleotide in the operation (e) as a primer, a large amount of the specific sequence of the nucleic acid can be easily obtained. Further, by using the product of the operation (e) and / or the amplified product obtained by repeating the operation (d) and the operation (e) as a sample nucleic acid, the operation (a) and subsequent steps are performed to obtain a larger amount of the amplified product be able to. Further, the first oligonucleotide and / or the second oligonucleotide used in the operation (f ′) is different from the first oligonucleotide and / or the second oligonucleotide used in the first operation (a). Good. Further, the first oligonucleotide and / or the second oligonucleotide used in the operation (g) may be different from the first oligonucleotide and / or the second oligonucleotide used in the first operation (a). Good.
At this time, by replacing the first and / or second oligonucleotide of the operation (e) with a different oligonucleotide, an amplified product of the specific nucleic acid sequence with higher specificity can be obtained.

Examples of the method of detecting the specific nucleic acid of the present invention include a method of previously labeling the first oligonucleotide and / or the second orthonucleotide, a method of detecting using a labeled nucleic acid probe, and the like. In the method of pre-labeling the first oligonucleotide and / or the second oligonucleotide, among known labels such as radioisotope, enzyme, fluorescent substance, luminescent substance, biotin, hapten, etc., oligonucleotide hybridization or It is preferable to use a substance that does not affect the ligation reaction or the like. Examples of these techniques include the method disclosed in Japanese Patent Application Laid-Open No. 2-2934 as a label for ligation reaction and the method disclosed in Japanese Patent Application Laid-Open No. 1-252300 as a label for extension reaction. These are detected by a general method. In the method using a labeled nucleic acid probe, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, biotin, a hapten or the like is used as a labeled substance.

[0018]

According to the amplification method of the present invention, the amplification reaction is carried out only when the first oligonucleotide is annealed to a specific nucleic acid sequence to be extended and then linked to the second oligonucleotide. Therefore, two specificities are required, the specificity depending on the nucleotide sequence of the oligonucleotide and the specificity satisfying the condition for linking the extension product and the oligonucleotide, and the nonspecific reaction is suppressed accordingly. Moreover, in the repetitive reaction of the operation (d) and the operation (e), one first oligonucleotide is used as a primer, so that even when mishybridization is performed, significant amplification of the non-specific product is not performed. High S / N (Signal /
Noise) ratio is obtained. Also, elongation and ligation reaction is heat resistant D
Using NA polymerase or thermostable DNA ligase, the reaction proceeds simply by changing the temperature of the reaction solution, and amplification can be easily performed. Furthermore, the amplification method of the present invention can increase the S / N (Signal / Noise) ratio without amplification of the probe remaining due to mismatch or non-specific hybridization, as compared with the amplification method using a probe. .. The detection method of the present invention can effectively detect a specific nucleic acid sequence in a sample.

[0019]

EXAMPLES The effects of the present invention will be further clarified by showing Examples and Comparative Examples of the present invention below, but the scope of the present invention is not limited by these Examples. (Reference Example 1) Synthesis of various oligonucleotides Using ABI DNA Synthesizer Model 391, various oligonucleotides having the following sequences were synthesized by the phosphoamidite method. 1) First oligonucleotide: This oligonucleotide has a sequence complementary to the nucleotide sequences from 483 to 504 of Vibrio parahaemolyticus TDH (Thermostable Direct Haemolysin) gene (Sequence Listing 1). 2) Second oligonucleotide: This oligonucleotide has a sequence complementary to the 52nd to 78th nucleotide sequence of the Vibrio parahaemolyticus TDH (Thermostable Direct Haemolysin) gene, a spacer, and a sequence complementary to the first oligonucleotide. Row table 2). In addition, a phosphate group is bound to the 5'end. 3) Third oligonucleotide: This oligonucleotide has a sequence complementary to the nucleotide sequences 400 to 421 of the Vibrio parahaemolyticus TDH (Thermostable Direct Haemolysin) gene (Sequence Listing 3). 4) Fourth oligonucleotide: This oligonucleotide has a sequence complementary to the nucleotide sequence from the 144th to 170th nucleotides of the Vibrio parahaemolyticus TDH (Thermostable Direct Haemolysin) gene, a spacer, and a sequence complementary to the third oligonucleotide. Row table 4). In addition, a phosphate group is bound to the 5'end. 5) Fifth oligonucleotide: This oligonucleotide is an oligonucleotide probe (24mer) (sequence list 5) complementary to the 103rd to 126th sequences of the Vibrio parahaemolyticus TDH (Thermostable Direct Hemolycin) gene. Also,
If necessary, the 5'phosphate group is labeled with ³² P.

The method is 0.2 μM according to the ABI company's manual.
Performed on a scale. Deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C overnight. Purification was carried out by FPLC (Pharmacia) on a reverse phase column. If necessary, the synthesized oligonucleotide had a phosphate group bound to the 5'end by the following method. Oligonucleotide 5-20 pmoles 10x protruding end kinase buffer 10 μl 1 mM ATP 1 μl T4 polynucleotide kinase (Toyobo) 10 units Add water to make 100 μl of the total volume and incubate at 37 ° C for 1 hour. Here, 10 × protruding end kinase buffer means 0.5M Tris-HCl (pH8.0) 0.1M MgCl ₂ 0.1M 2-mercaptoethanol.

Example 1 Amplification Method of Specific Nucleic Acid Operation (a) 40 pmol of the first oligonucleotide of Reference Example 1, 0.4 pmol of the second oligonucleotide, a genome isolated and partially purified from cultured cells of TDH-producing Vibrio parahaemolyticus. Nucleic acid 1ng and 100pg
And were both added to 20 μl of reaction solution. After keeping at 94 ° C for 5 minutes, it was kept at 50 ° 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% TritonX-100 2 mM ATP, GTP, CTP, TTP 100 μM NAD procedure (b) Reaction above Add 4 units of Tth DNA Polymerase (Toyobo) and heat-resistant DNA ligase (EPICENTRE TECHNOLOGIES) to the solution.
0 unit was added, and the mixture was kept warm at 65 ° C for 2 minutes and then kept at 50 ° C for 3 minutes to extend the oligonucleotide and carry out a ligation reaction. Operation (c) Using the extension and ligation reaction products obtained in operation (b) as a template, an amplification reaction was performed according to the following amplification cycle. Amplification cycle Denaturation 94 ° C 60 seconds Annealing 50 ° C 120 seconds Extension reaction 75 ° C 90 seconds Cycle number 30 times (d) After that, electrophoresis was performed on agarose gel and the synthesized DNA was confirmed by ethidium bromide staining (Fig. 3). ). As a result, DNA was synthesized near 480mer. This indicates that the first oligonucleotide was extended and ligated with the second oligonucleotide, and a DNA molecule was synthesized using this extension and ligation product as a template.

Example 2 Amplification Method of Specific Nucleic Acid from Amplification Product Operation (a) 40 pmol of the third oligonucleotide of Reference Example 1, 0.4 pmol of the fourth oligonucleotide, and 1 amplification product obtained in Example 1
Dilute the solution to 100,000 times with 1 μl and 0.1 μl, and add 2
Added to 0 μl reaction. Hold at 94 ℃ for 5 minutes, then at 50 ℃
It was kept warm 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% TritonX-100 2 mM ATP, GTP, CTP, TTP 100 μM NAD procedure (b) Reaction above Add 4 units of Tth DNA Polymerase (Toyobo) and heat-resistant DNA ligase (EPICENTRE TECHNOLOGIES) to the solution.
0 unit was added, and the mixture was kept warm at 65 ° C for 2 minutes and then kept at 50 ° C for 3 minutes to extend the oligonucleotide and carry out a ligation reaction. Operation (c) Using the extension and ligation reaction products obtained in operation (b) as a template, an amplification reaction was performed according to the following amplification cycle. Amplification cycle Denaturation 94 ° C 60 seconds Annealing 50 ° C 120 seconds Extension reaction 75 ° C 90 seconds Cycle number 30 times (d) After that, electrophoresis was performed on agarose gel to confirm the synthesized DNA by ethidium bromide staining (Fig. 4). ). As a result, DNA was synthesized near the 305mer. This indicates that the third oligonucleotide was extended and ligated with the fourth oligonucleotide, and a DNA molecule was synthesized using this extension and ligation product as a template.

(Example 3) The amplification generation part of the agarose gel obtained in the operation (d) of Example 1 was transferred onto a nylon membrane Hybond-n (manufactured by Amwesherm) by Southern blotting. Nylon membrane 6 x SSC, 5 x Danehart's solution, 1 mM
EDTA, 10 μl of boiled salmon semen DNA (average 500 bases)
After pre-hybridizing in 100 μl of a solution containing 60 μl at 60 ° C. for 1 hour, a probe labeled with ³² P at the 5′-terminal phosphate group of the fifth oligonucleotide prepared in Reference Example 1 was added to the above solution at Hybridized for hours. 6 x SSC at 60 ℃
The Nakade nylon membrane was thoroughly washed and then dried. Adhere X-ray film (New AIF RX made by Fuji Photo Industry Co., Ltd.)
Photosensitized at ℃ for 24 hours. The result was that the film was exposed in the same place as the DNA observed by the ethidium bromide staining method. This indicates that the DNA detected by the ethidium bromide staining method is a product obtained by accurately amplifying the target nucleic acid. It also shows that a trace amount of target nucleic acid could be easily detected (Fig. 5).

[0024]

[Sequence listing] SEQ ID NO: 1 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..22 Method by which the characteristics were determined: S Other characteristics: Vibrio parahaemolyticus TDH (Thermostable Direct Haem
(olysin) gene has a sequence complementary to the nucleotide sequence from the 483rd position to the 504th position. Sequence ACTACCACTC TCATATGCTT CT 22

SEQ ID NO: 2 Sequence length: 54 Sequence type: Nucleic acid Topology: Single-stranded Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..27 Method of determining features: S, etc. Features: Vibrio parahaemolyticus TDH (Thermostable Direct Haem
(olysin) gene has a sequence complementary to the 52nd to 78th nucleotide sequences. Location: 28..32 Other features: spacer Location: 33..54 Other features: having a sequence complementary to the first oligonucleotide. Sequence CTCAAAAGCA GATGTTTTGA ATGCAGCAAA AAAGAAGCAT ATGAGAGTGG TAGT 54

SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method of determining: S Other features: Vibrio parahaemolyticus TDH (Thermostable Direct Haem
olysin) has a sequence complementary to the nucleotide sequence from the 400th position to the 421st position of the gene. Array CTTCACCAAC AAAGTTAGCT AC 22

SEQ ID NO: 4 Sequence length: 54 Sequence type: Nucleic acid Topology: Single-stranded Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..27 Method of determining features: S, etc. Features: Vibrio parahaemolyticus TDH (Thermostable Direct Haem
olysin) has a sequence complementary to the nucleotide sequence from the 144th position to the 170th position of the gene. Location: 28..32 Other features: Spacer Location: 33..54 Other features: Having a sequence complementary to the third oligonucleotide. Sequence ACATTGACCG GAGCTTGGGT ATTAAAAAAA AAGTAGCTAA CTTTGTTGGT GAAG 54

SEQ ID NO: 5 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features: Location: 1..24 Method for determining characteristics: S Other characteristics: Vibrio parahaemolyticus TDH (Thermostable Direct Haem
(olysin) gene has a complementary sequence to the 103rd to 126th nucleotide sequences of the gene. Sequence AAACAATATC TCATCAGAAC CGGG 24

[Brief description of drawings]

FIG. 1 is a diagram showing an example of an oligonucleotide of the present invention.

FIG. 2 is a diagram schematically showing the principle of the present invention.

FIG. 3 shows an electrophoretic pattern of the DNA synthesized in Example 1.

FIG. 4 shows the electrophoretic pattern of the DNA synthesized in Example 2.

FIG. 5 shows a photosensitive pattern of an X-ray film exposed in Example 3. The light-sensitive part shows the same part as the DNA observed by the ethidium bromide staining method of Example 1.

[Explanation of symbols]

In FIG. 1, A is a base sequence homologous to a part of the specific nucleic acid sequence, and B is
Is a base sequence complementary to a sequence 3 ′ downstream of a part of the specific nucleic acid sequence. In FIG. 2, 1 is a specific nucleic acid, 2 is a first oligonucleotide,
3 refers to the second oligonucleotide. In FIG. 3, lane 1 corresponds to the markers 2, 3 and 4 corresponding to 1 ng, 100 pg and 0 g of the nucleic acid sample of Example 1, respectively. In FIG. 4, lane 1 is a marker, 2, 3, 4 and 5 are amplification products of Example 1 and 1 μl of nucleic acid sample of Example 2, respectively.
, 0.1 μl and 0 μl.

Claims (12)

[Claims] 1. At least a base sequence A and a base sequence B
Which comprises a base sequence B complementary to a part of the specific nucleic acid sequence in order from the 5 ′ end to the 3 ′ end, and a 3 ′ downstream sequence of the specific nucleic acid sequence. An oligonucleotide having a base sequence A homologous to. 2. A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following operations. Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. 3. A method for amplifying at least one specific nucleic acid sequence contained in a sample, comprising the following steps. Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. And operation (f): the amplification product obtained in operation (e) is separated from the template synthesized in operation (d) and the single-stranded molecule generated in operation (e) is used as a template, and the first oligonucleotide The synthesis of the nucleotide extension product using as a primer is repeated at least once. 4. A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following steps. Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): Operation (c) The linked products are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. And operation (f ′): the above-mentioned operations (a) to (e) are repeated at least once for the amplification product obtained in operation (e). 5. A method for amplifying at least one specific nucleic acid sequence contained in a sample, which comprises the following steps: Operation (a): a first oligonucleotide complementary to a part of a specific nucleic acid sequence and a base sequence B complementary to another part of the specific nucleic acid sequence from the 5 ′ end to the 3 ′ end, A second oligonucleotide having at least a base sequence A complementary to the first oligonucleotide is mixed with a sample and reacted, and the first oligonucleotide and the second oligonucleotide are annealed to the specific nucleic acid sequence in the sample. Operation (b): Using the first oligonucleotide of the annealed product obtained in the operation (a) as a primer, a nucleotide extension reaction is performed up to the 5'end of the second oligonucleotide of the annealed product. Operation (c): The 3'end of the polynucleotide generated by extension in the operation (b) is linked to the 5'end of the second oligonucleotide of the annealed product. Operation (d): The products ligated in operation (c) are separated from the template in which they were synthesized to produce single chain molecules. And operation (e): using the single-stranded molecule generated in operation (d) as a template and using the first oligonucleotide as a primer, a nucleotide extension product is synthesized. Procedure (f): The amplification product obtained in the procedure (e) was separated from the template synthesized in the procedure (d) and the single-stranded molecule generated in the procedure (e) was used as a template, and the first oligonucleotide was added. The synthesis of the nucleotide extension product used as the primer is repeated at least once. And operation (g): the above-mentioned operations (a) to (e) or operations (a) to (f) are repeated at least once for the amplification product obtained in operation (f). 6. The first oligonucleotide and / or the second oligonucleotide used in the operation (f ′) is the first oligonucleotide and / or the second oligonucleotide used in the first operation (a). 5. The method for amplifying a nucleic acid sequence according to claim 4, which is different from 7. The first oligonucleotide and / or the second oligonucleotide used in step (g) is the first oligonucleotide.
The method for amplifying a nucleic acid sequence according to claim 5, which is different from the first oligonucleotide and / or the second oligonucleotide used in the operation (a). 8. A first oligonucleotide and / or a second oligonucleotide according to claim 2, a ligase, a nucleic acid polymerase and / or a reverse transcriptase, four kinds of deoxynucleotide triphosphates, and a reaction buffer. A reagent kit for amplifying a target nucleic acid, comprising: 9. The operation (a) (b) according to claim 2, wherein the first oligonucleotide and / or the second oligonucleotide labeled with the first oligonucleotide and / or the second oligonucleotide according to claim 2 is used. (C)
(D) and (e), or the operation (a) according to claim 3.
(B) (c) (d) (e) and / or (f) or operation (a) (b) (c) (d) (e) according to claim 4.
A method for detecting a nucleic acid sequence, which comprises detecting the target nucleic acid sequence by measuring the label after carrying out (f ′) and (f ′). 10. The operation (a) (b) according to claim 9.
(C) (d) and (e), or (a) (b) (c)
(D) (e) and (f), or (a) (b) (c)
(D) After carrying out (e) and (f '), the product is added with a labeled oligonucleotide probe capable of hybridizing with the sequence to be detected and / or its variant, and the target nucleic acid sequence. A method for detecting a nucleic acid sequence, which comprises detecting 11. A first oligonucleotide and / or a second oligonucleotide labeled with the first oligonucleotide and / or the second oligonucleotide according to claim 2, a ligase, a nucleic acid polymerase and / or a reverse transcriptase, and 4 kinds. A reagent kit for detecting a target nucleic acid, comprising a deoxynucleotide triphosphate, a reaction buffer, and a system for detecting a label. 12. The first oligonucleotide and / or the second oligonucleotide according to claim 2, a ligase,
A reagent kit for detecting a target nucleic acid sequence comprising a nucleic acid polymerase and / or a reverse transcriptase, four kinds of deoxynucleotide triphosphates, a reaction buffer, a labeled oligonucleotide probe and a system for detecting a label.