JPH10276776A - Reversibly inactivated heat-resistant dna polymerase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new reversibly inactivated heat-resistant DNA polymerase modified with a dicarboxylic acid anhydride, not generating a nonspecific band, not forming the dimer of a primer, and useful as a reagent for amplifying nucleic acids, etc. SOLUTION: This new reversibly inactivated heat-resistant DNA polymerase modified with a dicarboxylic acid anhydride can eliminate problems on PCR, such as the generation of back grounds (nonspecific band) due to nonspecific amplification and the formation of a primer dimer, etc. The heat-resistant DNA polymerase is obtained by separating the chromosomal DNA of Thermus thermophilus HB8 by a conventional method, cleaving with a restriction enzyme, binding to a DNA, transforming Escherichia coli, selecting a colony having a DNA polymerase activity to obtain a gene, expressing the gene, and subsequently reacting the obtained heat-resistant DNA polymerase with 2,3- dimethylmaleic anhydride, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thermostable DNA polymerase reversibly inactivated, and more particularly to a thermostable DNA polymerase reversibly inactivated by being modified with dicarboxylic anhydride. And a nucleic acid amplification method using the enzyme and a reagent therefor.

[0002]

2. Description of the Related Art Conventionally, polymerase chain reaction (PC)
Many studies have been made on thermostable DNA polymerases used in techniques for amplifying nucleic acids such as R). The thermostable DNA polymerase used in the PCR reaction is mainly a DNA polymerase derived from Thermusthermophilus (Tth polymerase) or a thermostable DNA polymerase.
DNA polymerase (Taq polymerase) derived from Aquaticus (Thermus aquaticus) and the like have been used. Further, a DNA polymerase derived from a hyperthermophilic archaeon, such as a thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase, WO
92/9689, JP-A-5-328969), heat resistance D derived from Thermococcus litoralis
NA polymerase (Tli polymerase, JP-A-6-7160), thermostable DNA polymerase (KOD polymerase) derived from Pyrococcus sp. KOD1, and the like are known.

[0003] However, many nucleic acid amplification methods using these thermostable DNA polymerases have problems of background (non-specific band) generation due to non-specific amplification and formation of primer dimer. I have.
In these amplification reactions, in many cases, a reaction solution is prepared, and by the time the reaction temperature reaches the annealing temperature of the primer, the primer is annealed to a non-specific site (mispriming) and synthesis by DNA polymerase is started. It is said that this is the cause.

In order to overcome such problems, DNA
A method in which one of the components essential for synthesis (DNA polymerase, magnesium, etc.) is added to the reaction solution after reaching the annealing temperature (hot start PCR, Chou et al.,
Nucleic Acids Res, Vol.20p.1717-1723 (1992)) has been developed. However, this method requires a very troublesome operation of opening the lid of the reaction tube set in the reaction apparatus and adding the reagent thereto, and has the drawback of causing cross-contamination.

[0005] Recently, several reagents have been developed to make the hot start PCR method easier. For example, wax that melts at high temperature and becomes liquid (trade name Ampli
-Wax Perkin Elmer) or an antibody against DNA polymerase (Tth start, Taqstart Clontech) is used. In a method using wax, a layer is formed with this wax, and a solution containing a primer and dNTP is put in a lower layer, a DNA polymerase and a solution containing a template DNA are put in an upper layer, and the reaction is started. Both can be separated, and the background due to mispriming can be reduced. In addition, in the method using an antibody, since the polymerase activity is suppressed by the antibody, the DNA synthesis reaction does not occur until the temperature at which the antibody is deactivated (usually 70 ° C. or lower, see JP-A-6-209775). To
As in the case of using wax, the background can be reduced. However, a method using wax requires a complicated operation for forming a wax layer. When an antibody is used, the antibody is generally very expensive, and there is a problem in industrial use.

[0006]

Recently, a DNA polymerase whose polymerase activity is released by high temperature has been developed (trade name: AmpliTaq Gold, manufactured by PerkinElmer). When this enzyme is used, hot start can be performed without using wax or antibodies as described above. However, this enzyme requires a heat treatment time of 9 to 12 minutes at 92 to 95 ° C. in order to perform PCR efficiently. In general, when DNA is treated at a high temperature of 90 ° C. or more for a long time, decomposition such as depurination occurs, and particularly in the case of a long target, there is a problem that the amplification efficiency is not sufficient. Therefore, a novel thermostable DNA polymerase that solves these problems has been long-awaited.

[0007]

Means for Solving the Problems In view of the above, the present inventors have proposed a method for amplifying non-specific by-products and primer dimers.
In order to overcome the problems in R and increase the accuracy and sensitivity of PCR, the inventors have conducted intensive studies to find an improved thermostable DNA polymerase. As a result, by modifying the thermostable DNA polymerase with dicarboxylic anhydride, the polymerase activity was improved. Is reversibly inactivated (ie, the relative activity after chemical modification is reduced to 0 to 25% relative to the unmodified enzyme, but the recovery of the relative activity after heat treatment is 50 to 50%).
100%).
Furthermore, it has been found that by performing PCR using the enzyme, it is possible to amplify a long target DNA with a low background and complete the present invention.

[0008] That is, the present invention is a reversibly inactivated thermostable DNA polymerase modified with a dicarboxylic anhydride.

The present invention is a method for synthesizing a nucleic acid, comprising reacting the above-mentioned thermostable DNA polymerase with DNA as a template in the presence of a primer and dNTP to extend the primer and synthesize the DNA.

According to the present invention, (1) double-stranded DNA is converted into single-stranded DNA if necessary, and
The reaction of the heat-resistant DNA polymerase according to any one of claims 1 to 4 in the presence of at least two primers and dNTP to perform chain extension to obtain a double-stranded DNA, and then (2) strand separation Then, (3) using the separated single-stranded DNA as a template, reacting the above-mentioned thermostable DNA polymerase in the presence of at least two primers and dNTP, and further performing the above steps (1) to (3)
And a method for amplifying a nucleic acid.

[0011] The present invention is a reagent for nucleic acid synthesis containing a primer, dNTP and the above-mentioned thermostable DNA polymerase.

The present invention is a reagent for nucleic acid synthesis containing a primer, dNTP and the above-mentioned thermostable DNA polymerase and a reagent for nucleic acid detection containing a detection probe.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The thermostable DNA polymerase used in the present invention is a DNA polymerase having a so-called thermostability of 70 to 100 ° C., as long as it has thermostability that can be used for PCR. It is not something to be done. Examples of such an enzyme include Tth polymerase, Taq polymerase, Pfu polymerase, KOD polymerase, Tli polymerase, a DNA polymerase derived from Thermotoga maritima, and a DNA polymerase derived from Mormosipo africanus. These thermostable DNA polymerases are not limited to those produced by natural microorganisms and the like, but also include those produced by gene recombination techniques. Furthermore, a thermostable DNA produced from a gene obtained by modifying a gene encoding an enzyme collected from a natural microorganism or the like.
Polymerases such as ΔTth, ΔTaq and the like are also included. These thermostable DNA polymerases are usually not reversibly inactivated. Not reversibly inactivated means that the relative activity at room temperature and the relative activity after heat treatment hardly change.

The dicarboxylic anhydride used in the present invention is not particularly limited, but includes citraconic anhydride, 2-methylmaleic anhydride, 2,3-dimethylmaleic anhydride,
Maleic anhydrides such as cis aconitic anhydride, succinic anhydrides such as tetrafluorosuccinic anhydride, exoses-3,6-endoxo- △ -tetrahydrophthalic anhydride, exosis 3,6-endoxohexahydrophthalic anhydride And the like. As the dicarboxylic anhydride, succinic anhydride, maleic anhydride or a derivative thereof, for example, 2-methylmaleic anhydride, 2,2
3-dimethylmaleic anhydride or tetrafluorosuccinic anhydride is preferred.

[0015] The reversibly inactivated heat-resistant DN of the present invention.
As a method for producing A polymerase, first, a solution containing a heat-resistant DNA polymerase is dialyzed against a buffer such as a sodium carbonate buffer having a pH of 8.5 to 10 to obtain a concentrated solution. A solution containing the above acid anhydride is added to the enzyme solution, and the mixture is reacted at 4 to 40 ° C., preferably at room temperature, for 5 minutes to 1 hour. In addition, since the pH may decrease during the reaction, it is preferable to maintain the pH by adding sodium hydroxide or the like as needed.

The reversibly inactivated thermostable DNA polymerase modified with the dicarboxylic anhydride obtained according to the present invention has a relative activity of 0 to 25% relative to the unmodified enzyme after chemical modification. Active, but at 92 ° C 5
Heat treatment was performed for 10 minutes to confirm the recovery of the relative activity. As a result, about 50 to 100% of the activity of the unmodified enzyme was maintained.

In the present invention, DNA is synthesized by reacting the reversibly inactivated thermostable DNA polymerase with DNA as a template in the presence of a primer and dNTP to extend the primer. Primers are about 15-30 oligonucleotides having a sequence complementary to the template. The reaction conditions are about pH 8.0 to
At 9.0 (value at room temperature), heat at about 85-95 ° C. Also, depending on the thermostable DNA polymerase to be used, necessary buffers, metal ions and other reagents such as BSA, a surfactant, and salts such as ammonium sulfate are added.

According to the present invention, (1) double-stranded DNA is converted into single-stranded DNA if necessary, and
The above-mentioned heat-resistant DNA polymerase is reacted in the presence of at least two primers and dNTP to perform chain extension to obtain double-stranded DNA. Then, (2) strand separation is performed, and then (3) separated. Using the single-stranded DNA as a template and the above-mentioned reversibly inactivated thermostable DNA polymerase in the presence of at least two primers and dNTP, and further comprising the steps (1) to (3)
Is repeated to amplify the nucleic acid.

The nucleic acid synthesis reagent of the present invention comprises a primer,
dNTPs and the reversibly inactivated heat-resistant DN described above
Contains A polymerase. If necessary, buffers, metal ions and other reagents such as BSA, surfactants and salts such as ammonium sulfate are contained.

The reagent for detecting nucleic acid of the present invention comprises a primer,
dNTPs and the reversibly inactivated heat-resistant DN described above
It contains a nucleic acid synthesis reagent containing A polymerase and a detection probe. The detection probe is about 15 to 100 oligonucleotides having a sequence complementary or homologous to the base sequence of the nucleic acid to be detected.
The method for detecting a nucleic acid is performed on a nucleic acid amplified using the reversibly inactivated thermostable DNA polymerer of the present invention,
The detection probe is allowed to react, and the detection probe that has hybridized or the detection probe that has not hybridized is measured. A labeling substance such as an enzyme, a fluorescent substance or a radioactive substance or a reagent capable of reacting with a substance having the labeling substance such as biotin or avidin may be bound to the detection probe. Detection of these labeling substances follows a conventionally known method.

The reversibly inactivated heat-resistant D of the present invention
When PCR is performed using NA polymerase, there is an advantage that the amount of primer dimers is smaller than when PCR is performed using a DNA polymerase that is not chemically modified. Also, as in the present invention, AmpliTaq Gold, an enzyme whose activity is restored by heat treatment
In the case of using, the amplification product could not be confirmed only after treatment at 94 ° C for 1 minute, while using the reversibly inactivated thermostable DNA polymerase of the present invention,
A sufficient amplification product can be obtained even with a treatment for 1 minute. Also, 9
Even if the treatment is carried out for minutes, the amplification product is more when the reversibly inactivated thermostable DNA polymerase of the present invention is used. In addition, the heat-resistant D of the present invention is reversibly inactivated.
When PCR is performed using NA polymerase, amplification of a desired long-chain nucleic acid is possible.

[0022]

Next, the present invention will be described with reference to examples, but these examples do not limit the present invention in any way.

Reference Example 1 Purification of Tth Polymerase The chromosomal DNA of Thermos thermophilus HB8 was isolated by the following method. First, 150 ml of 1.5%
Polypeptone, 1.5% yeast extract, 0.2% NaCl
After shaking culture at 70 ° C. overnight in a liquid medium consisting of (pH 7.5), the cells were collected by centrifugation (8000 rpm, 10 minutes). 10 mM Tris-HCl (pH 8.0), 1 mM ED
Suspend in 24 ml of a solution containing TA, add 3 ml of lysozyme solution (20 mg / ml), then add 10%
6 ml of the SDS solution was added. Add 30 ml of phenol and mix gently with stirring at 10,000 rpm, 1
The aqueous layer and the solvent layer were separated by centrifugation for 5 minutes, and the aqueous layer was separated. This operation was performed again, and a double volume of ethanol was gently overlaid on the aqueous layer, and the DNA was sprinkled on the glass rod while slowly stirring with a glass rod to separate the DNA. This was added to 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
Was dissolved in a solution containing (hereinafter referred to as TE). This was treated with an equal volume of a chloroform / phenol solution, and the aqueous layer was separated by centrifugation, twice the amount of ethanol was added, the DNA was separated again by the above method, and dissolved with 2 ml of TE.

1 μg of the DNA obtained in the above step was digested with restriction enzyme H
IndIII (manufactured by Toyobo Co., Ltd.) was reacted at 37 ° C. for 15 minutes with 5 units to carry out limited digestion.
One unit of A ligase (manufactured by Toyobo) was reacted at 16 ° C. for 12 hours to ligate DNA. The ligated DNA was transformed using competent cells of Escherichia coli JM109.
About 1 × 10 ⁶ transformant colonies were obtained per μg of DNA used. The resulting colony was 50 μg /
L medium (1% polypeptone, 0.
5% yeast extract, 0.5% sodium chloride, 0.25m
(MIPTG) at 37 ° C. for 18 hours, the cells were crushed, and the heat-resistant DNA polymerase activity was measured using a liquid treated at 75 ° C. for 30 minutes according to the activity measurement method described in the following purification step.

Heat-resistant DNA of about 3,000 colonies
When the polymerase activity was measured, two heat-resistant DNs
A colony having A polymerase activity was obtained. In these plasmids, an inserted DNA fragment of about 6 kbp containing one HindIII site was present, and this plasmid was designated as pUPLO.

Next, the inserted DNA fragments of about 2.8 kbp and 3.2 kbp were cut out from pUPL0 with HindIII and introduced into pUC19, respectively.
1 and pUPL02 were constructed. Each was transformed into Escherichia coli JM109, and after culturing the transformants, the activity of the thermostable DNA polymerase was measured by the above method, and no activity was observed. Then p
A 1.4 kbp fragment obtained by treating UPL02 with PstI and HindIII was introduced into pUC19, and
3 was constructed.

Further, a 3.2 kbp fragment obtained by treating pUPL01 with HindIII was digested with HindIII of pUPL03.
It was introduced into the dIII site to construct pUPL1. This p
A strain (Escherichia coli JM109 (pUPL1)) in which Escherichia coli JM109 was transformed with UPL1 had about twice the productivity of thermostable DNA polymerase than Thermos thermophilus HB8.

The obtained Escherichia coli JM109 (pUPL1)
Culture and purification were carried out using
2 ml of a solution containing 0000 units / ml of Tth DNA polymerase was obtained. First, the sterilized 100
TB medium (ColdSpri) containing μg / ml ampicillin
ng Habour Laboratory, Molecular Cloning, p. A.
6L) was dispensed into a 10L jar fermenter (two jar fermenters were used). This medium contained 100 μg / ml ampicillin in advance.
Escherichia coli JM109 cultured in 0 ml of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, Gibco) for 16 hours at 30 ° C.
(PUPL1) (using a 500 ml Sakaguchi flask) was inoculated, and cultured with aeration and agitation at 35 ° C. for 12 hours. The cells were recovered from the culture solution by centrifugation, and 400 ml of a disruption buffer (10 mM Tris-HCl (pH 8.0), 80 m
M KCl, 5 mM 2-mercaptoethanol, 1 m
After suspension in MEDTA), the cells were disrupted by sonication to obtain a cell lysate. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Further, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate fractionation, and heparin sepharose chromatography (Pharmacia) were performed. Finally, a shape buffer (50 mM) was used.
Tris-HCl (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 50% glycerin)
Was replaced with

The DNA polymerase activity in the above purification step was measured by the following procedure. When the enzyme activity is high, the storage buffer (50 mM Tris-HCl (Ph
8.5), 50 mM potassium chloride, 1 mM dithiothreitol, 50% glycerin, 0.1% Tween 20,
The sample was diluted with 0.1% nonidet P40) for measurement.

(Reagent) A. B. 670 mM Tris-HCl (pH 8.8) 166 mM ammonium sulfate 67 mM magnesium chloride 10 mM 2-mercaptoethanol 1 μg / μl activated calf thymus DNA 2 mM dNTP (250 cpm / pmol [ ³
H] dTTP) E. 20% trichloroacetic acid (containing 2 mM pyrophosphate) 1μg / μl carrier calf thymus DNA

(Method) 5 μl each of solution A, solution B and solution C and 30 μl of sterile water were added to an Eppendorf tube and mixed by stirring, and then the cell lysate or 5 μl of the above heat-treated solution was added.
And react at 75 ° C. for 10 minutes. Thereafter, the mixture is cooled on ice, 50 μl of Solution E and 100 μl of Solution D are added, and the mixture is further cooled on ice for 10 minutes. This solution was filtered through a glass filter (Whatman GF / C filter), washed sufficiently with solution D and cold ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (manufactured by Packard) to incorporate nucleotides into template DNA. Is measured. One unit of the enzymatic activity was the amount of the enzyme that incorporated 10 nmol of nucleotides into the acid-insoluble fraction per 30 minutes under these conditions.

Example 1 Chemical Modification of Tth Polymerase (Part 1) The Tth polymerase obtained in Reference Example 1 was chemically modified by the following procedure . That is, 5 ml of the enzyme solution was added to a sodium carbonate buffer (50 mM sodium carbonate (pH 9.3), 10 ml).
Dialysis was performed against 0 mM NaCl). 500ml
Was changed three times every 6 hours. This operation reduced the liquid volume to 10.5 ml. 5 ml of this enzyme solution
20 μl of a 1M 2,3-dimethyl maleic anhydride solution (manufactured by Aldrich) (solvent: N-methyl-2-pyrrolidone (manufactured by Nacalai Tesque)) was added to the mixture while gradually stirring. Then, it was left at room temperature for 5 minutes (this chemically modified product was designated as enzyme A).

Example 2 Chemical Modification of Tth Polymerase (Part 2) Using 100 μl of 1M 2,3-dimethyl maleic anhydride solution (manufactured by Aldrich), p
A chemically modified Tth polymerase was obtained in the same manner as in Example 1 except that H was adjusted to be 8.5 or less (this chemically modified product is referred to as enzyme B).

Example 3 Confirmation of Activity of Chemically Modified Product The activity of the chemically modified Tth polymerase obtained in Examples 1 and 2 was confirmed by the method described in Reference Example 1. As a control, Tth polymerase that had just been replaced with the sodium carbonate buffer obtained in Example 1 was used (this enzyme is referred to as unmodified Tth). At the time of activity measurement, the enzyme solution was diluted 1000-fold with a storage buffer. As a result, the unmodified Tth
15% enzyme A and 7% enzyme B relative to polymerase
(See FIG. 1).

Example 4 Confirmation of Recovery of Activity of Chemically Modified Products The enzymes A, B and unmodified Tth obtained in Examples 1 and 2 were dialyzed against a storage buffer. 500 ml of buffer was changed three times every 6 hours. In addition, the liquid volume was reduced to 3 ml by this operation. Next, p
Heat treatment was performed in three kinds of buffers having different H, and the recovery of the activity was confirmed. pH 8.0, pH 8.3 and pH 8.8
(PH at room temperature) in buffer (10 mM Tris-HC)
1, 80 mM potassium chloride, 1.5 mM magnesium chloride, 0.5 mg / ml BSA, 0.1% sodium cholate, 0.1% Triton X-100) 1
5 μl of enzyme diluted 100-fold with storage buffer to 00 μl
In addition, it was processed at 92 ° C. 1. After 0, 5, and 10 minutes
Samples were taken at 5 μl each and the activity was measured by the method described in Reference Example 1. The result is shown in FIG. As shown in FIG. 2, the activity of the chemically modified enzymes A and B was restored by the heat treatment. In contrast, the enzyme which had not been subjected to the chemical modification treatment hardly changed.

Example 5 PCR using chemically modified Tth polymerase PCR was performed using the chemically modified DNA polymerase obtained in Examples 1 and 2. That is, 50 μl of a reaction solution (10 mM Tris-HCl (pH 8.8), 80 m
M potassium chloride, 1.5 mM magnesium chloride,
0.5 mg / ml BSA, 0.1% sodium cholate, 0.1% Triton X-100, 0.2 mM
dNTP, 100 ng genomic DNA from human placenta
(Manufactured by Clonetech) 10 picomoles of Sequence Listings 1 and 2
2.5 units of enzyme A or enzyme B were added to the primers described) to perform a PCR reaction. The enzymatic activity was determined by the method described in Reference Example 1 after treatment at 80 ° C. for 30 minutes in the pH 8.0 buffer described in Example 4. The model PJ2000 manufactured by PerkinElmer was used as the thermal cycler. The reaction conditions were as follows: treatment at 94 ° C. for 1 minute;
° C, 30 seconds → 55 ° C, 1 minute → 72 ° C, 1 minute 30
Cycled.

As a comparison, unmodified Tth polymerase,
Tth start (Tth antibody, Clontech)
AmpliTaq G, Tth polymerase mixed with
Old (Perkin Elmer) was also subjected to a PCR reaction in the same manner. However, the composition of the reaction solution was in accordance with each instruction manual. AmpliTaq Gold is 9
The treatment at 4 ° C. was performed for 1 minute and 9 minutes. After reaction, 5μ
An agarose gel electrophoresis was performed on 1 l of the reaction solution, and amplification of a target of about 0.4 kb was confirmed.

FIG. 3 shows the results of agarose gel electrophoresis. As a result, when PCR was performed using the chemically modified enzymes A and B, the amount of primer dimer was smaller than that obtained by PCR using unmodified Tth polymerase. In AmpliTaq Gold, 94
Amplification products could not be confirmed only after treatment at 1 ° C. for 1 minute. In addition, the amount of the amplification product after the treatment for 9 minutes is as follows.
Less than in case B.

Example 6 Long PCR using chemically modified Tth polymerase PCR was carried out using the chemically modified DNA polymerase obtained in Examples 1 and 2. That is, 50 μl of a reaction solution (10 mM Tris-HCl (pH 8.8), 80 m
M potassium chloride, 1.5 mM magnesium chloride,
0.5 mg / ml BSA, 0.1% sodium cholate, 0.1% Triton X-100, 0.2 mM
dNTP, 100 ng of human placenta-derived genomic DNA (manufactured by Clontech), 10 picomoles of the primers described in Sequence Tables 3 and 4, 0.15 units of Pfu DNA polymerase (manufactured by Stratagene)) and enzyme A or enzyme B Was added to perform a PCR reaction. The model PJ2000 manufactured by PerkinElmer was used as the thermal cycler. The reaction conditions were as follows: treatment at 94 ° C. for 1 minute;
30 ° C., 30 seconds → 68 ° C., 6 minutes.

As a comparison, a PCR reaction was carried out for unmodified Tth polymerase in the same manner. After completion of the reaction, 5 μl of the reaction solution was subjected to agarose gel electrophoresis to obtain about 8.
The amplification of the 5 kb target was confirmed. FIG. 3 shows the results of agarose gel electrophoresis. As a result, when PCR was performed using the chemically modified enzymes A and B, amplification of the target 8.5 kb target was confirmed. When PCR was performed using unmodified Tth polymerase, amplification of the target could not be confirmed.

[0041]

EFFECT OF THE INVENTION The reversibly inactivated heat resistance D of the present invention
By using NA polymerase, it is possible to efficiently amplify a nucleic acid without requiring a complicated operation and without using an expensive antibody. Also, the use of the reversibly inactivated thermostable DNA polymerases of the present invention allows amplification and detection of small amounts of target nucleic acids by the polymerase chain reaction (PCR).
Furthermore, non-specific by-products, primer dimers, and the like are not amplified.

[0042]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA GAAGAGCCAA GGACAGGTAC 20

SEQ ID NO: 2 Sequence length: 21 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA GGAAAATAGA CCAATAGGCA G21

SEQ ID NO: 3 Sequence length: 35 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA TGATAGGCAC TGACTCTCTG TCCCTTGGGC TGTTT 35

SEQ ID NO: 4 Sequence length: 35 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA ACATGATTAG CAAAAGGGCC TAGCTTGGAC TCAGA 35

[Brief description of the drawings]

FIG. 1 is a diagram showing a decrease in the activity of a chemically modified DNA polymerase.

FIG. 2 shows the recovery of the activity of a chemically modified DNA polymerase.

FIG. 3. P using chemically modified DNA polymerase
It is a photograph replacing a figure by electrophoresis showing a result of CR.

FIG. 4. P using chemically modified DNA polymerase
It is a photograph replacing a figure by electrophoresis showing a result of CR.

Claims (8)

[Claims] 1. A reversibly inactivated thermostable DNA polymerase modified with a dicarboxylic anhydride. 2. The method of claim 1, wherein the dicarboxylic anhydride is succinic anhydride,
2. A maleic anhydride or a derivative thereof.
The thermostable DNA polymerase according to the above. 3. The heat-resistant composition according to claim 2, wherein the derivative of succinic anhydride or maleic anhydride is 2-methylmaleic anhydride, 1,2-dimethylmaleic anhydride or tetrafluorosuccinic anhydride. Sex DNA polymerase. 4. The thermostable DNA polymerase according to claim 1, wherein the thermostable DNA polymerase is Tth polymerase, Taq polymerase, Pfu polymerase, KOD polymerase, Tli polymerase or a mutated DNA polymerase thereof. 5. A method using DNA as a template, a primer and d
A nucleic acid synthesis method comprising reacting the heat-resistant DNA polymerase according to any one of claims 1 to 4 in the presence of NTP to extend a primer and synthesize DNA. 6. The method according to claim 1, wherein the double-stranded DNA is, if necessary, a single-stranded DNA, and the single-stranded DNA is used as a template in the presence of at least two primers and dNTP.
After reacting with the heat-resistant DNA polymerase according to any one of (1) to (4) to obtain a double-stranded DNA by chain elongation,
The heat-resistant DNA polymerase according to any one of claims 1 to 4, wherein (2) strand separation is performed, and (3) the separated single-stranded DNA is used as a template in the presence of at least two primers and dNTP. And further repeating the above steps (1) to (3). 7. The primer, dNTP and claim 1
A reagent for nucleic acid synthesis containing the thermostable DNA polymerase according to any one of claims 4 to 4. 8. The primer, dNTP and claim 1
5. A reagent for nucleic acid synthesis containing the thermostable DNA polymerase according to any one of 4 and a reagent for nucleic acid detection containing a detection probe.