JP3463780B2 - DNA polymerase composition for nucleic acid amplification - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA polymerase composition for nucleic acid amplification, a nucleic acid amplification reagent containing the composition, and a nucleic acid amplification method using the reagent.

[0002]

2. Description of the Related Art The polymerase chain reaction (PC
There have been many studies on thermostable DNA polymerases used in techniques for amplifying nucleic acids such as R). Thermostable DNA polymerases used in the PCR reaction are mainly DNA polymerases derived from Thermus thermophilus (Tth polymerase) and DNA polymerases derived from Thermus aquaticus (Taq polymerase). Have been used. Also,
DNA polymerase derived from hyperthermophilic archaeon, for example, thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase, WO92 / 096
89, JP-A-5-328969), Thermus litoralis (The
thermostable DNA polymerase from rmococcus litoralis)
(Tli polymerase, JP-A-6-7160) and the like are known.

The present inventors have found that Pyrococcus sp. Thermostable DNA polymerase derived from KOD1 (KOD polymerase
7-298879 bulletin). Furthermore, the present inventors have found that Pyrococcus sp. Succeeded in producing a modified enzyme in which the 3′-5 ′ exonuclease activity was reduced to at least 5% or less as compared with the enzyme before modification while maintaining the DNA synthesis rate and thermostability of the KOD1-derived polymerase. did. The enzyme has a DNA synthesis rate of at least 30 bases / sec and a pH of 8.8 (25 ° C).
It is difficult to measure pH at 95 ° C. ) Is a thermostable DNA polymerase capable of retaining 60% or more of residual activity after treatment at 95 ° C. for 6 hours, and having a 3′-5 ′ exonuclease activity of at least 5 as compared with the enzyme before modification. It is an enzyme that has decreased to less than or equal to%. It was also found that the use of the enzyme increases the amplification efficiency as compared with the use of the enzyme before modification.

On the other hand, as one of the methods for amplifying long-chain nucleic acid, Taq polymerase (KlenTaq-278) lacking 3'-5 'exonuclease activity and Pfu polymerase or Tli polymerase having 3'-5' exonuclease activity. Alternatively, a method of performing PCR using a DNA polymerase composition in which these mutant enzymes are mixed has been reported (Barns, WM (1994) Proc. Natl. Acad. Sci. U.
SA 91, 2216-2220). In addition, Tth polymerase which does not show 3′-5 ′ exonuclease activity and Pfu polymerase or Tli polymerase which shows 3′-5 ′ exonuclease activity, Thermotoga maritima (Thermotog
a method of performing PCR using a polymerase composition in which a thermostable DNA polymerase derived from A. maritima) is mixed (Japanese Patent Laid-Open No. 8-38198).

[0005]

However, although these compositions have improved amplification efficiency as compared with the case of using one kind of DNA polymerase, they are stable in heat and DN.
Since two kinds of DNA polymerases having different A synthesis rates are used, the amplification efficiency cannot be said to be sufficient, and a method having a higher amplification efficiency has been desired.

[0006]

Therefore, as a result of intensive studies, the present inventors have found that a DNA polymerase composition for amplifying a nucleic acid, which has a smaller amount than the first DNA polymerase when measured with a first DNA polymerase and a polymerase activity unit. Of the second DNA polymerase, and the 3′-5 ′ exonuclease activity is higher than that of the DNA polymerase having substantially the same thermostability and DNA synthesis rate, specifically the first DNA polymerase. 0 to 5% modified thermostable DNA polymerase, and wherein said second DNA polymerase has a naturally occurring 3'-5 'exonuclease activity, or the naturally occurring enzyme, 3'-5 'exonuclease activity is 100
By using a DNA polymerase composition that is a DNA polymerase selected from the group consisting of modified DNA polymerases that are ~ 6%, P with excellent amplification efficiency can be obtained.
The present invention has been achieved by finding that CR can be performed.

That is, the present invention is modified thermostable having 3'-5 'exonuclease activity, which is 0 to 5%, as compared with thermostable DNA polymerase having 3'-5' exonuclease activity before modification. 100% to 6% as compared with a DNA polymerase (first DNA polymerase) and a thermostable DNA polymerase having a 3′-5 ′ exonuclease activity or a thermostable DNA polymerase having a 3′-5 ′ exonuclease activity before modification. A modified thermostable DNA polymerase having a 3'-5 'exonuclease activity (second DNA polymerase), comprising a first DNA polymerase and a second DNA
DNA in which the polymerase is at least 30 bases / second
95 at a synthesis rate of pH 8.8 (measured value at 25 ° C)
A DNA polymerase composition for nucleic acid amplification, which has a thermostability capable of retaining a residual activity of 60% or more after a treatment at 6 ° C for 6 hours.

Further, the present invention is a nucleic acid amplification method characterized by reacting a primer, dNTP and the above DNA polymerase composition using DNA as a template to extend the primer to synthesize a DNA primer extension product. .

Further, the present invention provides two primers, one of which is complementary to the DNA extension product of the other primer, dNTP and the above DNA polymerase composition, a divalent ion, a monovalent ion and a buffer. It is a reagent for nucleic acid amplification containing.

[0010]

DETAILED DESCRIPTION OF THE INVENTION

The first DNA polymerase of the present invention is 0 to 5%, preferably 1% compared to the thermostable DNA polymerase having 3'-5 'exonuclease activity before modification.
It is an enzyme having a 3'-5 'exonuclease activity reduced to less than or equal to%.

As such a first DNA polymerase, an amino acid sequence in which at least one of the 141st, 142nd, 143rd, 210th and 311th amino acids of the amino acid sequence of SEQ ID NO: 2 is replaced with another amino acid. There are enzymes that have. Examples thereof include an enzyme in which the 141st aspartic acid of SEQ ID NO: 2 is replaced with alanine, an enzyme in which the 143rd glutamic acid of SEQ ID NO: 2 is replaced with alanine, and the 141st aspartic acid and 143th glutamic acid of SEQ ID NO: 2. With the alanine substituting, the enzyme with the 210th asparagine of SEQ ID NO: 2 substituted with aspartic acid, 311 of SEQ ID NO: 2
An enzyme in which the second tyrosine is replaced with phenylalanine can be mentioned. It also includes an enzyme in which isoleucine at the 142nd position in SEQ ID NO: 142 is replaced with arginine.

The first DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
It has a 3'-5 'exonuclease activity that is 0-5%. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours.

The first DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
It has a 3'-5 'exonuclease activity that is 0-5%. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: First of the amino acid sequences set forth in SEQ ID NO: 2
Amino acid sequence in which at least one of the 41st, 142nd, 143rd, 210th and 311th amino acids has been replaced with another amino acid

The first DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
It has a 3'-5 'exonuclease activity that is 0-5%. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88 to 90 KDa Amino acid sequence: 141st aspartic acid of SEQ ID NO: 2 to alanine, 142nd isoleucine to arginine, 143rd glutamic acid to alanine,
An amino acid sequence in which the 141st aspartic acid and the 143rd glutamic acid are replaced with alanine, the 210th asparagine is replaced with aspartic acid, and the 311th tyrosine is replaced with phenylalanine.

The second DNA polymerase of the present invention has a 3 '
Compared with a thermostable DNA polymerase having a 5 ′ exonuclease activity or a thermostable DNA polymerase having a 3′-5 ′ exonuclease activity before modification, 1
3'-5 'which is from 0 to 6%, preferably from 90 to 30%
Modified thermostable DN having exonuclease activity
A polymerase. Examples of such a second DNA polymerase include an enzyme having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid obtained by substituting the 140th, 142th and 144th amino acids of the amino acid sequence shown in SEQ ID NO: 2 with another amino acid. There are enzymes that have sequences. Examples thereof include an amino acid sequence in which the 142nd isoleucine of SEQ ID NO: 2 is replaced with aspartic acid, glutamic acid, asparagine, glutamine or lysine, or an enzyme in which the 144th threonine is replaced with valine.

The second DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has DNA synthesis activity and has 3′-5 ′ exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: amino acid sequence set forth in SEQ ID NO: 2

The second DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
100% to 6%, preferably 90 to 30% 3'-
It has a 5'exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Amino acid sequence: the amino acid sequence present in the exo 1 (EXO1) region of the amino acid sequence of SEQ ID NO: 2, X ₁ D
An amino acid sequence in which at least one amino acid of X ₁ , X ₂ and X _{3 in} the X ₂ EX ₃ motif is replaced with another amino acid. DNA having 3′-5 ′ exonuclease activity
A highly conserved amino acid region for this exonuclease is known in the amino acid sequence of polymerase (EXO I, EXO II, EXO III, FIG. 4). Exo I
It is known that the X ₁ DX ₂ EX ₃ motif exists in the (EXO1) region, and these amino acids, D (aspartic acid) and E (glutamic acid), are essential for exonuclease activity.

The second DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
100% to 6%, preferably 90 to 30% 3'-
It has a 5'exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Amino acid sequence: 140th, 142nd and 1st of SEQ ID NO: 2
Amino acid sequence in which the 44th amino acid is replaced with another amino acid

The second DNA polymerase of the present invention is a modified thermostable DNA having the following physicochemical properties.
There is a polymerase. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
100% to 6%, preferably 90 to 30% 3'-
It has a 5'exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: The amino acid sequence in which the 142nd isoleucine of SEQ ID NO: 2 is replaced with aspartic acid, glutamic acid, asparagine, glutamine, or lysine, or the 144th threonine Amino acid sequence substituted with valine

First DNA polymerase and second DNA
DNA synthesis rate of polymerase is at least 30 bases /
Seconds, preferably 100-120 bases / second, pH
It is a thermostable DNA polymerase capable of maintaining an activity of 60% or more after treatment at 8.8 (measured value at 25 ° C) at 95 ° C for 6 hours. The first DNA polymerase and the second DNA polymerase of the present invention are preferably KOD polymerase or a mutant of the enzyme.

In the present invention, the activity of the second DNA polymerase is preferably smaller than that of the first DNA polymerase, and the amount of the second DNA polymerase is 0.02 to 0.1 unit per 2.5 units of the first DNA polymerase. Is preferred.

As a method for producing these modified enzymes, for example, a mutation is introduced into a gene encoding a natural type KOD polymerase, and 3'-5 'as compared with the natural type KOD polymerase by a protein engineering method. There is a method for producing a novel enzyme with reduced exonuclease activity.

Although the gene encoding the KOD polymerase for introducing the mutation is not particularly limited, one embodiment of the present invention is a Pyrococcous sp. KO
The gene described in Sequence Listing / SEQ ID NO: 3 derived from D was used.

In another embodiment of the present invention, a mutation is introduced into the gene encoding the amino acid sequence set forth in SEQ ID NO: 1 so that the 3'-5 'exonuclease activity is reduced as compared to the native KOD polymerase. Produce a new enzyme.

As a method for introducing a mutation into the natural KOD polymerase gene, any known method can be used. For example, natural KOD polymerase gene DNA
A method of protein engineering, such as a PCR method or a site-directed mutagenesis method can be used, such as a method of contacting a drug as a mutation source or a method of ultraviolet irradiation. In addition, since the gene repair mechanism has been deleted, it is possible to introduce a mutation in vivo using Escherichia coli in which a gene mutation frequently occurs. Chameleon site-dire used in the present invention
What is the cted mutagenesis kit (manufactured by Stratagene)?
(1) A plasmid in which the gene of interest is inserted is denatured, and the plasmid is annealed with a mutant primer and a selective primer. (2) Then D with DNA polymerase
After NA synthesis is performed, a ligation reaction is performed with a ligase. (3) Cleave the plasmid with a restriction enzyme that is not present in the selection primer but is present in the plasmid serving as a template to cleave DNA having no mutation inserted therein. (Four)
The remaining plasmid is then transformed into E. coli. (Five)
This is a method in which a mutant plasmid is prepared from the transformant and (3) and (4) are repeated to obtain a plasmid having the desired mutation inserted therein.

The modified polymerase gene obtained as described above can be used for example as pLED-M1, pBluescr.
After being inserted into a vector such as ipt and transformed into, for example, Escherichia coli, it is spread on an agar medium containing a drug such as ampicillin to form colonies. A colony is inoculated into a nutrient medium such as LB medium or 2 × YT medium, and the colony is incubated at 37 ° C. for 12 to 2
After culturing for 0 hour, the cells are crushed to extract a crude enzyme solution. Any known method may be used as a method for disrupting the cells, and for example, a physical disruption method such as ultrasonication or glass beer disruption, or a lytic enzyme such as lysozyme may be used. This crude enzyme is heat treated, for example at 80 ° C. for 3
Treatment for 0 minutes to inactivate the polymerase derived from the host,
NA polymerase activity is measured. Next, the 3'-5 'exonuclease activity was measured, and the activity ratio of both was compared with that of natural KOD polymerase to obtain 3'-5'.
Enzymes with reduced exonuclease activity can be screened.

Purified D from the strain selected by the above method
As a method for obtaining NA polymerase, any known method may be used, and examples thereof include the following method. After the cells obtained by culturing in a nutrient medium are collected, they are crushed and extracted by an enzymatic or physical crushing method to obtain a crude enzyme solution. The crude enzyme extract thus obtained is heat-treated, for example, at 80 ° C. for 30 minutes, and then the KOD polymerase fraction is collected by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as Sephadex G-25 (Pharmacia Biotech) gel filtration. After this operation, Q Sepharose,
A purified enzyme preparation can be obtained by separating and purifying by column chromatography using heparin sepharose or the like. The purified enzyme preparation was purified by SDS-PAGE to the extent that it showed a substantially single band.

In the present invention, the DNA synthesis activity means the deoxyribonucleic acid obtained by covalently bonding the α-phosphate of deoxyribonucleoside 5'-triphosphate to the 3'-hydroxyl group of the oligonucleotide or polynucleotide annealed to the template DNA. The activity of catalyzing the reaction of introducing deoxyribonucleoside 5′-monophosphate into the template in a template-dependent manner.

In the activity measuring method, when the enzyme activity is high, the sample is diluted with a storage buffer for measurement. In the present invention, the following solution A (25 μl), solution B and solution C (5 μl) and sterilized water (10 μl) were added to an Eppendorf tube and mixed by stirring, and then the above enzyme solution (5 μl) was added to the solution at 75 ° C. for 1 hour.
React for 0 minutes. Then, the mixture is ice-cooled, 50 μl of E liquid and 100 μl of D liquid are added, and the mixture is stirred and ice-cooled for 10 minutes. This solution was filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard) to incorporate nucleotides into the template DNA. taking measurement. One unit of the enzyme activity is the amount of the enzyme that incorporates 10 nmol of nucleotide into the acid-insoluble fraction per 30 minutes under this condition. A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ³ H] dTTP) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 μg / μl carrier DNA

In the present invention, the 3'-5 'exonuclease activity means that the 3'terminal region of DNA is excised and 5'
-Refers to the activity of releasing mononucleotides. The activity is measured by 50 μl of reaction solution (120 mM Tris-HCl (pH 8.8 at
25 ℃), 10mM KCl, 6mM ammonium sulfate, 1mM MgCl ₂ ,
0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) is dispensed into a 1.5 ml Eppendorf tube, and a DNA polymerase is added. 75 ° C
After reacting for 10 minutes, stop the reaction by cooling with ice,
Next, 50 μl of 0.1% BSA is added as a carrier, and further 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution is added and mixed. After leaving it on ice for 15 minutes, it is centrifuged at 12,000 rpm for 10 minutes to separate the precipitate. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard) to measure the amount of nucleotides released in the acid-soluble fraction.

In the present invention, the DNA synthesis rate means the number of DNAs synthesized per unit time. The measurement method is D
NA polymerase reaction solution (20 mM Tris-HCl (pH 7.5), 8 mM
Magnesium chloride, 7.5 mM dithiothreitol, 100 μ
g / ml BSA, 0.1 mM dNTP, 0.2 μCi [α- ³² P] dCTP) was added to the primer-annealed M13mp181 single-stranded D
React with NA at 75 ° C. Stop the reaction by adding an equal volume of the reaction stop solution (50 mM sodium hydroxide, 10 mM EDTA, 5% Ficoll, 0.05% bromophenol blue). The DNA synthesized in the above reaction is fractionated by alkaline agarose gel electrophoresis, then the gel is dried and autoradiography is performed. Labeled λ / HindIII is used as a DNA size marker. The rate of DNA synthesis is determined by measuring the size of the synthesized DNA using the band of this marker as an index.

In the present invention, the thermal stability means a pH of 8.
8 (measured value at 25 ° C.) means residual activity after treatment at 95 ° C. for 6 hours.

The thermostable DNA polymerase of the present invention before modification is Pyrococcus sp., Which is one of the hyperthermophilic archaeon isolated in Kobojima, Kagoshima Prefecture. It is an enzyme derived from KOD. The mycological properties of KOD producing the enzyme are described in JP-A-7-298879. The enzyme is produced by culturing the above strain. The enzyme has the following physicochemical properties. Action: It has DNA synthesis activity and has 3′-5 ′ exonuclease activity. DNA synthesis rate: at least 120 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: amino acid sequence set forth in SEQ ID NO: 2

In the nucleic acid amplification method of the present invention, the above DNA polymerase composition is used, using DNA as a template, primers, and four types of deoxyribonucleotide triphosphates (dN).
TP) is reacted to extend the primer to synthesize a DNA primer extension product.

In the PCR method, which is one of the nucleic acid amplification methods of the present invention, first, when a nucleic acid in a sample, particularly a long-chain nucleic acid, is double-stranded, it is denatured by heat to form a single-stranded. Incomplete strand separation of long nucleic acids will interfere with primer annealing and extension reactions. Then, using the single strand as a template, a primer complementary to the template, preferably a primer in which one is complementary to the DNA extension product of the other and dNTP are used in a PCR reaction solution using the DNA polymerase composition of the present invention. React with.

The reaction temperature uses a two-step temperature cycle, in which a high temperature at which the nucleic acid to be amplified is denatured and a low temperature at which the primer anneals to the denatured nucleic acid to cause primer extension are alternately repeated. Usually, 94 ° C for 0.5 to 1 minute → 68 ° C for 0.5 to 10 minutes is repeated 25 to 40 times. The two primers anneal to opposite ends of the template nucleic acid sequence, and the extension product of each primer is a complementary copy of the template nucleic acid sequence and hybridizes to the other primer when separated from its complement. Anneal to the template nucleic acid in such a way that it can. The reaction time is preferably sufficient for the extension reaction to complete chain synthesis. For amplification of nucleic acids longer than 20 kb,
Annealing and extension times of at least 10-20 minutes are preferred.

Long-chain nucleic acids are preferably protected against degradation during amplification, eg using glycerol, dimethylsulfoxide (DMSO) and the like.

The presence of misincorporated nucleotides prematurely terminates strand synthesis, reduces the number of template strands for the next round of amplification, and reduces the efficiency of amplification of long nucleic acids. However, in the present invention, due to the presence of a small amount of 3'-5 'exonuclease activity in the reaction solution in addition to the DNA polymerase activity, erroneous incorporated nucleotides are removed during the synthesis of the primer extension product. However, the predominant polymerase activity allows complete strand synthesis.

The pH and composition of the reaction buffer, the salts (divalent and monovalent ions), and the design of the primers are important for the amplification efficiency of long-chain nucleic acids. The PCR reagents are usually prepared at room temperature before the denaturation step, which may cause the binding of the primer to another primer or some homologous nucleic acid sequences. The formation of extension products from this non-specific primer binding will also reduce the amplification efficiency of the long chain products. In order to prevent such specific binding, a so-called hot start method in which an enzyme is added after the temperature becomes high is preferable.

In order to maintain the activity of the DNA polymerase of the present invention, divalent ions such as magnesium ions and monovalent ions such as ammonium ions and / or potassium ions are preferably present together. The nucleic acid amplification reaction solution contains a buffer solution and these ions, BSA, a nonionic surfactant,
For example, Triton X-100 and buffer may be present.

The nucleic acid amplification reagent of the present invention comprises two kinds of primers, one of which is complementary to the DNA extension product of the other, dNTP and the above DNA polymerase composition, magnesium ion and ammonium ion and / or Or it contains potassium ions, BSA and nonionic surfactants and buffers. In the present invention,
The activity of the second DNA polymerase is preferably lower than the activity of the first DNA polymerase, and it is preferable that the second DNA polymerase is 0.02 to 0.1 unit per 2.5 units of the first DNA polymerase. The reagent of the present invention may optionally contain an auxiliary solubilizer such as glycerin, DMSO, polyethylene glycol and the like.

As the buffer, Tris buffer, tris (hydroxymethyl) methylglycine (tricine buffer), N-bis (hydroxyethyl) glycine (bicine buffer), etc. are used. Optimal buffer and pH
Depends on the DNA polymerase used. In the present invention, when KOD polymerase and a mutant of the enzyme are used, the pH is 7.5 to 9.2 (at 25 ° C, 10 mM.
˜50 mM, preferably 20-120 mM. The divalent cation is preferably magnesium ion, and magnesium chloride or the like is used. The concentration is preferably 1-2 mM. The monovalent cation is preferably an ammonium ion or potassium ion, and ammonium sulfate, potassium glutamate, potassium acetate and the like are used.
It is preferable that their concentration is 2 to 50 mM. The primers are preferably two types of oligonucleotides, one of which is complementary to the DNA extension product of the other. The concentration is preferably 0.2 to 1 μM.

Next, the present invention will be described in detail with reference to examples. Reference Example 1 of DNA polymerase gene derived from hyperthermophilic archaeon KOD
Cloning The hyperthermophilic archaeon KOD1 strain isolated in Kobojima, Kagoshima Prefecture was cultured at 95 ° C., and then the cells were collected. Chromosomal DNA of the hyperthermophilic archaeon KOD strain was prepared from the obtained bacterial cells by a conventional method. Pyrococcus furi
osus) -derived DNA polymerase (Pfu polymerase) based on the amino acid sequence of the conserved region, two primers (5'-
GGATTAGTATAGTGCCAATGGSSGGCGA-3 'and 5'-GAGGGCAGA
AGTTTATTCCGAGCTT-3 ') was synthesized. A PCR reaction was carried out using these two types of primers and the prepared DNA as a template.

After the nucleotide sequence of the PCR amplified DNA fragment was determined and the amino acid sequence was determined, Southern hybridization was carried out on the KOD1 strain chromosomal DNA restriction enzyme treated product using this amplified DNA fragment as a probe, and D
The size of the fragment encoding NA polymerase was determined (approximately 4-7 Kbp). Further, a DNA fragment of this size was recovered from an agarose gel, inserted into a plasmid pBS (manufactured by Stratagene), and E. coli was extracted from these mixtures.
(E. coli JM109) was transformed to prepare a library. Colony hybridization was performed using the probe used for Southern hybridization, and a clone strain (E. coli) that was considered to contain a DNA polymerase gene derived from the KOD1 strain was obtained from the above library.
JM109 / pSBKOD1) was obtained.

The obtained clone strain, (E.coli JM109 / pSBK
The plasmid and pSBKOD1 were recovered from OD1) and the nucleotide sequence was determined according to a conventional method. Further, the amino acid sequence was deduced from the determined nucleotide sequence. DNA derived from KOD1 strain
The polymerase gene consists of 5010 bases, and 1670
1 amino acid was encoded (SEQ ID NO: 1).

In order to create the complete polymerase gene, two intervening sequences (1374-2453 bp: 27) were used.
08-4316bp) was removed by PCR fusion method. In the PCR fusion method, using the plasmid recovered from the clone strain as a template, 3 sets of primers were combined, and each PCR was performed to amplify 3 fragments excluding intervening sequences. At this time, the primers used for PCR were designed so that the sequence similar to that of the binding partner would be present on the side binding to other fragments. Separate restriction enzyme sites (N-terminal side: Eco
RV, C-terminal side: BamHI) was designed to be created. Next, among the PCR-amplified fragments, the fragment located in the center of the structure and the fragment located on the N-terminal side were mixed, and PCR was performed using each fragment as a primer. Similarly, structurally, the fragment located at the center and the fragment located at the C-terminal side were mixed, and PCR was performed using each fragment as a primer. Using the two types of fragments thus obtained, P
Perform CR to remove intervening sequences and add EcoR to the N-terminus
A complete gene fragment encoding the DNA polymerase derived from the KOD1 strain having BamHI sites at the V and C ends was obtained. Furthermore, an expression vector capable of inducing the same gene with a T7 promoter, NcoI / Ba of pET-8c
Using the mHI site and the restriction enzyme site created earlier,
Subcloning and recombinant expression vector (pET-
pol) was obtained. In addition, E. coli BL21 (DE
3) / pET-pol has been deposited with the Institute of Biotechnology (FERM BP-5513).

Reference Example 2 Subcloning of KOD polymerase gene In order to modify the thermostable DNA polymerase, the KOD polymerase gene was excised from the plasmid pET-pol and subcloned into pBluescript. That is, pET-pol is a restriction enzyme, XbaI and B
Cut with amHI (manufactured by Toyobo), KOD of about 2.3 kb
The polymerase gene was excised. Next, this DNA fragment was digested with XbaI and BamHI using a ligation kit (Ligation high manufactured by Toyobo) to obtain a plasmid p.
Ligated with Bluescript SK (-). next,
Commercially available competent cell (Toyobo's competent high JM
109) was used for transformation. LB agar medium containing 100 μg / ml of ampicillin (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, Gibco) at 35 ° C. 16
After culturing for a period of time, a plasmid was prepared from the obtained colony. Furthermore, the partial base sequence was confirmed to obtain a plasmid pKOD1 containing the KOD polymerase gene.

Reference Example 3 Preparation of modified gene (DA) and modified thermostable DNA
Purification of Limerase (DA) Using the plasmid pKOD1 obtained in Reference Example 2, a plasmid having a modified thermostable DNA polymerase gene in which the 141st aspartic acid of KOD polymerase shown in Sequence Listing 2 was replaced with alanine was prepared. Did (pKO
DDA). Made by Chameleon site-directed mutagenes
The is kit (manufactured by Stratagene) was used. The method was performed according to the instruction manual. The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 7 was used. The mutants were confirmed by decoding the nucleotide sequence. Escherichia coli JM109 was transformed with the obtained plasmid to obtain JM109 (pKO
DDA) was obtained.

TB medium containing 100 μg / ml ampicillin that had been sterilized (Molecular Cloning, p.A.2)
6L was dispensed into a 10L jar fermenter. 50 ml of LB medium (1% bactotryptone, containing 100 μg / ml ampicillin in advance in this medium)
Escherichia coli JM109 (pKODDA) (500 ml Sakaguchi flask used) was inoculated with 0.5% yeast extract, 0.5% sodium chloride (manufactured by Gibco) at 30 ° C for 16 hours and inoculated with aeration stirring at 35 ° C for 12 hours. did.
The bacterial cells were recovered from the culture solution by centrifugation, and 400 ml of disruption buffer solution (10 mM Tris-HCl (pH8.0), 80 mM KCl, 5 mM 2-
After suspending in mercaptoethanol and 1 mM EDTA), the cells were disrupted by ultrasonic treatment to obtain a cell disruption solution. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Nucleic acid removal treatment with polyethyleneimine, ammonium sulfate fractionation, and heparin sepharose chromatography were performed, and finally the storage buffer (50 mM Tris-
Replaced with HCl (pH8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0.1% Tween20, 0.1% nonidet P40, 50% glycerin, and modified thermostable DNA polymerase (D
A) was obtained. The DNA polymerase activity in the above purification step was measured by the following procedure. When the enzyme activity was high, the sample was diluted with the storage buffer for measurement.

(Reagent) A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ^3] H] dTTP) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 μg / μl carrier DNA

(Method) Solution A (25 μl), solution B and solution C (5 μl each) and sterilized water (10 μl) were added to an Eppendorf tube and mixed with stirring, and then 5 μl of the above enzyme solution was added to the mixture.
React for 10 minutes at 5 ° C. Then, cool with ice, and E solution 50μ
1, and 100 μl of D solution were added, and the mixture was stirred and ice-cooled for 10 minutes. This liquid was added to a glass filter (Whatman GF
/ C filter), thoroughly washed with solution D and ethanol, the radioactivity of the filter was measured with a liquid scintillation counter (manufactured by Packard), and nucleotide incorporation into the template DNA was measured. One unit of enzyme activity was defined as the amount of enzyme that incorporated 10 nmole of nucleotide into the acid-insoluble fraction per 30 minutes under these conditions.

Reference Example 4 Preparation of mutant (EA) gene and modified thermostable DNA
Purification of Limerase By the same method as in Reference Example 3, a plasmid having a modified thermostable DNA polymerase gene in which glutamic acid at position 143 of KOD polymerase shown in Sequence Listing 2 was replaced with alanine was prepared (pKODEA). The primer described in SEQ ID NO: 5 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 8 was used. Further, a modified heat-resistant DN was prepared by the same purification method as in Reference Example 3.
A polymerase (EA) was obtained.

Reference Example 5 Preparation of mutant (DEA) gene and modified thermostable DNA
Purification of polymerase In the same manner as in Reference Example 3, modified thermostable DN in which 141st aspartic acid and 143rd glutamic acid of KOD polymerase shown in Sequence Listing 2 were replaced with alanine
A plasmid having the A polymerase gene was prepared (p
KODDEA). The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 6 was used. Further, by the same purification method as in Reference Example 3, a modified thermostable DNA polymerase (DE
A) was obtained.

Reference Example 6 Preparation of mutant (ND) gene and modified thermostable DNA
Purification of Limerase By the same method as in Reference Example 3, a plasmid having a modified thermostable DNA polymerase gene in which the 210th asparagine of KOD polymerase shown in Sequence Listing 2 was replaced with aspartic acid was prepared (pKODND). The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer described in SEQ ID NO: 9 was used. Further, a modified thermostable DNA polymerase (ND) was obtained by the same purification method as in Reference Example 3.

Reference Example 7 Preparation of mutant (YF) gene and modified thermostable DNA
Purification of Limerase By the same method as in Reference Example 3, a plasmid having a modified thermostable DNA polymerase gene in which the 311th tyrosine of KOD polymerase shown in Sequence Listing 2 was replaced with phenylalanine was prepared (pKODYF). The primer described in SEQ ID NO: 4 was used as a selection primer.
As the mutant primer, the primer described in SEQ ID NO: 10 was used. Further, a modified thermostable DNA polymerase (YF) was obtained by the same purification method as in Reference Example 3.

Reference Example 8 Exonuclease of modified thermostable DNA polymerase
Confirmation of activity The exonuclease activity of the modified thermostable DNA polymerases (DA, EA, DEA, ND and YF) obtained in Reference Examples 3 to 7 above was measured by the following method. As a control, a natural KOD polymerase (manufactured by Toyobo) was used. 50 μl of reaction solution (120 mM Tris-HCl (pH 8.8 at 25
℃), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl ₂ ,
0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) was dispensed into a 1.5 ml Eppendorf tube, and 25 units, 50 units, and 100 units of DNA polymerase were added, respectively. In addition, natural type KO
D polymerase was used in 0.25 units, 0.5 units and 1 unit. After reacting at 75 ° C for 10 minutes, the reaction was stopped by cooling with ice, and then 0.1% BSA was used as a carrier.
50 μl, and then 10% trichloroacetic acid, 2%
100 μl of sodium pyrophosphate solution was added and mixed.
After leaving it on ice for 15 minutes, it was centrifuged at 12,000 rpm for 10 minutes to separate the precipitate. The radioactivity of 100 μl of the supernatant was measured with a liquid scintillation counter (manufactured by Packard) to measure the amount of nucleotides released in the acid-soluble fraction. FIG. 1 shows the polymerase activity of each DNA polymerase and the DNA degradation rate. This result shows the modified heat resistance D
Exonuclease activity could not be detected in the three polymerases of NA polymerase (DEA, DA, EA). Further, the modified thermostable DNA polymerase (ND) exhibited an exonuclease activity of about 0.1% of the natural KOD polymerase, and the modified thermostable DNA polymerase (YF) exhibited an exonuclease activity of about 0.01%.

Reference Example 9 Confirmation of Thermostability The thermostability of the modified thermostable DNA polymerases (DA, EA, DEA, ND and YF) obtained in Reference Examples 3 to 7 were measured by the following method. 5 units of purified modified DNA polymerase was added to 100 μl of a buffer solution (20 mM Tris-HCl).
pH8.8 at 25 ℃, 10mM potassium chloride, 10mM ammonium sulfate, 2mM magnesium sulfate, 0.1% Triton X-100,
0.1 mg / ml BSA, 5 mM 2-mercaptoethanol) was mixed and pre-incubated at 95 ° C. Samples were taken from this mixed solution over time, and the polymerase activity was measured by the method described in Reference Example 3. For comparison, Taq polymerase (manufactured by Toyobo) and natural KOD polymerase (manufactured by Toyobo) were subjected to the same operation. As shown in FIG. 2, all the modified thermostable DNA polymerases showed residual activity of 60% or more after treatment at 95 ° C. for 6 hours, like the natural KOD polymerase. In contrast, Taq polymerase had a residual activity of 15% or less.

Reference Example 10 Measurement of DNA Synthesis Rate The DNA synthesis rates of the modified thermostable DNA polymerases (DA, EA, DEA, ND and YF) obtained in Reference Examples 3 to 7 were measured by the following method. Purified modified DNA
1 unit of polymerase was added to 10 μl of reaction solution (20 mM Tris-HCl
(pH7.5), 8 mM magnesium chloride, 7.5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNTP, 0.2 μCi [α-
³² P] dCTP) and 75 μl of M13mp181 single-stranded DNA annealed with 0.2 μg of the primer of SEQ ID NO: 15
The reaction was carried out at 20 ° C. for 20 seconds, 40 seconds, and 60 seconds. To stop the reaction, use the same amount of reaction stop solution (50mM sodium hydroxide, 10mM EDT
A, 5% Ficoll, 0.05% bromophenol blue) was added. For comparison, Taq polymerase (manufactured by Toyobo) and natural KOD polymerase (manufactured by Toyobo) were subjected to the same operation.

After the DNA synthesized in the above reaction was fractionated by alkaline agarose gel electrophoresis, the gel was dried and subjected to autoradiography. Labeled λ / HindIII was used as a DNA size marker. The rate of DNA synthesis was determined by measuring the size of the synthesized DNA using the band of this marker as an index. As a result, all modified polymerases had a synthesis rate of about 120 bases / second, similar to the natural KOD polymerase. In contrast, Taq polymerase had a synthesis rate of about 60 bases / sec.

Reference Example 11 Preparation of modified gene (IN) and modified thermostable DNA
Purification of polymerase Using the plasmid pKOD1 obtained in Reference Example 2, K
X ₁ DX ₂ present in the EXO1 region of OD polymerase
Of EX ₃ motif, to produce plasmid with a modified thermostable DNA polymerase gene was replaced with isoleucine X ₂ asparagine (pKODIN). A chameleon site-directed mutagenesis kit (manufactured by Stratagene) was used for preparation. The method was performed according to the instruction manual. The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer shown in SEQ ID NO: 15 was used. The mutants were confirmed by decoding the nucleotide sequence. E. coli JM10 with the obtained plasmid
9 was transformed to obtain JM109 (pKODIN).

TB medium containing sterilized 100 μg / ml ampicillin (Molecular Cloning, p.A.2)
6L was dispensed into a 10L jar fermenter. 50 ml of LB medium (1% bactotryptone, containing 100 μg / ml ampicillin in advance in this medium)
Escherichia coli JM109 (pKODIN) (500 ml Sakaguchi flask used), which had been cultivated in 0.5% yeast extract, 0.5% sodium chloride, manufactured by Gibco Co., Ltd. at 30 ° C. for 16 hours, was inoculated and aerated with stirring at 35 ° C. for 12 hours. did.
The bacterial cells were recovered from the culture solution by centrifugation, and 400 ml of disruption buffer solution (10 mM Tris-HCl (pH8.0), 80 mM KCl, 5 mM 2-
After suspending in mercaptoethanol and 1 mM EDTA), the cells were disrupted by ultrasonic treatment to obtain a cell disruption solution. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Nucleic acid removal treatment with polyethyleneimine, ammonium sulfate fractionation, and heparin sepharose chromatography were performed, and finally the storage buffer (50 mM Tris-H
Cl (pH8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0.1% Tween20, 0.1% nonidet P40, 50% glycerin), and a modified thermostable DNA polymerase (I
N) was obtained. The DNA polymerase activity in the above purification step was measured by the following procedure. When the enzyme activity was high, the sample was diluted with the storage buffer for measurement.

(Reagent) A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ^3] H] dTTP D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 μg / μl carrier DNA

(Method) Solution A (25 μl), solution B and solution C (5 μl) and sterilized water (10 μl) were added to an Eppendorf tube, and the mixture was stirred and mixed.
React at 10 ° C for 10 minutes. Then, cool with ice, and E solution 50μ
1, and 100 μl of solution D are added, and the mixture is stirred and ice-cooled for another 10 minutes. This solution was added to a glass filter (Whatman GF /
C filter), thoroughly washed with solution D and ethanol, the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard), and the incorporation of nucleotide into the template DNA was measured by 1 unit of this enzyme activity. Under the conditions, the amount of the enzyme that incorporates 10 nmole of nucleotide into the acid-insoluble fraction per 30 minutes was used.

Reference Example 12 Preparation of mutant (IE) gene and modified thermostable DNA
Purification of Polymerase In the same manner as in Reference Example 11, E of KOD polymerase
Among the X ₁ DX ₂ EX ₃ motifs present in the XO1 region, a thermostable DNA polymerase gene was prepared by replacing isoleucine in X ₂ with glutamic acid (pKODI
E). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer described in SEQ ID NO: 16 was used. Further, a modified thermostable DNA polymerase (IE) was obtained by the same purification method as in Reference Example 11.

Reference Example 13 Construction of mutant (IQ) gene and modified thermostable DNA
Purification of Limerase By the same method as in Reference Example 11, E of KOD polymerase
Of the X ₁ DX ₂ EX ₃ motifs present in the XO1 region, a thermostable DNA polymerase gene was prepared by replacing the isoleucine of X ₂ with glutamine (pKODI
Q). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer shown in SEQ ID NO: 17 was used. Further, a modified thermostable DNA polymerase (IQ) was obtained by the same purification method as in Reference Example 11.

Reference Example 14 Preparation of mutant (ID) gene and modified thermostable DNA
Purification of Limerase By the same method as in Reference Example 11, E of KOD polymerase
Among the X ₁ DX ₂ EX ₃ motifs present in the XO1 region, a thermostable DNA polymerase gene was produced by replacing isoleucine in X ₂ with aspartic acid (pKODI
D). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer shown in SEQ ID NO: 18 was used. Further, a modified thermostable DNA polymerase (ID) was obtained by the same purification method as in Reference Example 11.

Reference Example 15 Preparation of mutant (TV) gene and modified thermostable DNA
Purification of Limerase X existing in EX01 region was treated in the same manner as in Reference Example 11.
_{Among the 1} DX ₂ EX ₃ motifs, a thermostable DNA polymerase gene was prepared by replacing tyrosine of X ₃ with valine (pKODTV). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer shown in SEQ ID NO: 19 was used. Further, a modified thermostable DNA polymerase (TV) was obtained by the same purification method as in Reference Example 11.

Reference Example 16 Preparation of mutant (IK) gene and modified thermostable DNA
Purification of Limerase X existing in the EXO1 region was treated in the same manner as in Reference Example 11.
Of X in the ₁ DX ₂ EX ₃ motif, a thermostable DNA polymerase gene was prepared by replacing isoleucine in X ₂ with lysine (pKODIK). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer shown in SEQ ID NO: 21 was used. Furthermore,
A modified thermostable DNA polymerase (IK) was obtained by the same purification method as in Reference Example 11.

Reference Example 17 Preparation of mutant (IR) gene and modified thermostable DNA
Purification of Limerase X existing in the EXO1 region was treated in the same manner as in Reference Example 11.
Of the X in the ₁ DX ₂ EX ₃ motif, a thermostable DNA polymerase gene was prepared by replacing the isoleucine of X ₂ with arginine (pKODIR). The primer described in SEQ ID NO: 14 was used as the selection primer. As the mutant primer, the primer described in SEQ ID NO: 20 was used. Further, a modified heat-resistant DN was prepared by the same purification method as in Reference Example 11.
A polymerase (IR) was obtained.

Reference Example 18 Exonuclease of modified thermostable DNA polymerase
Confirmation of activity The exonuclease activity of the modified thermostable DNA polymerases (IN, IE, IQ, ID, YV, IK and IR) obtained in Reference Examples 11 to 17 above was measured by the following method. As a control, a natural KOD polymerase (manufactured by Toyobo) was used. 50 μl of reaction solution (120 mM Tris-HCl
(pH8.8 at 25 ℃), 10mM KCl, 6mM ammonium sulfate,
1 mM MgCl ₂ , 0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) was dispensed into a 1.5 ml Eppendorf tube, and 0.5 units, 1 unit, and 1 unit of the above DNA polymerase were added, respectively. 0.5 unit was added and reacted at 75 ° C. for 10 minutes. The reaction was stopped by cooling with ice, then 50 ml of 0.1% BSA was added as a carrier, and 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution was added and mixed. After leaving it on ice for 15 minutes, it was centrifuged at 12,000 rpm for 10 minutes to separate the precipitate. The radioactivity of 100 μl of the supernatant was measured with a liquid scintillation counter (manufactured by Packard) to measure the amount of nucleotides released in the acid-soluble fraction.

FIG. 5 shows the polymerase activity of each DNA polymerase and the DNA decomposition rate. Further natural type KOD
The ratio of exonuclease activity with polymerase is shown in FIG. Thus, according to the present invention, 3'-of various strengths
It was shown that a thermostable DNA polymerase having 5 ′ exonuclease activity was obtained. About 95% IN, about 76% IE, and about 64 IQ against the 3'-5 'exonuclease activity of native KOD polymerase.
%, ID about 52%, TV about 48%, IK about 30%
%, IR had about 0% of the same activity.

Reference Example 11 Modification of DNA synthesis by PCR of modified DNA polymerase
Measurement of accuracy The accuracy of DNA synthesis by PCR was measured for native KOD polymerase, modified thermostable DNA polymerases IE, ID, IK, IR, and Taq polymerase by the following method. Plasmid pUR288 (Current
Protocols in Molecular Biology 1.5.6) was digested with the restriction enzyme ScaI. PCR was performed using 1 ng of this plasmid. After completion of the reaction, 5 μl of the reaction solution was subjected to agarose gel electrophoresis to confirm amplification of a target of about 5.3 kb. The remaining reaction solution was treated with phenol / chloroform and then precipitated with ethanol. After drying the precipitate, 50 μl of High buffer (50 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT). Furthermore, 10 units of a restriction enzyme ScaI (manufactured by Toyobo) was added, and the mixture was reacted at 37 ° C. for 16 hours. The target amplification product was separated by agarose gel electrophoresis, and the agarose in that portion was cut out. DNA was purified from this agarose using GeneClean 2 (BIO101). 10 ng of the purified DNA was diluted to 10 μl with TE buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA), and the ligation kit (manufactured by Toyobo Ligation h
10 μl of the reaction solution of (IG) was added and reacted at 16 ° C. for 30 minutes. Next, a commercially available competent cell (Toyobo co
Transformation was performed using mpetent high JM109).

100 μg / ml ampicillin, 1 mM
Isopropylthio-β-galactoside (IPTG, manufactured by Nacalai Tesque, Inc.), 0.7% 5-bromo-4-chloro-
3-indolyl-β-D-galactoside (X-gal
(Nacalai Tesque) LB agar medium (1%
Bactrypton, 0.5% yeast extract,
0.5% sodium chloride, 1.5% agar, manufactured by Gibco)
After culturing at 35 ° C for 16 hours, the colonies were counted.
The lacZ gene (β-galactosidase) is present in pUR288. Therefore, when the DNA synthesis during PCR is performed correctly, blue colonies are formed on the agar medium. On the contrary, when an error occurs during DNA synthesis and the β-galactosidase activity encoded by the lacZ gene is reduced or deleted, light blue or white colonies are formed. Table 1 shows the mutation rate (%) when each enzyme was used with the light blue colony and the white colony as mutant colonies.

[0075]

[Table 1]

As is clear from Table 1, the modified thermostable DNA polymerases IE, ID, IK obtained by the present invention,
Although IR is inferior to natural KOD polymerase,
Lower mutation rate than Taq polymerase, ie DNA
The accuracy of synthesis was high.

[0077]Example 1 PCR using a DNA polymerase composition (human genome
) Modified thermostable DNA polymerase (DA, EA, DE
A, ND, YF) and native KOD polymerase
Was used to perform PCR. 50 ml reaction solution (120 mM Tr
is-HCl (pH 8.8 at 25 ℃), 10mM KCl, 6mM ammonium sulfate
1 mM MgCl_2, 0.2 mM dNTP, 0.1% Triton X-100, 0.00
1% BSA, 30 ng of human placenta-derived genomic DNA (clone
(Manufactured by Tech Co., Ltd.), 10 pmol of the sequence listings 11 and 12
2.5 units of ND as a primer) and KOD polymerase
0.05 unit was added to carry out PCR reaction. Thermal service
IKER is model PJ2000 manufactured by Perkin Elmer
Was used. The reaction conditions are 94 ° C, 30 seconds → 68 ° C, 3
Minutes were run for 30 cycles. Modified heat-resistant DN for comparison
A polymerase (ND), Taq polymerase (Toyobo
Commercially available), a commercially available DNA polymerase mixture (ExTaq
(Manufactured by Takara Shuzo) and Advantage Tth (clone
(Manufactured by Tech Co., Ltd.) was also subjected to PCR reaction in the same manner. However
However, for the reaction solution composition, use the buffer solution attached to the commercial product.
Use the same amount of genomic DNA and primers as above
It was After the reaction was completed, agarose gel was added to 5 μl of the reaction solution.
Electrophoreses to confirm amplification of a target of about 4 kb
did. The results of agarose gel electrophoresis are shown in FIG.
As a result, the modified thermostable DNA polymerase (ND)
PCR with a mixture of native KOD polymerases
Better than commercial polymerase mix when performed
It was amplification.

[0078]

INDUSTRIAL APPLICABILITY In the present invention, the length of the amplifiable template nucleic acid can be greatly increased by using both the 3′-5 ′ exonuclease activity and the polymerase activity. Further, by using a mixture of two or more kinds of DNA polymerases having almost the same thermostability and DNA synthesis rate and different only in 3′-5 ′ exonuclease activity, nucleic acid amplification with excellent amplification efficiency is performed. You can

[0079]

[Sequence list]

SEQ ID NO: 1 Array length: 5342 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomic DNA Origin: Hyperthermophilic archaeon Share name: KOD1 Sequence features 156-5165 P CDS 1374-2453 intervening sequence 2708-4316 Intervening sequence Array GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TGCCGGTTTT 60 ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TACAATAAAG 120 CCTGGATTGT TCTACAAGAT TATGGGGGAT GAAAG ATG ATC CTC GAC ACT GAC 173                                        Met Ile Leu Asp Thr Asp                                          1 5 TAC ATA ACC GAG GAT GGA AAG CCT GTC ATA AGA ATT TTC AAG AAG GAA 221 Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu              10 15 20 AAC GGC GAG TTT AAG ATT GAG TAC GAC CGG ACT TTT GAA CCC TAC TTC 269 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Thr Phe Glu Pro Tyr Phe          25 30 35 TAC GCC CTC CTG AAG GAC GAT TCT GCC ATT GAG GAA GTC AAG AAG ATA 317 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Glu Val Lys Lys Ile      40 45 50 ACC GCC GAG AGG CAC GGG ACG GTT GTA ACG GTT AAG CGG GTT GAA AAG 365 Thr Ala Glu Arg His Gly Thr Val Val Thr Val Lys Arg Val Glu Lys  55 60 65 70 GTT CAG AAG AAG TTC CTC GGG AGA CCA GTT GAG GTC TGG AAA CTC TAC 413 Val Gln Lys Lys Phe Leu Gly Arg Pro Val Glu Val Trp Lys Leu Tyr                  75 80 85 TTT ACT CAT CCG CAG GAC GTC CCA GCG ATA AGG GAC AAG ATA CGA GAG 461 Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu              90 95 100 CAT GGA GCA GTT ATT GAC ATC TAC GAG TAC GAC ATA CCC TTC GCC AAG 509 His Gly Ala Val Ile Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys         105 110 115 CGC TAC CTC ATA GAC AAG GGA TTA GTG CCA ATG GAA GGC GAC GAG GAG 557 Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro Met Glu Gly Asp Glu Glu     120 125 130 CTG AAA ATG CTC GCC TTC GAC ATT GAA ACT CTC TAC CAT GAG GGC GAG 605 Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu 135 140 145 150 GAG TTC GCC GAG GGG CCA ATC CTT ATG ATA AGC TAC GCC GAC GAG GAA 653 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu                 155 160 165 GGG GCC AGG GTG ATA ACT TGG AAG AAC GTG GAT CTC CCC TAC GTT GAC 701 Gly Ala Arg Val Ile Thr Trp Lys Asn Val Asp Leu Pro Tyr Val Asp             170 175 180 GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG CGC TTC CTC CGT GTT GTG 749 Val Val Ser Thr Glu Arg Glu Met Ile Lys Arg Phe Leu Arg Val Val         185 190 195 AAG GAG AAA GAC CCG GAC GTT CTC ATA ACC TAC AAC GGC GAC AAC TTC 797 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe     200 205 210 GAC TTC GCC TAT CTG AAA AAG CGC TGT GAA AAG CTC GGA ATA AAC TTC 845 Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe 215 220 225 230 GCC CTC GGA AGG GAT GGA AGC GAG CCG AAG ATT CAG AGG ATG GGC GAC 893 Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp                 235 240 245 AGG TTT GCC GTC GAA GTG AAG GGA CGG ATA CAC TTC GAT CTC TAT CCT 941 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro             250 255 260 GTG ATA AGA CGG ACG ATA AAC CTG CCC ACA TAC ACG CTT GAG GCC GTT 989 Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val         265 270 275 TAT GAA GCC GTC TTC GGT CAG CCG AAG GAG AAG GTT TAC GCT GAG GAA 1037 Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu     280 285 290 ATA ACA CCA GCC TGG GAA ACC GGC GAG AAC CTT GAG AGA GTC GCC CGC 1085 Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn Leu Glu Arg Val Ala Arg 295 300 305 310 TAC TCG ATG GAA GAT GCG AAG GTC ACA TAC GAG CTT GGG AAG GAG TTC 1133 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe                 315 320 325 CTT CCG ATG GAG GCC CAG CTT TCT CGC TTA ATC GGC CAG TCC CTC TGG 1181 Leu Pro Met Glu Ala Gln Leu Ser Arg Leu Ile Gly Gln Ser Leu Trp             330 335 340 GAC GTC TCC CGC TCC AGC ACT GGC AAC CTC GTT GAG TGG TTC CTC CTC 1229 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu         345 350 355 AGG AAG GCC TAT GAG AGG AAT GAG CTG GCC CCG AAC AAG CCC GAT GAA 1277 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu     360 365 370 AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT GAA GGA GGC TAT GTA AAA 1325 Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr Glu Gly Gly Tyr Val Lys 375 380 385 390 GAG CCC GAG AGA GGG TTG TGG GAG AAC ATA GTG TAC CTA GAT TTT AGA 1373 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg                 395 400 405 TGC CAT CCA GCC GAT ACG AAG GTT GTC GTC AAG GGG AAG GGG ATT ATA 1421 Cys His Pro Ala Asp Thr Lys Val Val Val Lys Gly Lys Gly Ile Ile             410 415 420 AAC ATC AGC GAG GTT CAG GAA GGT GAC TAT GTC CTT GGG ATT GAC GGC 1469 Asn Ile Ser Glu Val Gln Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly         425 430 435 TGG CAG AGA GTT AGA AAA GTA TGG GAA TAC GAC TAC AAA GGG GAG CTT 1517 Trp Gln Arg Val Arg Lys Val Trp Glu Tyr Asp Tyr Lys Gly Glu Leu     440 445 450 GTA AAC ATA AAC GGG TTA AAG TGT ACG CCC AAT CAT AAG CTT CCC GTT 1565 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 455 460 465 470 GTT ACA AAG AAC GAA CGA CAA ACG AGA ATA AGA GAC AGT CTT GCT AAG 1613 Val Thr Lys Asn Glu Arg Gln Thr Arg Ile Arg Asp Ser Leu Ala Lys                 475 480 485 TCT TTC CTT ACT AAA AAA GTT AAG GGC AAG ATA ATA ACC ACT CCC CTT 1661 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Ile Ile Thr Thr Pro Leu             490 495 500 TTC TAT GAA ATA GGC AGA GCG ACA AGT GAG AAT ATT CCA GAA GAA GAG 1709 Phe Tyr Glu Ile Gly Arg Ala Thr Ser Glu Asn Ile Pro Glu Glu Glu         505 510 515 GTT CTC AAG GGA GAG CTC GCT GGC ATA CTA TTG GCT GAA GGA ACG CTC 1757 Val Leu Lys Gly Glu Leu Ala Gly Ile Leu Leu Ala Glu Gly Thr Leu     520 525 530 TTG AGG AAA GAC GTT GAA TAC TTT GAT TCA TCC CGC AAA AAA CGG AGG 1805 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Lys Lys Arg Arg 535 540 545 550 ATT TCA CAC CAG TAT CGT GTT GAG ATA ACC ATT GGG AAA GAC GAG GAG 1853 Ile Ser His Gln Tyr Arg Val Glu Ile Thr Ile Gly Lys Asp Glu Glu                 555 560 565 GAG TTT AGG GAT CGT ATC ACA TAC ATT TTT GAG CGT TTG TTT GGG ATT 1901 Glu Phe Arg Asp Arg Ile Thr Tyr Ile Phe Glu Arg Leu Phe Gly Ile             570 575 580 ACT CCA AGC ATC TCG GAG AAG AAA GGA ACT AAC GCA GTA ACA CTC AAA 1949 Thr Pro Ser Ile Ser Glu Lys Lys Gly Thr Asn Ala Val Thr Leu Lys         585 590 595 GTT GCG AAG AAG AAT GTT TAT CTT AAA GTC AAG GAA ATT ATG GAC AAC 1997 Val Ala Lys Lys Asn Val Tyr Leu Lys Val Lys Glu Ile Met Asp Asn     600 605 610 ATA GAG TCC CTA CAT GCC CCC TCG GTT CTC AGG GGA TTC TTC GAA GGC 2045 Ile Glu Ser Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 615 620 625 630 GAC GGT TCA GTA AAC AGG GTT AGG AGG AGT ATT GTT GCA ACC CAG GGT 2093 Asp Gly Ser Val Asn Arg Val Arg Arg Ser Ile Val Ala Thr Gln Gly                 635 640 645 ACA AAG AAC GAG TGG AAG ATT AAA CTG GTG TCA AAA CTG CTC TCC CAG 2141 Thr Lys Asn Glu Trp Lys Ile Lys Leu Val Ser Lys Leu Leu Ser Gln             650 655 660 CTT GGT ATC CCT CAT CAA ACG TAC ACG TAT CAG TAT CAG GAA AAT GGG 2189 Leu Gly Ile Pro His Gln Thr Tyr Thr Tyr Gln Tyr Gln Glu Asn Gly         665 670 675 AAA GAT CGG AGC AGG TAT ATA CTG GAG ATA ACT GGA AAG GAC GGA TTG 2237 Lys Asp Arg Ser Arg Tyr Ile Leu Glu Ile Thr Gly Lys Asp Gly Leu     680 685 690 ATA CTG TTC CAA ACA CTC ATT GGA TTC ATC AGT GAA AGA AAG AAC GCT 2285 Ile Leu Phe Gln Thr Leu Ile Gly Phe Ile Ser Glu Arg Lys Asn Ala 695 700 705 710 CTG CTT AAT AAG GCA ATA TCT CAG AGG GAA ATG AAC AAC TTG GAA AAC 2333 Leu Leu Asn Lys Ala Ile Ser Gln Arg Glu Met Asn Asn Leu Glu Asn                 715 720 725 AAT GGA TTT TAC AGG CTC AGT GAA TTC AAT GTC AGC ACG GAA TAC TAT 2381 Asn Gly Phe Tyr Arg Leu Ser Glu Phe Asn Val Ser Thr Glu Tyr Tyr             730 735 740 GAG GGC AAG GTC TAT GAC TTA ACT CTT GAA GGA ACT CCC TAC TAC TTT 2429 Glu Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe         745 750 755 GCC AAT GGC ATA TTG ACC CAT AAC TCC CTG TAC CCC TCA ATC ATC ATC 2477 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile     760 765 770 ACC CAC AAC GTC TCG CCG GAT ACG CTC AAC AGA GAA GGA TGC AAG GAA 2525 Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu 775 780 785 790 TAT GAC GTT GCC CCA CAG GTC GGC CAC CGC TTC TGC AAG GAC TTC CCA 2573 Tyr Asp Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro                 795 800 805 GGA TTT ATC CCG AGC CTG CTT GGA GAC CTC CTA GAG GAG AGG CAG AAG 2621 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys             810 815 820 ATA AAG AAG AAG ATG AAG GCC ACG ATT GAC CCG ATC GAG AGG AAG CTC 2669 Ile Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu         825 830 835 CTC GAT TAC AGG CAG AGG GCC ATC AAG ATC CTG GCA AAC AGC ATC CTA 2717 Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Ile Leu     840 845 850 CCC GAG GAA TGG CTT CCA GTC CTC GAG GAA GGG GAG GTT CAC TTC GTC 2765 Pro Glu Glu Trp Leu Pro Val Leu Glu Glu Gly Glu Val His Phe Val 855 860 865 870 AGG ATT GGA GAG CTC ATA GAC CGG ATG ATG GAG GAA AAT GCT GGG AAA 2813 Arg Ile Gly Glu Leu Ile Asp Arg Met Met Glu Glu Asn Ala Gly Lys                 875 880 885 GTA AAG AGA GAG GGC GAG ACG GAA GTG CTT GAG GTC AGT GGG CTT GAA 2861 Val Lys Arg Glu Gly Glu Thr Glu Val Leu Glu Val Ser Gly Leu Glu             890 895 900 GTC CCG TCC TTT AAC AGG AGA ACT AAC AAG GCC GAG CTC AAG AGA GTA 2909 Val Pro Ser Phe Asn Arg Arg Thr Asn Lys Ala Glu Leu Lys Arg Val         905 910 915 AAG GCC CTG ATT AGG CAC GAT TAT TCT GGC AAG GTC TAC ACC ATC AGA 2957 Lys Ala Leu Ile Arg His Asp Tyr Ser Gly Lys Val Tyr Thr Ile Arg     920 925 930 CTG AAG TCG GGG AGG AGA ATA AAG ATA ACC TCT GGC CAC AGC CTC TTC 3005 Leu Lys Ser Gly Arg Arg Ile Lys Ile Thr Ser Gly His Ser Leu Phe 935 940 945 950 TCT GTG AGA AAC GGG GAG CTC GTT GAA GTT ACG GGC GAT GAA CTA AAG 3053 Ser Val Arg Asn Gly Glu Leu Val Glu Val Thr Gly Asp Glu Leu Lys                 955 960 965 CCA GGT GAC CTC GTT GCA GTC CCG CGG AGA TTG GAG CTT CCT GAG AGA 3101 Pro Gly Asp Leu Val Ala Val Pro Arg Arg Leu Glu Leu Pro Glu Arg             970 975 980 AAC CAC GTG CTG AAC CTC GTT GAA CTG CTC CTT GGA ACG CCA GAA GAA 3149 Asn His Val Leu Asn Leu Val Glu Leu Leu Leu Gly Thr Pro Glu Glu         985 990 995 GAA ACT TTG GAC ATC GTC ATG ACG ATC CCA GTC AAG GGT AAG AAG AAC 3197 Glu Thr Leu Asp Ile Val Met Thr Ile Pro Val Lys Gly Lys Lys Asn     1000 1005 1010 TTC TTT AAA GGG ATG CTC AGG ACT TTG CGC TGG ATT TTC GGA GAG GAA 3245 Phe Phe Lys Gly Met Leu Arg Thr Leu Arg Trp Ile Phe Gly Glu Glu 1015 1020 1025 1030 AAG AGG CCC AGA ACC GCG AGA CGC TAT CTC AGG CAC CTT GAG GAT CTG 3293 Lys Arg Pro Arg Thr Ala Arg Arg Tyr Leu Arg His Leu Glu Asp Leu                 1035 1040 1045 GGC TAT GTC CGG CTT AAG AAG ATC GGC TAC GAA GTC CTC GAC TGG GAC 3341 Gly Tyr Val Arg Leu Lys Lys Ile Gly Tyr Glu Val Leu Asp Trp Asp             1050 1055 1060 TCA CTT AAG AAC TAC AGA AGG CTC TAC GAG GCG CTT GTC GAG AAC GTC 3389 Ser Leu Lys Asn Tyr Arg Arg Leu Tyr Glu Ala Leu Val Glu Asn Val         1065 1070 1075 AGA TAC AAC GGC AAC AAG AGG GAG TAC CTC GTT GAA TTC AAT TCC ATC 3437 Arg Tyr Asn Gly Asn Lys Arg Glu Tyr Leu Val Glu Phe Asn Ser Ile     1080 1085 1090 CGG GAT GCA GTT GGC ATA ATG CCC CTA AAA GAG CTG AAG GAG TGG AAG 3485 Arg Asp Ala Val Gly Ile Met Pro Leu Lys Glu Leu Lys Glu Trp Lys 1095 1100 1105 1110 ATC GGC ACG CTG AAC GGC TTC AGA ATG AGA AAG CTC ATT GAA GTG GAC 3533 Ile Gly Thr Leu Asn Gly Phe Arg Met Arg Lys Leu Ile Glu Val Asp                 1115 1120 1125 GAG TCG TTA GCA AAG CTC CTC GGC TAC TAC GTG AGC GAG GGC TAT GCA 3581 Glu Ser Leu Ala Lys Leu Leu Gly Tyr Tyr Val Ser Glu Gly Tyr Ala             1130 1135 1140 AGA AAG CAG AGG AAT CCC AAA AAC GGC TGG AGC TAC AGC GTG AAG CTC 3629 Arg Lys Gln Arg Asn Pro Lys Asn Gly Trp Ser Tyr Ser Val Lys Leu         1145 1150 1155 TAC AAC GAA GAC CCT GAA GTG CTG GAC GAT ATG GAG AGA CTC GCC AGC 3677 Tyr Asn Glu Asp Pro Glu Val Leu Asp Asp Met Glu Arg Leu Ala Ser     1160 1165 1170 AGG TTT TTC GGG AAG GTG AGG CGG GGC AGG AAC TAC GTT GAG ATA CCG 3725 Arg Phe Phe Gly Lys Val Arg Arg Gly Arg Asn Tyr Val Glu Ile Pro 1175 1180 1185 1190 AAG AAG ATC GGC TAC CTG CTC TTT GAG AAC ATG TGC GGT GTC CTA GCG 3773 Lys Lys Ile Gly Tyr Leu Leu Phe Glu Asn Met Cys Gly Val Leu Ala                 1195 1200 1205 GAG AAC AAG AGG ATT CCC GAG TTC GTC TTC ACG TCC CCG AAA GGG GTT 3821 Glu Asn Lys Arg Ile Pro Glu Phe Val Phe Thr Ser Pro Lys Gly Val             1210 1215 1220 CGG CTG GCC TTC CTT GAG GGG TAC TCA TCG GCG ATG GCG ACG TCC ACC 3869 Arg Leu Ala Phe Leu Glu Gly Tyr Ser Ser Ala Met Ala Thr Ser Thr         1225 1230 1235 GAA CAA GAG ACT CAG GCT CTC AAC GAA AAG CGA GCT TTA GCG AAC CAG 3917 Glu Gln Glu Thr Gln Ala Leu Asn Glu Lys Arg Ala Leu Ala Asn Gln     1240 1245 1250 CTC GTC CTC CTC TTG AAC TCG GTG GGG GTC TCT GCT GTA AAA CTT GGG 3965 Leu Val Leu Leu Leu Asn Ser Val Gly Val Ser Ala Val Lys Leu Gly 1255 1260 1265 1270 CAC GAC AGC GGC GTT TAC AGG GTC TAT ATA AAC GAG GAG CTC CCG TTC 4013 His Asp Ser Gly Val Tyr Arg Val Tyr Ile Asn Glu Glu Leu Pro Phe                 1275 1280 1285 GTA AAG CTG GAC AAG AAA AAG AAC GCC TAC TAC TCA CAC GTG ATC CCC 4061 Val Lys Leu Asp Lys Lys Lys Asn Ala Tyr Tyr Ser His Val Ile Pro             1290 1295 1300 AAG GAA GTC CTG AGC GAG GTC TTT GGG AAG GTT TTC CAG AAA AAC GTC 4109 Lys Glu Val Leu Ser Glu Val Phe Gly Lys Val Phe Gln Lys Asn Val         1305 1310 1315 AGT CCT CAG ACC TTC AGG AAG ATG GTC GAG GAC GGA AGA CTC GAT CCC 4157 Ser Pro Gln Thr Phe Arg Lys Met Val Glu Asp Gly Arg Leu Asp Pro     1320 1325 1330 GAA AAG GCC CAG AGG CTC TCC TGG CTC ATT GAG GGG GAC GTA GTG CTC 4205 Glu Lys Ala Gln Arg Leu Ser Trp Leu Ile Glu Gly Asp Val Val Leu 1335 1340 1345 1350 GAC CGC GTT GAG TCC GTT GAT GTG GAA GAC TAC GAT GGT TAT GTC TAT 4253 Asp Arg Val Glu Ser Val Asp Val Glu Asp Tyr Asp Gly Tyr Val Tyr                 1355 1360 1365 GAC CTG AGC GTC GAG GAC AAC GAG AAC TTC CTC GTT GGC TTT GGG TTG 4301 Asp Leu Ser Val Glu Asp Asn Glu Asn Phe Leu Val Gly Phe Gly Leu             1370 1375 1380 GTC TAT GCT CAC AAC AGC TAC TAC GGT TAC TAC GGC TAT GCA AGG GCG 4349 Val Tyr Ala His Asn Ser Tyr Tyr Gly Tyr Tyr Gly Tyr Ala Arg Ala         1385 1390 1395 CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC GTA ACG GCC TGG GGA AGG 4397 Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser Val Thr Ala Trp Gly Arg     1400 1405 1410 GAG TAC ATA ACG ATG ACC ATC AAG GAG ATA GAG GAA AAG TAC GGC TTT 4445 Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile Glu Glu Lys Tyr Gly Phe 1415 1420 1425 1430 AAG GTA ATC TAC AGC GAC ACC GAC GGA TTT TTT GCC ACA ATA CCT GGA 4493 Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly                 1435 1440 1445 GCC GAT GCT GAA ACC GTC AAA AAG AAG GCT ATG GAG TTC CTC AAC TAT 4541 Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Met Glu Phe Leu Asn Tyr             1450 1455 1460 ATC AAC GCC AAA CTT CCG GGC GCG CTT GAG CTC GAG TAC GAG GGC TTC 4589 Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe         1465 1470 1475 TAC AAA CGC GGC TTC TTC GTC ACG AAG AAG AAG TAT GCG GTG ATA GAC 4637 Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp     1480 1485 1490 GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT GAG ATT GTG AGG CGT GAC 4685 Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp 1495 1500 1505 1510 TGG AGC GAG ATA GCG AAA GAG ACG CAG GCG AGG GTT CTT GAA GCT TTG 4733 Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Leu                 1515 1520 1525 CTA AAG GAC GGT GAC GTC GAG AAG GCC GTG AGG ATA GTC AAA GAA GTT 4781 Leu Lys Asp Gly Asp Val Glu Lys Ala Val Arg Ile Val Lys Glu Val             1530 1535 1540 ACC GAA AAG CTG AGC AAG TAC GAG GTT CCG CCG GAG AAG CTG GTG ATC 4829 Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile         1545 1550 1555 CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC TAC AAG GCA ACC GGT CCC 4877 His Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro     1560 1565 1570 CAC GTT GCC GTT GCC AAG AGG TTG GCC GCG AGA GGA GTC AAA ATA CGC 4925 His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Val Lys Ile Arg 1575 1580 1585 1590 CCT GGA ACG GTG ATA AGC TAC ATC GTG CTC AAG GGC TCT GGG AGG ATA 4973 Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile                 1595 1600 1605 GGC GAC AGG GCG ATA CCG TTC GAC GAG TTC GAC CCG ACG AAG CAC AAG 5021 Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Lys             1610 1615 1620 TAC GAC GCC GAG TAC TAC ATT GAG AAC CAG GTT CTC CCA GCC GTT GAG 5069 Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu         1625 1630 1635 AGA ATT CTG AGA GCC TTC GGT TAC CGC AAG GAA GAC CTG CGC TAC CAG 5117 Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln     1640 1645 1650 AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG CTG AAG CCG AAG GGA ACT 5165 Lys Thr Arg Gln Val Gly Leu Ser Ala Trp Leu Lys Pro Lys Gly Thr 1655 1660 1665 1670 TGACCTTTCC ATTTGTTTTC CAGCGGATAA CCCTTTAACT TCCCTTTCAA AAACTCCCTT 5225 TAGGGAAAGA CCATGAAGAT AGAAATCCGG CGGCGCCCGG TTAAATACGC TAGGATAGAA 5285 GTGAAGCCAG ACGGCAGGGT AGTCGTCACT GCCCCGAGGG TTCAACGTTG AGAAGTT 5342

SEQ ID NO: 2 Array length: 774 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile   1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg              20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile          35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr      50 55 60 Val Lys Arg Val Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Val  65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile                  85 90 95 Arg Asp Lys Ile Arg Glu His Gly Ala Val Ile Asp Ile Tyr Glu Tyr             100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro         115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr     130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Val                 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys             180 185 190 Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr         195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu     210 215 220 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile                 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr             260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu         275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn     290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ala Gln Leu Ser Arg Leu                 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu             340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala         355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr     370 375 380 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His                 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp             420 425 430 Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe         435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys     450 455 460 Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr                 485 490 495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser             500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile         515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe     530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Asn Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu                 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys             580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu         595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala     610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro                 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp             660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala         675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu     690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln                 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys             740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp         755 760 765 Leu Lys Pro Lys Gly Thr     770

SEQ ID NO: 3 Array length: 2325 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomic DNA Array ATGATCCTCG ACACTGACTA CATAACCGAG GATGGAAAGC CTGTCATAAG AATTTTCAAG 60 AAGGAAAACG GCGAGTTTAA GATTGAGTAC GACCGGACTT TTGAACCCTA CTTCTACGCC 120 CTCCTGAAGG ACGATTCTGC CATTGAGGAA GTCAAGAAGA TAACCGCCGA GAGGCACGGG 180 ACGGTTGTAA CGGTTAAGCG GGTTGAAAAG GTTCAGAAGA AGTTCCTCGG GAGACCAGTT 240 GAGGTCTGGA AACTCTACTT TACTCATCCG CAGGACGTCC CAGCGATAAG GGACAAGATA 300 CGAGAGCATG GAGCAGTTAT TGACATCTAC GAGTACGACA TACCCTTCGC CAAGCGCTAC 360 CTCATAGACA AGGGATTAGT GCCAATGGAA GGCGACGAGG AGCTGAAAAT GCTCGCCTTC 420 GACATTCAAA CTCTCTACCA TGAGGGCGAG GAGTTCGCCG AGGGGCCAAT CCTTATGATA 480 AGCTACGCCG ACGAGGAAGG GGCCAGGGTG ATAACTTGGA AGAACGTGGA TCTCCCCTAC 540 GTTGACGTCG TCTCGACGGA GAGGGAGATG ATAAAGCGCT TCCTCCGTGT TGTGAAGGAG 600 AAAGACCCGG ACGTTCTCAT AACCTACAAC GGCGACAACT TCGACTTCGC CTATCTGAAA 660 AAGCGCTGTG AAAAGCTCGG AATAAACTTC GCCCTCGGAA GGGATGGAAG CGAGCCGAAG 720 ATTCAGAGGA TGGGCGACAG GTTTGCCGTC GAAGTGAAGG GACGGATACA CTTCGATCTC 780 TATCCTGTGA TAAGACGGAC GATAAACCTG CCCACATACA CGCTTGAGGC CGTTTATGAA 840 GCCGTCTTCG GTCAGCCGAA GGAGAAGGTT TACGCTGAGG AAATAACACC AGCCTGGGAA 900 ACCGGCGAGA ACCTTGAGAG AGTCGCCCGC TACTCGATGG AAGATGCGAA GGTCACATAC 960 GAGCTTGGGA AGGAGTTCCT TCCGATGGAG GCCCAGCTTT CTCGCTTAAT CGGCCAGTCC 1020 CTCTGGGACG TCTCCCGCTC CAGCACTGGC AACCTCGTTG AGTGGTTCCT CCTCAGGAAG 1080 GCCTATGAGA GGAATGAGCT GGCCCCGAAC AAGCCCGATG AAAAGGAGCT GGCCAGAAGA 1140 CGGCAGAGCT ATGAAGGAGG CTATGTAAAA GAGCCCGAGA GAGGGTTGTG GGAGAACATA 1200 GTGTACCTAG ATTTTAGATC CCTGTACCCC TCAATCATCA TCACCCACAA CGTCTCGCCG 1260 GATACGCTCA ACAGAGAAGG ATGCAAGGAA TATGACGTTG CCCCACAGGT CGGCCACCGC 1320 TTCTGCAAGG ACTTCCCAGG ATTTATCCCG AGCCTGCTTG GAGACCTCCT AGAGGAGAGG 1380 CAGAAGATAA AGAAGAAGAT GAAGGCCACG ATTGACCCGA TCGAGAGGAA GCTCCTCGAT 1440 TACAGGCAGA GGGCCATCAA GATCCTGGCA AACAGCTACT ACGGTTACTA CGGCTATGCA 1500 AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA CGGCCTGGGG AAGGGAGTAC 1560 ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT TTAAGGTAAT CTACAGCGAC 1620 ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG AAACCGTCAA AAAGAAGGCT 1680 ATGGAGTTCC TCAACTATAT CAACGCCAAA CTTCCGGGCG CGCTTGAGCT CGAGTACGAG 1740 GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT ATGCGGTGAT AGACGAGGAA 1800 GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG ACTGGAGCGA GATAGCGAAA 1860 GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG GTGACGTCGA GAAGGCCGTG 1920 AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG AGGTTCCGCC GGAGAAGCTG 1980 GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA AGGCAACCGG TCCCCACGTT 2040 GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GTCAAAATAC GCCCTGGAAC GGTGATAAGC 2100 TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG CGATACCGTT CGACGAGTTC 2160 GACCCGACGA AGCACAAGTA CGACGCCGAG TACTACATTG AGAACCAGGT TCTCCCAGCC 2220 GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG ACCTGCGCTA CCAGAAGACG 2280 AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA CTTGA 2325

SEQ ID NO: 4 Array length: 24 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTTTTGCTCA GATCTTCTTT CCTG 24

SEQ ID NO: 5 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CAGGAAAGAA GATCTGAGCA AAAG 24

SEQ ID NO: 6 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTGAAAATGC TCGCCTTCGC GATTGCAACT CTCTAC 36

SEQ ID NO: 7 Array length: 33 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTGAAAATGC TCGCCTTCGC GATTGAAACT CTCT 34

SEQ ID NO: 8 Array length: 30 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array GCCCTCGTGG TAGAGAGTTG CAATGTCGAA 30

SEQ ID NO: 9 Array length: 32 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CGGACGTACT GATAACGTAC GACGGTGACA AC 32

SEQ ID NO: 10 Array length: 33 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CCTTGAGAGA GTCGCGCGCT TCTCGATGGA AGA 33

SEQ ID NO: 11 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array TGGCTAGCCA AGGAACCACC AGTTGATTAG CAGAG 35

SEQ ID NO: 12 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array ATAAGAGGTC CCAAGACTTA GTACCTGAAG GGTGA 36

SEQ ID NO: 13 Array length: 24 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CGCCAGGGTT TTCCCAGTCA CGAC 24

SEQ ID NO: 14 Array length: 24 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA CTTTTGCTCA GATCTTCTTT CCTG 24

SEQ ID NO: 15 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AGCTGAAAAT GCTAGCCTTC GACAATGAAA CTCTCT 36

SEQ ID NO: 16 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AGCTGAAAAT GCTAGCCTTC GACGAAGAAA CTCTCT 36

SEQ ID NO: 17 Array length: 33 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA GAAAATGCTC GCCTTTGATC AAGAAACTCT CTA 33

SEQ ID NO: 18 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AGCTGAAAAT GCTAGCCTTC GACGATGAAA CTCTCT 36

SEQ ID NO: 19 Array length: 30 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA CGCCTTCGAC ATTGAAGTAC TCTACCATGA 30

SEQ ID NO: 20 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AGCTGAAAAT GCTAGCCTTC GACAGAGAAA CTCTCT 36

SEQ ID NO: 21 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AGCTGAAAAT GCTAGCCTTC GACAAAGAAA CTCTCT 36

SEQ ID NO: 22 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AAAAAGTACT CACCAGTCAC AGAAAAGCAT CTTAC 35

SEQ ID NO: 23 Array length: 34 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA AAAAAGTACT CAACCAAGTC ATTCVTGAGA ATAGT 34

[Brief description of drawings]

FIG. 1 is a diagram showing a polymerase activity and a DNA degradation rate of a modified DNA polymerase.

FIG. 2 is a diagram showing the thermostability of a modified DNA polymerase.

FIG. 3 PCR using DNA polymerase composition
It is a photograph of electrophoresis showing the results of (human genome).

FIG. 4: Thermostable DNA polymerase exo (EXO)
It is a figure which shows the amino acid sequence of a area | region.

FIG. 5 is a diagram showing the polymerase activity and DNA degradation rate of modified DNA polymerases.

FIG. 6 shows the ratio of exonuclease activity with natural KOD polymerase.

─────────────────────────────────────────────────── ─── Continuation of front page (72) Yoshihisa Kawamura 10-24 Toyocho, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Institute (72) Masahiro Takagi 1-3 Aoyamadai, Suita City, Osaka Prefecture C-58 -207 (72) Inventor Tadayuki Imanaka 2-28-11 Fujishirodai, Suita City, Osaka Prefecture (56) References JP-A-8-38198 (JP, A) JP-A-7-298879 (JP, A) International Publication 95/016028 (WO, A1) International Publication 92/009689 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) BIOSIS / WPI (DIALOG) PubMed

Claims (25)

(57) [Claims] 1. A sequence number as the first DNA polymerase
No. 141, 142, 14 of the amino acid sequence described in No. 2
At least one of the amino acids 3, 210 and 311
Has an amino acid sequence in which one is replaced with another amino acid
Thermostability with previous 3'-5 'exonuclease activity
3'-, which is 0-5% compared to DNA polymerase
Modified thermostability with 5'exonuclease activity
Amino acid of SEQ ID NO: 2 including a DNA polymerase and as the second DNA polymerase
Sequence and has 3'-5 'exonuclease activity
Thermostable DNA polymerase or
At least the 140, 142 and 144 amino acids
Also has an amino acid sequence in which one is replaced with another amino acid,
Resistance to 3'-5 'exonuclease activity before modification
100-6% compared to thermophilic DNA polymerase
Modified with 3'-5 'exonuclease activity
A first DNA polymer containing a thermostable DNA polymerase
Enzyme and the second DNA polymerase are at least 30
DNA synthesis rate of bases / second, pH 8.8 (at 25 ° C
Measured value of 60% or more after treatment at 95 ° C for 6 hours
That it has the thermal stability of being able to retain active
A characteristic DNA polymerase composition for amplifying nucleic acid. 2. The activity of the second DNA polymerase is the first
The DNA polymerase composition for amplifying nucleic acid according to claim 1, which has a smaller activity than that of the DNA polymerase. 3. The DNA polymerase composition for nucleic acid amplification according to claim 1, wherein the second DNA polymerase is 0.02 to 0.1 unit per 2.5 units of the first DNA polymerase. 4. The 3'-5 'exonuclease activity of the first DNA polymerase is higher than the 3'-5' exonuclease activity of the thermostable DNA polymerase before modification,
The DN according to claim 1, which has been reduced to about 1% or less.
A polymerase composition. 5. The first DNA polymerase further comprises:
Modified thermostable DNA polymer having physicochemical properties
The DNA polymerase composition according to claim 1, which is a protease. Optimum temperature: Approx. 75 ° C Molecular weight: 88 to 90 KDa 6. The first DNA polymerase is SEQ ID NO: 2.
The DNA according to claim 1, which is a thermostable DNA polymerase in which the 141st aspartic acid is replaced with alanine.
Polymerase composition. 7. The first DNA polymerase is SEQ ID NO: 2.
2. The DNA according to claim 1, which is a thermostable DNA polymerase in which the 142nd isoleucine is replaced with arginine.
Polymerase composition. 8. The first DNA polymerase is SEQ ID NO: 2.
2. The DNA polymerase composition according to claim 1, which is a thermostable DNA polymerase in which the 143rd glutamic acid is replaced with alanine. 9. The first DNA polymerase is SEQ ID NO: 2.
2. The DNA polymerase composition according to claim 1, which is a thermostable DNA polymerase in which the 141st aspartic acid and the 143rd glutamic acid are replaced with alanine. 10. The DNA polymerase composition according to claim 1, wherein the first DNA polymerase is a thermostable DNA polymerase in which the 210th asparagine of SEQ ID NO: 2 is replaced with aspartic acid. 11. The D according to claim 1, wherein the first DNA polymerase is a thermostable DNA polymerase in which the 311th tyrosine of SEQ ID NO: 2 is replaced with phenylalanine.
NA polymerase composition. 12. The second DNA polymerase further comprises:
Modified thermostable DNA polymer having physical and chemical properties
A DNA polymerase composition according to claim 1, which is an enzyme.
object. Optimum temperature: Approx. 75 ° C Molecular weight: 88 to 90 KDa 13. The DNA polymerase composition according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which the 142nd isoleucine of SEQ ID NO: 2 is replaced with aspartic acid. 14. The heat-resistant DNA polymerase according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which the 142nd isoleucine of SEQ ID NO: 2 is replaced with glutamic acid.
NA polymerase composition. 15. The heat-resistant DNA polymerase according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which the 142nd isoleucine of SEQ ID NO: 2 is replaced with asparagine.
NA polymerase composition. 16. The DN according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which isoleucine at the 142nd position in SEQ ID NO: 2 is replaced with glutamine.
A polymerase composition. 17. The DNA polymerase composition according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which the 142nd isoleucine of SEQ ID NO: 2 is substituted with lysine. 18. The DN according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which isoleucine at the 142nd position in SEQ ID NO: 2 is replaced with arginine.
A polymerase composition. 19. The DNA polymerase composition according to claim 1, wherein the second DNA polymerase is a thermostable DNA polymerase in which the 144th threonine of SEQ ID NO: 2 is replaced with valine. 20. A first DNA polymerase and a second D.
All of the NA polymerases have the following physicochemical properties
Is a modified thermostable DNA polymerase with quality
The DNA polymerase composition according to claim 1. Optimum temperature: Approx. 75 ° C Molecular weight: 88 to 90 KDa 21. A primer and dN using DNA as a template
TP and reacting the reagent containing a nucleic acid amplification DNA polymerase composition of any one of claims 1 to 20, and extending the primer, the nucleic acid amplification method, wherein the synthesis of DNA primer extension product .. 22. The nucleic acid amplification method according to claim 21, wherein the primers are two types of oligonucleotides, one of which is complementary to the DNA extension product of the other primer. Claim 23. repeated heating and cooling 22
The described nucleic acid amplification method. 24. The method of claim 1 for amplifying nucleic acids DNA polymerase composition of any one of claims 20, wherein, divalent ions,
A reagent for nucleic acid amplification containing a monovalent ion, a primer, dNTP and a buffer solution. 25. The DNA polymerase composition for nucleic acid amplification according to any one of claims 1 to 20 , magnesium ion and ammonium ion and / or potassium ion, and one primer DN of the other primer.
Two primers, dNT, which are complementary to the A extension product
A reagent for nucleic acid amplification containing P, BSA, a nonionic surfactant and a buffer.