JPH1175847A - Amplification of nucleic acid utilizing monoclonal antibody specific to dna polymerase originating from hyperthermophilic archaebacterium pyrococcus sp.kod1 strain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject new antibody composed of a monoclonal antibody specific to a DNA polymerase originating from a hyperthermophilic archaebacterium, inhibiting the activity of the above enzyme at a specific temperature or below, restoring the activity at or above the specific temperature and useful, for improving the efficiency of PCR. SOLUTION: This new monoclonal antibody has specificity to a DNA polymerase (KOD DNA polymerase) originating from a hyperthermophilic archaebacterium Pyrococcus sp.KOD1 strain. It inhibits the KOD DNA polymerase activity at <60 deg.C and restores the activity by the irreversible inactivation of the antibody at >60 deg.C. It belongs to IgG1 class and is useful for preventing the lowering of amplification efficiency caused by non-specific amplification and the formation of a primer dimer causing the troubles in PCR (polymerase chain reaction). The antibody can be produced by immunizing a mouse with KOD-DNA polymerase to obtain a hybridoma and culturing the hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a hyperthermophilic archaeon Py.
rococcus sp. The present invention relates to a monoclonal antibody specific to a KOD1 strain-derived DNA polymerase, a hot start PCR method using the antibody, and a reagent therefor.

[0002]

2. Description of the Related Art The PCR (polymerase chain reaction) method developed in 1986 is capable of rapidly and specifically detecting and quantifying an extremely small amount of nucleic acid. It has become one of.

[0003]

However, this P
The CR method is not without its problems. In particular, non-specific amplification of non-target nucleic acids may be seen. In many cases of this non-specific amplification, after preparing a reaction solution and setting it in a thermal cycler, a part of the 3 ′ end of the primer is replaced with the template D before the reaction temperature reaches the annealing temperature.
Annealed to NA and DNase by DNA polymerase
Start of A amplification (mispriming)
Is thought to be the cause.

As a method for solving this problem of non-specific amplification, a hot start PCR method has been developed. As a hot start PCR method, wax (Ampli-
Wax ^™ PCR Gem, Perkin-Elmer
Using a monoclonal antibody of heat-resistant DNA polymerase (TaqStart ^™ , Clontech)
Is known (JP-A-6-209775).
No.).

[0005] The method using a wax utilizes the property that the wax is solid at room temperature, but is dissolved and becomes liquid under PCR reaction conditions. Therefore, a solid layer is formed with this wax, and a lower layer containing a liquid containing a primer and a upper layer containing a liquid containing a heat-resistant DNA polymerase separates the two, thereby preventing the above-described mispriming. In this method, when the temperature reaches 70 ° C. or higher in the first PCR cycle, the wax dissolves, so that the upper and lower layers are mixed and DNA amplification starts.

When a monoclonal antibody of a thermostable DNA polymerase is used,
When added to the CR reaction solution, the action of the thermostable DNA polymerase is suppressed, and the above-described mispriming is prevented. Enter the first PCR cycle, temperature is 70 ° C
At this point, the monoclonal antibody is irreversibly denatured and DNA amplification begins.

[0007] On the other hand, many improvements have been made so far on thermostable DNA polymerases used for PCR. When the PCR method was first developed, Thermu
A PolI-type thermostable DNA polymerase such as Taq DNA polymerase derived from Saquaticus and Tth DNA polymerase derived from Thermus thermophilus has often been used.

However, these enzymes have problems such as poor fidelity of amplification and difficulty in amplifying long target nucleic acids. Recently, in addition to DNA polymerase activity,
Α-type thermostable D having 3 ′ → 5 ′ exonuclease
NA polymerase, such as the hyperthermophilic archaeon Pyroco
ccus sp. KOD DNA polymerase derived from the KOD1 strain (Toyobo) or Pyrococcus fu
riosus Pfu DNA polymerase (St
ratagene) is used for amplification requiring high fidelity. In addition, a mixed-type enzyme in which a PolI-type heat-resistant DNA polymerase and an α-type heat-resistant DNA polymerase are mixed at an appropriate ratio is used for amplifying a long target nucleic acid. In particular, it has been demonstrated that KOD DNA polymerase exhibits excellent PCR characteristics by combining high accuracy and extension ability. However, there has been no report on a monoclonal antibody against α-type thermostable DNA polymerase or a PCR method using the monoclonal antibody.

[0009]

Means for Solving the Problems The present inventors have proposed the above-mentioned KO.
The mouse was immunized with D DNA polymerase to obtain a hybridoma, a monoclonal antibody specific to KOD DNA polylase was obtained, and a monoclonal antibody inhibiting KOD DNA polymerase could be selected from among them. In addition, KOD DNA polymerase, which is an enzyme having excellent PCR characteristics, was subjected to hot start PCR using a monoclonal antibody, and the amplification efficiency of the target fragment could be increased in a short reaction time. Furthermore, a modified form of KOD DNA polymerase, KOD
Hot start PCR using a monoclonal antibody to Dash DNA polymerase is also possible,
Hot start PC using conventional DNA polymerase
It was also found that the amplification efficiency was superior to that of R, and the present invention was completed.

[0010] That is, the present invention relates to a hyperthermophilic archaeon Pyrococcus sp. It is a monoclonal antibody specific to the DNA polymerase derived from the KOD1 strain. a) has the ability to inhibit KOD DNA polymerase activity at temperatures below 60 ° C. and is irreversibly inactivated at temperatures above 60 ° C.
Restore D DNA polymerase activity. b) IgG
Antibodies that are one class.

[0011] The present invention also relates to the hyperthermophilic archaeon Pyroco.
ccus sp. It is a nucleic acid amplification reagent containing the KOD1 strain-derived DNA polymerase and the above monoclonal antibody.

[0012] The present invention also provides at least two kinds of primers, deoxyribonucleoside-5'-phosphate, and hyperthermophilic primordial Pyrococcus sp. D derived from KOD1 strain
It is a nucleic acid amplification reagent containing NA polymerase and the above monoclonal antibody.

[0013] The present invention further provides a sample containing a target nucleic acid,
At least two primers, deoxyribonucleoside-5'-phosphate, hyperthermophilic primordial Pyrococcus
sp. The DNA polymerase derived from the KOD1 strain and the reagent for nucleic acid amplification containing the above monoclonal antibody are mixed at room temperature, and the resulting mixture is heated to at least 60 ° C.
Inactivate the monoclonal antibody specific for KOD DNA polymerase and form a primer extension product, then heat to 90 ° C. or higher to separate the primer extension product and further cool to 50-70 ° C. To form a primer extension product, and repeating the heating and cooling.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The hyperthermophilic archaeon of the present invention Pyroco
ccus sp. The KOD1 strain is a coccus having a diameter of about 1 μm isolated from sulfur dioxide at Kotakarajima, Kagoshima Prefecture, and has a plurality of polar flagella and is heat-resistant. it is conceivable that.

The DNA polymerase derived from the KOD1 strain of the present invention refers to the hyperthermophilic archaeon Pyrococcus sp. K
DNA polymerase collected from OD1, recombinant DNA polymerase obtained by introducing a gene encoding the enzyme into another host cell, or DN modified with the gene
A polymerase.

The hyperthermophilic archaeon Pyrococcus s
p. The DNA polymerase collected from KOD1 has the following physicochemical properties. Action: It has DNA synthesis activity and 3'-5 'exonuclease activity. DNA synthesis rate: at least 30 bases / second. Optimum pH: 6.5-7.5 (at 75 ° C.) Optimum temperature: 75 ° C. Molecular weight: 88-90 Kda The enzyme has high accuracy of DNA synthesis, high elongation rate, and high heat resistance. It is characterized by the following.

In addition, the hyperthermophilic archaeon Pyrococcus
sp. A recombinant DNA polymerase obtained by introducing a gene encoding the KOD DNA polymerase of KOD1 into another host cell and culturing the same is described in detail in JP-A-7-298879.

Further, the DNA polymerase having the gene modified is an enzyme having a reduced 3'-5 'exonuclease activity, and examples thereof include those having the following properties. Action: has DNA synthesis activity, compared to the enzyme before modification
It has a 3'-5 'exonuclease activity of 5% or less. DNA synthesis rate: at least 30 bases / second. Optimum pH: 6.5-7.5 (at 75 ° C) Optimum temperature: 75 ° C Molecular weight: 88-90 Kda

The method for producing the modified KOD DNA polymerase is described in detail in Japanese Patent Application No. 8-198911.

Examples of the modified KOD DNA polymerase include those described in Japanese Unexamined Patent Publication No. 7-298879.
Nos. 141 and 14 of the amino acid sequence set forth in SEQ ID NO: 2
At least one of the amino acids 3, 210 and 311;
An enzyme having an amino acid sequence in which one is replaced with another amino acid is exemplified. As a specific example, a modified DNA polymerase (DA) obtained by substituting the aspartic acid at position 141 of the KOD DNA polymerase of SEQ ID NO: 2 with alanine, and glutamic acid at position 143 of the KOD DNA polymerase of SEQ ID NO: 2 Modified DNA polymerase (EA) substituted with alanine, modified DNA polymerase (DEA) substituted with alanine for aspartic acid 141 and glutamic acid 143 of KOD DNA polymerase described in SEQ ID NO: 2, described in SEQ ID NO: 2 Modified KOD DNA polymerase obtained by substituting asparagine at position 210 for aspartic acid D
NA polymerase (ND), KOD set forth in SEQ ID NO: 2
Modified DNA polymerase (Y) in which tyrosine at position 311 of DNA polymerase is substituted with phenylalanine
F). Among these modified KOD DNA polymerases, three types, DA, DEA, and DE,
No 3'-5 'exonuclease activity is detected. ND or YF is about 0.1% of the enzyme before modification, respectively.
1%, 0.01% 3'-5 'exonuclease activity is detected.

The DNA polymerase derived from the KOD1 strain of the present invention includes a recombinant DNA polymerase obtained by introducing a gene encoding the enzyme into another host cell and a DNA polymerase obtained by modifying the gene, for example, 3′-DNA.
Mutant KOD lacking 5 'exonuclease activity
It may be a mixture of DNA polymerases. It is preferable that the ratio of the modified DNA polymerase to the DNA polymerase before modification is 1: 002 to 0.01. The mixture can amplify a longer target nucleic acid than KOD DNA polymerase before modification alone. In addition, it is composed of a single enzyme system, and is characterized by high accuracy of DNA synthesis, high elongation rate, and high heat resistance. Such a mixture includes KOD Dash DNA polymerase (manufactured by Toyobo).

[0022] The monoclonal antibody of the present invention comprises the KO
An antibody that specifically reacts with DNA polymerase (KOD DNA polymerase) derived from strain D1 and has the following properties: a) It has the ability to inhibit KOD DNA polymerase activity at a temperature lower than 60 ° C. Is irreversibly inactivated at elevated temperatures, resulting in KOD
Restore DNA polymerase activity. b) Antibodies that are of the IgG1 class.

The monoclonal antibody is prepared by a conventional method for preparing a monoclonal antibody. That is, K
The spleen cells of mice immunized with OD DNA polymerase and myeloma cells are subjected to cell fusion to prepare hybridomas. A colony in which production of an antibody against KOD DNA polymerase is recognized is cloned by a limiting dilution method to obtain a hybridoma cell line that produces a monoclonal antibody against KOD DNA polymerase.

Next, a cell line producing a monoclonal antibody having a function of inhibiting the DNA polymerase activity of KOD DNA polymerase is selected from the obtained hybridoma cell lines. In the present invention, KOD DNA
As a hybridoma cell line producing a monoclonal antibody that inhibits polymerase activity, 3G8 cell line and βG
One cell line was selected, but is not limited to monoclonal antibodies that inhibit the DNA polymerase activity of KOD DNA polymerase. In addition,
Mouse-mouse hybridoma 3G8 and mouse-mouse hybridoma βG1 have been respectively deposited at the Institute of Biotechnology and Industrial Technology (FERMBP-6056,
6057).

The monoclonal antibody of the present invention can be obtained not only by the PCR method using the KOD DNA polymerase before modification, but also by a KOD DNA polymerase before modification and a DNA polymerase modified with a gene encoding the enzyme, for example, 3 ′ → 5 ′. A mixture with a mutant KOD DNA polymerase lacking exonuclease activity, for example, K
It is also effective for PCR using OD Dash DNA polymerase (manufactured by Toyobo).

The nucleic acid amplifying reagent of the present invention comprises KOD DN
A polymerase and the above monoclonal antibody.
In one embodiment, at least two primers, deoxyribonucleoside-5'-phosphate, KOD
DNA polymerase and the above monoclonal antibodies. Further, it may contain a reagent on which DNA polymerase activity depends, for example, metal salts such as manganese and magnesium, and buffers.

Deoxyribonucleoside-5'-phosphate is, for example, dATP, dCTP, dTTP or dTTP.
GTP. Also encompasses analogs such as dITP or 7-aza-dGTP, dUTP and the like.

In the PCR method, at least two kinds of primers are oligonucleotides substantially complementary to the nucleic acid sequence to be amplified, and define both ends of the nucleic acid sequence to be amplified. It functions as a template for further synthesis when the synthesized extension product is separated from its complement. Generally, a primer is an oligonucleotide having 12 to 60 nucleotides. These primers are DNA
It is synthesized by a synthesizer or isolated from a biological source.

In the nucleic acid amplification reagent, the molar ratio of the monoclonal antibody to KOD DNA polymerase is about 1:
Desirably from 1 to about 500: 1. When the molar ratio of the monoclonal antibody is lower than that of KOD DNA polymerase, the activity is not inhibited, and a primer dimer is formed.

In a most preferred embodiment of the present invention, at least two primers, deoxyribonucleoside-
5′-phosphate, a recombinant DNA polymerase obtained by introducing a gene encoding the enzyme into another host cell, and a DNA polymerase obtained by modifying the gene, for example, 3 ′ →
Mutant KOD lacking 5 'exonuclease activity
A reagent for nucleic acid amplification comprising a mixture of DNA polymerase and the above monoclonal antibody.

The method for amplifying a target nucleic acid of the present invention comprises the steps of:
This is a so-called hot start PCR that inhibits the activity of primer annealing using a monoclonal antibody specific to a DNA polymerase. Specifically, a sample containing a target nucleic acid is provided with at least two types of primers, deoxyribonucleoside-5 ′. -Phosphate, KOD DNA
The nucleic acid amplification reagent containing the polymerase and the monoclonal antibody of the present invention is mixed at room temperature, and the resulting mixture is heated to at least 60 ° C. to inactivate the monoclonal antibody and to form a primer extension product. .

The hot start PCR of the present invention is KO
The D DNA polymerase and the monoclonal antibody bind at room temperature to make the KOD DNA polymerase inactive, but at the temperature at which the PCR reaction is started, for example, 75 ° C. or higher, the KOD DNA polymerase becomes active, and the normal This is a method in which the DNA synthesis reaction is started, the monoclonal antibody is denatured, and irreversibly inactivated.
The reaction proceeds.

Furthermore, in the present invention, a nucleic acid amplification reagent containing at least two kinds of primers, deoxyribonucleoside-5'-phosphate, KOD DNA polymerase and the monoclonal antibody of the present invention is added to a sample containing a target nucleic acid at room temperature. Mix and bring the resulting mixture to at least 60 ° C.
To inactivate the monoclonal antibody specific for KOD DNA polymerase and form the primer extension product, and then heat to 90 ° C. or higher to separate the primer extension product, and further to 50 ° C. ~ 70 ° C
To form an extension product. By repeating the heating and cooling, the target nucleic acid can be amplified exponentially.

[0034] The most preferred embodiment of the present invention is the KOD
A recombinant DNA polymerase obtained by introducing a gene encoding a DNA polymerase into another host cell and a DNA polymerase modified with the gene, for example, 3′-5 ′
Mutant KOD D lacking exonuclease activity
This is a method using a mixture of NA polymerases, which allows amplification of a target nucleic acid longer than recombinant DNA polymerase alone. Further, it is composed of a single enzyme system, and is characterized by high accuracy of DNA synthesis, high elongation rate, and high heat resistance.

In the present invention, by using the above enzyme mixture, an increase in amplification effect can be recognized even in long chain PCR using a DNA having a length of 10 kbp or more as a template.

[0036] The nucleic acid amplified by the nucleic acid amplification method of the present invention can be used to prepare cells, viruses, hair,
Specific nucleic acids in body fluids or materials containing detectable DNA or RNA can be detected or gene sequencing can be performed. Its use is extended to various ranges such as genetic diagnosis, unknown genetic research, and genetic testing.

[0037]

Next, the present invention will be described with reference to examples. Example 1 Preparation of Mouse Hybridoma Cell Line KOD was intraperitoneally injected into BalB / c mice (8-week-old female).
50 μg of an antigen preparation of DNA polymerase was injected.
An antigen preparation of KOD DNA polymerase was prepared from KOD DNA polymerase (manufactured by Toyobo Co., Ltd.) prepared from a recombinant E. coli, a phosphate buffer, and Freund's comp.
Let adjuvant was mixed and emulsified. Two and four weeks later, a further 50 μg each of the antigen preparation was injected and boosted.
Two weeks later, 100 µl of KOD DNA polymerase was used.
g were further injected and final immunized.

After blood loss of the immunized mouse, the mouse spleen cells were filtered through a nylon mesh, and the filtrate was centrifuged (900 rpm, 5 minutes). After removing the supernatant, Hem
lysis solution (155 mM NH ₄ Cl, 10 mM
KHCO ₃ , 1 mM NaN ₃ )
l was added. Immediately after mixing on an ice bath, 0.2 to 0.3 ml of FBS (fetal calf serum) was added. Further, 15 to 20 ml of Cell Glosser H solution (Sumitomo Pharmaceutical) was added to disperse the spleen cells, followed by centrifugation again. After removing the supernatant, 15-20 ml of Cell Glosser H solution was added again,
The spleen cells were dispersed.

Myeloma cells (P3X63Ag8U.
1, Dainippon Pharmaceutical) thawed the frozen cells, added 10 ml of Cell Glosser H solution (Sumitomo Pharmaceutical), mixed gently, and centrifuged (1,000 rpm, 5 minutes). The same operation was repeated for the sedimented cells. To the sedimented cells, a Cell Grosser H solution (Sumitomo Pharmaceutical) was added to make the total volume 15 ml.
O ₂ ). After the culture, centrifugation (900 rpm)
m, 5 minutes), and after removing the supernatant, 15 to 20 ml of Cell Glosser H solution (Sumitomo Pharmaceutical) was added to disperse the cells.

The densities of the spleen cells and myeloma cells were measured using a cell density cell counting system, and then mixed at a ratio of 5: 1 myeloma cells: spleen cells, followed by centrifugation (90%).
0 rpm for 5 minutes). After removing the supernatant, 0.6 to 0.7 ml of a polyethylene glycol 1500 solution (manufactured by Boehringer Mannheim) was added, followed by gentle stirring. After 1, 2, and 4 minutes while warming by hand, 2 ml of Cell Glosser H solution (Sumitomo Pharmaceutical) was added. Further, after adding 0.6 ml of FBS (fetal calf serum), centrifugation (900 rpm)
After 5 minutes), 40 to 50 ml of HAT medium was added. After gentle stirring, two drops were added to a 96-well microculture plate using a Pasteur pipette, and further a HAT medium was added one drop at a time.

The wells in which colonies were grown were placed in an EL.
When tested by the ISA method, several positive wells were found. After the colonies in the positive wells were replaced with HAT medium, cloning was performed by the limiting dilution method. That is, in a 96-well microculture plate,
The culture was performed by diluting the cells to an average of 1 cell or less per well. At this time, Balb / C mouse peritoneal macrophages were used as feeder cells to assist cell proliferation. As a result, 15 types of hybridoma cell lines (cell lines) producing monoclonal antibodies to KOD DNA polymerase were obtained.

Example 2 KOD DNA polymerase activity
About 15 types of monoclonal antibodies produced by hybridoma cell line study obtained sexual inhibition ability was examined inhibitory potency against KOD DNA polymerase. That is, KOD D
For 1 unit of NA polymerase (Toyobo)
The molar ratio of each of the 15 monoclonal antibodies was 1:
Mix for 10 minutes at room temperature in the range of 50 to 1: 500.
This mixture was added to the reaction solution (12 mM Tris-HCl, p
H8.8, 10 mM KCl, 6 mM (NH ₄ ) ₂ S
O ₂ , 0.1% Triton X-100, 0.001
% BSA, 1 mM MgCl ₂ , 12 μg of activated salmon sperm DNA), 0.1 mM dATP, dTTP, d
GTP and tritium labeled dCTP (200n
(Ci / nmol) and incubated at 42 ° C. for 2 hours 30 minutes, and then the DNA polymerase activity was measured.

The DNA polymerase activity was measured by measuring the incorporation of dCTP labeled with tritium using a liquid scintillation counter. The results are summarized in FIG. As is apparent from FIG.
Among the monoclonal antibodies, the 3G8 cell line (FE
RM BP-6056) and the βG1 cell line (FERM
BP-6057) produced monoclonal antibody 3G
8 and βG1 strongly inhibited KOD DNA polymerase activity.

When the isotypes of the monoclonal antibodies 3G8 and βG1 produced by the 3G8 cell line and the βG1 cell line were examined, both were found to be of the IgG1 type.

Example 3 KOD Dash DNA Poly
Addition of monoclonal antibody by PCR method using merase
The additive monoclonal antibodies βG1 and 3G8 were converted to KOD Da.
sh DNA polymerase (manufactured by Toyobo), mixed with PC
The effect of addition by the R method was confirmed. KOD Dash DN
10x reaction buffer 10μ attached to A polymerase
1, 2 mM dNTP, 20 μmol, 1 μl of the primers described in Sequence Listings 1 and 2, human placenta c
1 ng DNA (manufactured by Clontech), human genome D
100 ng of NA was added to the PCR reaction tube, and sterile distilled water was further added to make 98 μl. In addition, KO
A mixture of 2.5 units of D Dash DNA polymerase and 2 μg of monoclonal antibody 3G8 or βG1 mixed at room temperature for 10 minutes, respectively, was added to the PCR.
Was done.

The thermal cycler used was Model PJ2000 manufactured by PerkinElmer. The PCR conditions were 94 ° C., 15 seconds → 61 ° C., 30 seconds → 74 ° C.,
Zero seconds was repeated 35 cycles.

For comparison, Taq DNA polymerase, a mixture of Taq polymerase and Taq antibody (Taq start, Clontech), KOD D
PCR was performed in the same manner using only a mixture of ASH DNA polymerase (manufactured by Toyobo) and BSA (bovine serum albumin) and KOD Dash DNA polymerase without using an antibody. However, the reaction composition of Taq DNA polymerase followed the instruction manual (Clontech). The cycle conditions of Taq DNA polymerase were the same as those described above, except that 74 ° C. was changed to 72 ° C. The result is shown in FIG.

As is apparent from FIG. 2, KOD Das
h When DNA polymerase (manufactured by Toyobo Co., Ltd.) and the monoclonal antibodies 3G8 and βG1 were mixed and PCR was performed, the target 1.3 kb target was clearly better than the conditions using other monoclonal antibodies. Amplification was confirmed.

Example 4 KOD DNA polymerase
Effect of addition of monoclonal antibody in PCR used Monoclonal antibody 3G8 was mixed with KOD DNA polymerase (manufactured by Toyobo), and the effect of addition in PCR was confirmed. 10 μl of 10 × reaction buffer attached to KOD DNA polymerase, 10 μl of 2 mM dNTP, 1 μl of 20 pmol of primers described in Sequence Tables 3 and 4, human
100 ng of genomic DNA was added to a tube for PCR reaction, and sterile distilled water was further added to make 98 μl. PCR was performed by adding 2.5 units of KOD DNA polymerase and 2 μg of the monoclonal antibody 3G8 at room temperature for 10 minutes.

The thermal cycler used was Model PJ2000 manufactured by PerkinElmer. The reaction conditions were 9
After treatment at 5 ° C for 5 seconds, 30 seconds at 55 ° C → 3 seconds at 74 ° C
After treating for 0 second and further for 15 seconds at 95 ° C, 55
C., 30 seconds → 74 ° C., 30 seconds → 95 ° C., 15 seconds, 35 cycles were repeated.

For comparison, PCR was carried out using KOD DNA polymerase (manufactured by Toyobo Co., Ltd.) without using a monoclonal antibody, and in the same manner without using both. The result is shown in FIG.

When PCR was carried out by mixing KOD DNA polymerase and monoclonal antibody 3G8, it was clearly confirmed that the desired 555 bp target was properly amplified.

[0053]

EFFECT OF THE INVENTION By utilizing the monoclonal antibody of the present invention, the hyperthermophilic archaeon Pyrococcus sp. KOD1
Hot start P using DNA polymerase from strain
Performing CR makes it possible to efficiently amplify nucleic acids. In addition, amplification of nonspecific by-products and primer dimers can be suppressed. Furthermore, it becomes possible to amplify long-chain nucleic acids (> 10 kb).

[0054]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 27 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CCACCATCTC GGTCATCAGG ATTGCCT 27

SEQ ID NO: 2 Sequence length: 27 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence TTCTCATGGA AGCTATGGGT ATCACAT 27

SEQ ID NO: 3 Sequence length: 23 Sequence type: Nucleic acid (DNA) Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATGAACTCCT TCTCCACAAG CGC

SEQ ID NO: 4 Sequence length: 22 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GAAGAGCCCT CAGGCTGGAC TG

[Brief description of the drawings]

FIG. 1 is a graph showing the results of examining the ability to inhibit KOD DNA polymerase activity of 15 monoclonal antibodies produced by hybridoma cell lines.

FIG. 2. Monoclonal antibodies βG1 and 3G8
It is an electrophoresis photograph instead of a drawing showing the addition effect by the PCR method mixed with OD Dash DNA polymerase (manufactured by Toyobo Co., Ltd.).

FIG. 3. Monoclonal antibody 3G8 was converted to KOD DNA.
It is an electrophoresis photograph instead of a drawing showing the effect of addition in a PCR method mixed with a polymerase (manufactured by Toyobo).

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/50 G01N 33/53 D 33/53 33/569 F 33/569 33/577 B 33/577 C12N 15/00 C // (C12N 9/12 C12R 1:01) (C12P 21/08 C12R 1:91)

Claims (9)

[Claims] 1. A hyperthermophilic archaeon Pyro having the following properties:
coccus sp. A monoclonal antibody specific to a KOD1 strain-derived DNA polymerase (KOD DNA polymerase). a) having the ability to inhibit KOD DNA polymerase activity at temperatures below 60 ° C. and being irreversibly inactivated at temperatures above 60 ° C.
Restore DNA polymerase activity. b) Antibodies that are of the IgG1 class. 2. KOD DNA polymerase is a hyperthermophilic archaeon, Pyrococcus sp. The monoclonal antibody according to claim 1, which is a DNA polymerase collected from KOD1, a recombinant DNA polymerase obtained by introducing a gene encoding the enzyme into another host cell, or a DNA polymerase modified with the gene. 3. The monoclonal antibody according to claim 1, which is a monoclonal antibody secreted by the hybridoma cell line 3G8 or the hybridoma cell line βG1. 4. The monoclonal antibody according to claim 1, which is a monoclonal antibody secreted by the hybridoma cell line 3G8. 5. The monoclonal antibody according to claim 1, which is a monoclonal antibody secreted by the hybridoma cell line βG1. 6. The hyperthermophilic archaeon Pyrococcus s
p. KOD1 strain-derived DNA polymerase (KOD DN
A polymerase) and a reagent for nucleic acid amplification comprising the monoclonal antibody according to claim 1. 7. At least two kinds of primers, deoxyribonucleoside-5'-phosphate, hyperthermophilic archaea Pyr
ococcus sp. A nucleic acid amplification reagent comprising the KOD1 strain-derived DNA polymerase (KOD DNA polymerase) and the monoclonal antibody according to claim 1. 8. KOD DNA polymerase is a hyperthermophilic archaeon Pyrococcus sp. The nucleic acid amplification according to claim 6 or 7, which is a DNA polymerase collected from KOD1, a recombinant DNA polymerase obtained by introducing a gene encoding the enzyme into another host cell, or a DNA polymerase modified with the gene. For reagents. 9. The method according to claim 9, wherein the KOD DNA polymerase is Pyrococcus sp. A mixture of a recombinant DNA polymerase obtained by introducing a gene encoding a DNA polymerase collected from KOD1 into another host cell and a 3'-5 'exonuclease activity deficient mutant obtained by modifying the gene. Item 8. The nucleic acid amplification reagent according to Item 6 or 7.