JP2002253265A - Varied heat resistant dna polymerase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a heat resistant DNA polymerase enabling a PCR excellent in an amplification factor of a gene and accuracy of the amplification. SOLUTION: This heat resistant DNA polymerase has amino acids substituting histidine in a DXE sequence of an EXO1 region of a heat resistant DNA polymerase having activity of a 3'-5' exonuclease and a DX1 EX2 X3 X4 H (D: aspartic acid, E: glutamic acid, H: histidine, X1 , EX2 , X3 and X4 : are each an arbitrary amino acid) sequence comprising a peptide having length of four amino acids adjacent to the E of the sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a thermostable DNA polymerase having improved amplification efficiency and / or accuracy in polymerase chain reaction (PCR) and a method for producing the same. Furthermore, the present invention relates to a method for amplifying a nucleic acid using the thermostable DNA polymerase, and a reagent containing the thermostable DNA polymerase.

[0002]

2. Description of the Related Art In recent years, PCR has become one of the essential techniques in research and testing in the fields of biochemistry, molecular biology and clinicopathology. PCR is characterized by using a thermostable DNA polymerase, and the most frequently used DNA polymerases at present are mainly thermos DNA polymerases.
Heat resistance D from Aquaticus (Thermus aquaticus)
NA polymerase (Taq DNA polymerase)
Thermostability D from Thermoticus (Thermusthermophilus)
It is a heat-resistant DNA polymerase called Pol I type such as NA polymerase (Tth DNA polymerase). Pol I type DNA polymerases are characterized by high amplification efficiency and easy setting of conditions. However, the enzyme has a problem in that the fidelity of nucleic acid incorporation during amplification is poor, and is said to be unsuitable for cloning the amplified gene.

[0003] On the other hand, a DNA polymerase called α-form derived from hyperthermophilic archaeon, for example, a thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu DNA polymerase; WO92 / 9689; JP-A-5-328969). Publication), a thermostable DNA polymerase derived from Thermococcus litoralis (Ti (Vent) polymerase, JP-A-6-7160), Pyrococcus kodakaraensis KOD1 (former name:
Pyrococcus sp. KOD1) -derived thermostable DNA polymerase (KOD DNA polymerase; JP-A-7-29887)
No. 9) is also known. In recent reports, Pyrococcus kodakaraensis may be classified as Thermococcus. These α-type DNA polymerases have 3′-5 ′ exonuclease activity (Proof reading
Activity), and the accuracy of nucleic acid incorporation was determined by Taq DN
It is characterized by being superior to polI type DNA polymerases such as A polymerase.

[0004]

However, α-type D
There are problems such as insufficient amplification efficiency in PCR amplification using NA polymerase. In addition, many of these α-type DNA polymerases have a narrow range of optimum conditions such as a PCR reaction time, an enzyme amount, and a primer concentration. PCR in α-type DNA polymerase
It is conceivable that the above-mentioned problem in amplification involves the strength of 3'-5 'exonuclease activity. That is, at the time of PCR, primers and the like 3 ′
It is thought that the amplification efficiency in PCR is reduced by being deleted by -5 'exonuclease activity. The α-type DNA polymerase has a region exhibiting 3′-5 ′ exonuclease activity in a single protein,
Since there is a region showing DNA polymerase activity, both activities interact with each other, and it is considered that differences in the respective regions' affinity for nucleic acids and the like also affect PCR amplification.

[0005] In the amino acid sequence of the DNA polymerase responsible for the expression of 3'-5 'exonuclease activity, a highly conserved amino acid region thought to be involved in the expression of 3'-5' exonuclease activity. (EXOI
(Fig. 1), EXOII, EXOIII) are known to exist (Gene, 100, 27-38 (1991), Gene, 112, 139-).
144 (1992)). The EXO I region contains an XDXEX motif (D: aspartic acid, E: glutamic acid, X: any amino acid), and aspartic acid and glutamic acid are known to be essential for the expression of exonuclease activity (Kong et al.
(1993), Journal of Biological Chemistry, vol. 268, 1
965-1975). It has been reported that exonuclease activity can be deleted or reduced to 1 / 10,000 or less by replacing aspartic acid and glutamic acid in these amino acid sequences with a neutral amino acid, alanine (Kong et al. (1993), Journal of Biological Chemistry,
vol. 268,1965-1975). However, there has been a problem that by deleting or reducing the exonuclease activity to 1 / 10,000 or less, the accuracy at the time of DNA replication, which is a characteristic of α-type DNA polymerase, is also lost.

[0006] Further, attempts have been made to attenuate the 3'-5 'exonuclease activity stepwise by substituting the amino acid indicated by X in the XDXEX motif in KOD DNA polymerase with an arbitrary amino acid (particularly). JP-A-10-42871). According to this method, as 3′-5 ′ exonuclease activity decreases, PCR efficiency increases and fidelity in amplification increases.
Is simultaneously observed. Therefore, when this portion is replaced, the 3′-5 ′ is not impaired.
It is important to produce a variant enzyme with reduced exonuclease activity. However, an enzyme obtained by this method, for example, a mutant (IQ) in which the 142nd isoleucine from the 5 'end of KOD DNA polymerase is substituted with glutamine or a mutant in which lysine is substituted
(IK) did not always have a good amplification rate from low copy number DNA, and was not a mutant having excellent PCR efficiency (FIG. 3). Also, JP-A-10-4
According to Japanese Patent No. 2871, the 3'-5 'exonuclease activity (proof reading) is used as long as this method is used.
It is difficult to obtain a mutant with increased activity).

[0007]

Means for Solving the Problems The present inventors have prepared various mutants of KOD DNA polymerase in order to solve the present problem, and as a result of intensive studies, found that XDXEX in the EXOI region
By substituting the fourth histidine residue (147th; hereinafter also referred to as H) from the motif of glutamic acid with various amino acids, 3′-5 ′ exonuclease activity of various strengths, PCR efficiency and The present inventors have found that it is possible to obtain a thermostable DNA polymerase exhibiting accuracy, and arrived at the present invention (hereinafter, this histidine-containing motif is referred to as a DXEXXXH motif. DX ₁ EX ₂ X ₃ X ₄ H mochi −
And the notations such as array DX ₁ EX ₂ X ₃ X ₄ H are also synonymous. ).

That is, the present invention has the following configuration. 1. 3'-5 'consisting of 4 amino acids long peptide adjacent to E of DXE sequence and the sequence of the exonuclease I (Exol) region of thermostable DNA polymerase having exonuclease activity _{DX 1 EX 2 X 3 X 4} H (D : Aspartic acid, E: glutamic acid, H: histidine, X ₁ , X ₂ , X ₃ and X ₄ : any amino acid)
A thermostable DNA polymerase in which histidine is replaced by another amino acid in the sequence. 2. DX ₁ EX ₂ X ₃ X ₄ histidine aspartic acid H sequence, glutamic acid, tyrosine, alanine, lysine, and thermostable DNA polymerase of claim 1, wherein substituted with any amino acid selected from the group consisting of arginine . 3. The heat-resistant DNA polymerase according to claim 1, which has the following physicochemical properties. (1) DNA extension rate: at least 20 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has 10% or more DN as compared with the DNA polymerase before the heat treatment.
3. Retain A polymerase residual activity The thermostable DNA polymerase according to claim 3, which has the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DNase of 40% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 5. The thermostable DNA polymerase according to claim 4, which has the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DN of 60% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 6. 6. The heat-resistant DNA polymerase according to claim 5, wherein histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with any one of aspartic acid, glutamic acid, tyrosine, alanine, lysine, and arginine. 7. 7. The heat-resistant DNA polymerase according to claim 6, wherein histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with aspartic acid. 8. 7. The heat-resistant DNA polymerase according to claim 6, wherein histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with glutamic acid. 9. 7. The heat-resistant DNA polymerase according to claim 6, wherein histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with tyrosine. 10. In the amino acid sequence of SEQ ID NO: 2,
7. The thermostable DNA polymerase according to claim 6, wherein the histidine is substituted with alanine. 11. In the amino acid sequence of SEQ ID NO: 2,
7. The thermostable DNA polymerase according to claim 6, wherein the histidine is replaced with lysine. 12. In the amino acid sequence of SEQ ID NO: 2,
7. The thermostable DNA polymerase according to claim 6, wherein the second histidine is substituted with arginine. 13. DXE of exo I (EXOI) region of thermostable DNA polymerase having 3'-5 'exonuclease activity
DX ₁ EX ₂ X ₃ X ₄ H (D: aspartic acid, E: glutamic acid, H: histidine, X ₁ , X ₂ , X ₃ and X ₄₎ consisting of the sequence and a peptide having a length of 4 amino acids adjacent to E of the sequence : An arbitrary amino acid) a gene encoding a thermostable DNA polymerase in which histidine is replaced by another amino acid in the sequence. 14． 14. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 20 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has 10% or more DN as compared with the DNA polymerase before the heat treatment.
14. Retain A polymerase residual activity 14. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DNase of 40% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced by another amino acid. 16. 14. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DNase of 60% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced by another amino acid. 17． A recombinant vector comprising the gene according to any one of claims 13 to 16 inserted into an expression vector. 18. The genetic recombinant vector according to claim 17, wherein the vector is a vector derived from pLED-MI or pBluescript. 19. A transformant obtained by transforming a host cell with the recombinant vector according to claim 17. 20. The host cell is Escherichia coli
The transformant according to claim 19, which is E. coli). 21. A method for producing a thermostable DNA polymerase, comprising culturing the transformant according to claim 20, and collecting a thermostable DNA polymerase from the culture. 22. By reacting DNA as a template, at least one primer, dNTPs, and the thermostable DNA polymerase according to any one of claims 1 to 12,
A nucleic acid amplification or extension method comprising extending a primer to synthesize a DNA primer extension. 23. 23. The nucleic acid amplification method according to claim 22, wherein the primers are two kinds of oligonucleotides, one of which is complementary to the other DNA extension. 24. 23. The nucleic acid amplification method according to claim 22, wherein heating and cooling are repeated. 25. One primer is the DNA of the other primer
Two primers that are complementary to each other to the extension product, d
A nucleic acid amplification reagent comprising NTP, the thermostable DNA polymerase according to any one of claims 1 to 12, divalent ions, monovalent ions, and a buffer. 26. One primer is the DNA of the other primer
Two primers which are complementary to each other to the extension product, d
NTP, heat-resistant DN according to any one of claims 1 to 12.
A reagent for nucleic acid amplification, comprising: A polymerase, at least one selected from the group consisting of magnesium ions, ammonium ions and potassium ions, BSA (bovine serum albumin), a nonionic surfactant, and a buffer. . 27. One primer is the DNA of the other primer
Two primers which are complementary to each other to the extension product, d
NTP, heat-resistant DN according to any one of claims 1 to 12.
A polymerase, at least one selected from the group consisting of magnesium ions, ammonium ions, and potassium ions, BSA, a nonionic surfactant, a buffer, and the polymerase activity and 3′-5 ′ exonuclease of the thermostable DNA polymerase A nucleic acid amplification reagent comprising an antibody having an activity of suppressing at least one of the activities. 28. The heat-resistant DN according to claim 1.
A DNA polymerase composition comprising at least one A polymerase. 29. DNA as template, mutagenesis primer, d
NTP and heat resistance D according to any one of claims 1 to 12.
A gene mutation introduction method comprising synthesizing a DNA primer extension by extending a primer by reacting NA polymerase. 30. Mutation introduction primer, dNTP, and claim 1
13. A reagent for introducing a gene mutation, comprising the thermostable DNA polymerase according to any one of 12.

[0009] In the present invention, the DX of a thermostable DNA polymerase having 3'-5 'exonuclease activity is used.
By substituting H in the EXXXH motif with an acidic amino acid such as glutamic acid or aspartic acid, the 3′-5 ′ exonuclease activity can be reduced. In fact, the KOD DNA polymerase HE mutant (Histidine at position 147 was replaced with glutamic acid) and the HD mutant (Histidine at position 147 was replaced with aspartic acid) were 3'-5 'compared to wild-type KOD DNA polymerase.
Exonuclease activity is 25% and 6.25%, respectively.
(Fig. 2). In the present invention, 3 ′
By substituting histidine of the DXEXXXH motif of a thermostable DNA polymerase having -5 'exonuclease activity with an acidic amino acid such as glutamic acid or aspartic acid, or a neutral amino acid such as tyrosine or alanine, particularly from DNA having a low copy number. Can improve the efficiency of the PCR amplification. In fact, KOD DN
A polymerase HE mutant (Histidine at position 147 replaced with glutamic acid), HD mutant (Histidine at position 147 replaced with aspartic acid), HY mutant (147
Histidine is replaced with tyrosine) and HA variants
(The histidine at position 147 was replaced with alanine), it was possible to observe an increase in PCR efficiency (FIGS. 3 and 4). Of these mutants, particularly in HY, no remarkable decrease in exonuclease activity was observed (FIG. 2). Therefore, the histidine at position 147 of the DXEXXXH motif was used for PCR independently of the exonuclease activity. It suggests that it also plays a role in determining efficiency. In addition, in amplification of long-sized DNA, it was confirmed that the PCR efficiency was improved particularly in HE mutants and HD mutants substituted with acidic amino acids (FIG. 4).

Further, in the present invention, the DX of the thermostable polymerase having 3'-5 'exonuclease activity
By substituting H in the EXXXH motif with a basic amino acid such as lysine or arginine, the 3′-5 ′ exonuclease activity of the thermostable DNA polymerase and / or P
Accuracy at the time of CR can be improved. In fact, in the KOD DNA polymerase HK mutant (substitute histidine at position 147 with lysine) and the HR mutant (substitute histidine at position 147 with arginine) obtained in the examination of the present invention, the 3'-5 'exonuclease was used. A remarkable increase in activity could be confirmed (FIG. 2). In addition, both mutants were compared with the wild-type enzyme by PCR.
It was possible to confirm the improvement of the time accuracy (FIG. 5). Further, it can be easily predicted that a new function may be added by substituting the 147th histidine with an amino acid other than the above.

[0011]

DETAILED DESCRIPTION OF THE INVENTION The thermostable DNA polymerase of the present invention
Is a DNA having 3'-5 'exonuclease activity
Exo I (EXOI) region in the amino acid sequence of the polymerase
Region of the DXE sequence and the four amino acid long
DX made of peptide₁EX_TwoX_ThreeX_FourH (D: aspartic acid, E: g
Lutamic acid, H: histidine, X₁, X_Two, X_ThreeAnd X_Four: Any
In the amino acid sequence, H was replaced with another amino acid.
An enzyme characterized by the following. DX the array₁EX_TwoX_ThreeX_FourH and
The exo I (EXOI) region
Although the width of the area is slightly different, the DXE array
Wherein the C-terminus of the region is X _Two, X_Three, X_FourBeing one of
I have. Part of the exo I (EXOI) region and adjacent amino
DX consisting of acid sequence₁EX_TwoX_ThreeX_FourHeat resistant D with H motif
NA polymerase can be of any origin, for example,
KOD derived from Ilococcus kodakaraensis KOD1 strain
DNA polymerase from Pyrococcus furiosus
Thermostable DNA polymerase and Thermococcus li
Illustrative examples include heat-resistant DNA polymerase from Traris
It is. The DX₁EX_TwoX_ThreeX_FourThe specific sequence of the H motif is
"DIETLYH" can be mentioned. This array is Pyro
Coccus kodakaraensis KOD1 strain and Pyroco
In DNA polymerase from C. furiosus
Completely preserved, Thermococcus litoralis
In the conventional DNA polymerase, "DIETFYH"
Other than F, they are stored similarly (Fig. 1). The D
X₁EX_TwoX_ThreeX_FourAmong the H motifs, aspartic acid and glue
Mutants that intentionally substituted tamic acid with another amino acid
However, it is easily anticipated that the effects of the present invention will be similarly extended.
It can be said that this is included in the category of the present invention.
The “other amino acids” are not particularly limited,
Acidic amino acids such as aspartic acid and glutamic acid, tiro
Syn, alanine, glycine, valine, leucine, isolo
Isin, Serine, Proline, Asparagine, Glutami
, Threonine, cysteine, methionine, tryptophan
Neutral amino acids such as phenyl and phenylalanine;
Basic amino acids such as luginin can be exemplified.
You. Preferably, aspartic acid, glutamic acid, tiro
Syn, alanine, lysine, arginine.

In one embodiment of the present invention, 3'-5 '
DNA polymerase having exonuclease activity
An amino acid sequence containing an exoI (EXOI) region
Amino acid sequence DX₁EX_TwoX_ThreeX_FourH (D: aspartic acid, E: gluta
Minic acid, H: histidine, X₁, X _Two, X_ThreeAnd X_Four: Any amino
Acid) sequence, H is glutamic acid or aspartic acid
By replacing any acidic amino acids, the enzyme before modification
Significantly lower 3'-5 'exonuclease activity than
The following thermostable polymerases can be mentioned. Ma
In one embodiment of the present invention, 3'-5 'exonu
Amino acids of DNA polymerase having creatase activity
Amino acid sequence containing exo I (EXOI) region in the sequence
DX₁EX_TwoX_ThreeX_FourOf H, H is aspartic acid, glutamine
Substitution with acid, tyrosine and alanine increases nucleic acid
Examples of thermostable polymerases modified to be superior
Can be Further, as one embodiment of the present invention
Is a DNA having 3'-5 'exonuclease activity
Exo I (EXOI) region in the amino acid sequence of the polymerase
Region of the DXE sequence and the four amino acid long
DX made of peptide₁EX_TwoX_ThreeX_FourH (D: aspartic acid, E: g
Lutamic acid, H: histidine, X₁, X_Two, X_ThreeAnd X_Four: Any
H) in the amino acid sequence
By substituting with a basic amino acid, significantly 3′-
Increased 5 'exonuclease activity and / or accuracy
Heat-resistant polymerase. concrete
In the present invention, the 3'-5 'exonucleus is
The DX of a thermostable DNA polymerase having zease activity₁EX_TwoX_Three
X_FourH of the motif H to glutamic acid or aspartic acid
By substitution with an acidic amino acid of
Sonuclease activity can be reduced. In fact,
The KOD DNA polymerase HE mutant (human 147)
Glutamic acid was substituted for stidine and the HD mutant (1
Histidine at position 47 was replaced with aspartic acid)
3'-5'-d compared to native KOD DNA polymerase
The exonuclease activity is about 25% and 6.25%, respectively.
(Fig. 2).

In one embodiment of the present invention, 3′-5 ′
In the amino acid sequence of a DNA polymerase having an exonuclease activity, a DXE sequence of an exoI (EXOI) region and a D-amino acid sequence consisting of a 4-amino acid-long peptide adjacent to E in the sequence
X ₁ EX ₂ X ₃ X ₄ H (D: aspartic acid, E: glutamic acid, H: histidine, X ₁ , X ₂ , X ₃ and X ₄ : any amino acid) In the sequence, H is replaced with another amino acid And a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA elongation rate: at least 20 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase can retain 10% or more of the residual activity by treatment at 5 ° C. for 6 hours (ie, at 95 ° C. for 6 hours at pH 8.8 (measured at 25 ° C.)
Retains a DNA polymerase residual activity of 10% or more as compared to the DNA polymerase before the heat treatment. )

Further, as another embodiment of the present invention,
In the amino acid sequence of a DNA polymerase having 3'-5 'exonuclease activity, D in the exo I (EXOI) region
DX ₁ EX ₂ X ₃ X ₄ H consisting of an XE sequence and a peptide having a length of 4 amino acids adjacent to E of the sequence (D: aspartic acid, E: glutamic acid, H: histidine, X ₁ , X ₂ , X ₃ and X ₄ : Any amino acid) is an enzyme in which H is replaced with another amino acid in the sequence, and further has a modified heat-resistant D having the following physicochemical properties:
Regarding NA polymerase. (1) DNA synthesis rate: at least 30 bases / second (2) Thermal stability: retention of 40% or more of residual activity by treatment at pH 8.8 (measured at 25 ° C.) at 95 ° C. for 6 hours Can be.

Another embodiment of the present invention is a modified thermostable DNA polymerase having the following properties. (1) DNA synthesis rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
A residual activity of 60% or more can be maintained by treatment at 5 ° C. for 6 hours. (3) Optimum temperature: about 65 to 75 ° C. (measured by changing the temperature under the same composition as the thermal stability measurement; maximum value) (4) Molecular weight: 88 to 90 KDa (calculated value), and a sugar chain or deletion at a site other than histidine described in (5) below. ,
Insertion, or deletion, substitution, insertion of one or several amino acids,
There may be additions. (5) Amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which H is replaced by another amino acid.

[0016] Another embodiment of the present invention is a modified thermostable DNA polymerase having the following properties. (1) DNA synthesis rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
Can retain 60% or more of the residual activity by treatment at 5 ° C. for 6 hours. (3) Amino acid sequence: In the amino acid sequence of SEQ ID NO: 2, the 147th amino acid (that is, histidine) is replaced with another amino acid. Having an amino acid sequence substituted for

As a more specific example of the present invention, the histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with any of glutamic acid, aspartic acid, tyrosine, alanine, lysine and arginine. DNA polymerase can be mentioned. Here, in the present invention, the amino acid sequence set forth in SEQ ID NO: 2 comprises an amino acid sequence in which one or more amino acids among amino acids other than the 147th histidine residue have been deleted, substituted or added, And those having DNA polymerase activity. Preferably, one having one or more amino acids deleted, substituted or added and having a DNA polymerase activity, as long as the amino acid sequence has a homology of 95% or more with the amino acid sequence of SEQ ID NO: 2.

In the present invention, the DNA polymerase activity refers to the activity of deoxyribonucleic acid by covalently binding the α-phosphate of deoxynucleotide 5′-triphosphate to the 3′-hydroxyl group of an oligonucleotide or polynucleotide annealed to a template DNA. Refers to the activity of catalyzing a reaction for introducing a deoxyribonucleotide 5'-monophosphate in a template-dependent manner. The activity was measured using a storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM
M KCl, 1 mM dithiothreitol, 0.1%
Tween 20, 0.1% Nonidet P40,
The sample is diluted with 50% glycerin) and measured. In the present invention, 25 μl of solution A and 5 μl of solution B described below are used.
1, 5 μl of solution C, 10 μl of sterilized water, and 5 μl of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes.
Then, the mixture was cooled on ice, and 50 μl of solution E and 100 μl of solution D were added.
After stirring, the mixture is cooled on ice for another 10 minutes. This solution was filtered through a glass filter (GF / C filter manufactured by Whatman), washed thoroughly with solution D and ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (manufactured by Packard) to measure the incorporation of nucleotides into the template DNA. I do. One unit of the enzyme activity is 10 nm per 30 minutes under these conditions.
The amount of enzyme taken into the ol nucleotide in the acid-insoluble fraction (that is, the fraction that becomes insoluble when the solution D is added). A: 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA (bovine serum albumin) B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ 3H] dTT
P) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 mg / ml salmon sperm DNA

In the present invention, 3'-5 'exonuclease activity refers to the removal of the 3' terminal region of DNA and the
It refers to the activity of releasing a mononucleotide. The activity measurement is as follows. 50 μl reaction solution (120 mM Tr
is-hydrochloric acid buffer (pH 8.8, 25 ° C.), 10 mM KCl,
6 mM ammonium sulfate, 1 mM MgCl ₂ , 0.1% Tr
Iton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) is dispensed into a 1.5 ml microtube, and DNA polymerase is added. 10 at 75 ° C
After reacting for 1 minute, the reaction is stopped by ice cooling, then 50 μl of 0.1% BSA as a carrier is added, and 100 μl of 10% trichloroacetic acid, 2% sodium pyrophosphate solution is further added and mixed. After standing on ice for 15 minutes, the precipitate is separated by centrifugation at 12,000 rpm for 10 minutes. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard), and the amount of nucleotide released in the acid-soluble fraction is measured.

In the present invention, the DNA elongation rate refers to the number of synthesized DNA per unit time. The measuring method is DNA
Polymerase reaction solution (20 mM Tris-HCl buffer (pH
7.5), 8 mM magnesium chloride, 7.5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNT
P, 0.2 μCi [α-32P] dCTP) was mixed with primer-annealed M13mp18 single-stranded DNA at 75 ° C.
To react. The reaction was stopped by using an equal volume of a stop solution (50 mM sodium hydroxide, 10 mM EDTA, 5% Ficoll,
0.05% bromophenol blue). After fractionating the DNA synthesized by the above reaction by alkaline agarose gel electrophoresis, the gel is dried and subjected to autoradiography. Labeled λ / HindIII is used as a DNA size marker. The DNA elongation rate is determined by measuring the size of the synthesized DNA using the marker band as an index.

In the present invention, thermal stability (D after heat treatment)
The NA polymerase residual activity is defined as 5 units of polymerase in 100 μl of a buffer solution (20 mM Tris-HCl (p
H8.8; measured at 25 ° C.), 10 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X-100, 0.1 m
g / ml BSA, 5 mM 2-mercaptoethanol) and the residual activity in a treatment at 95 ° C. for 6 hours. Specifically, after the heat treatment, the activity is measured by measuring the DNA polymerase activity and comparing with the activity before the heat treatment.

In the present invention, the accuracy of DNA polymerase refers to the accuracy of incorporation of bases during DNA replication. In the present invention, the accuracy of the DNA polymerase is evaluated using the ribosome protein S12 (rpsL) gene involved in streptomycin resistance as an index. Streptomycin is an antibiotic that inhibits prokaryotic protein synthesis and binds to bacterial 30S ribosomal RNA (rRNA) to inhibit the initiation complex formation reaction of protein synthesis and causes misreading of the genetic code. In the streptomycin resistant mutant, a mutation is found in ribosomal protein S12. This mutation increases the translational fidelity of the ribosome and thus suppressor tRNA
Effects such as suppressing the reading of codons throughout
(pleiotropic effect). Therefore, when amplification is performed by PCR using the rpsL gene as a template, a mutation is introduced at a certain probability, and when it is a mutation at the amino acid level, the structure of the rpsL protein changes, and streptomycin cannot act on 30S ribosomal RNA. Something like that happens. Therefore, when the bacteria are transformed with the amplified plasmid DNA, the more mutations are introduced, the more frequently the streptomycin resistant bacteria appear.

The plasmid pMol 21 (Journal of Molecular
Biology (1999) 289, 835-850) is a plasmid containing the rpsL gene and the ampicillin resistance gene. A primer set for PCR amplification (one biotinylated and MluI restriction enzyme site introduced) was designed on the ampicillin resistance gene of this plasmid, and the total length of the plasmid was determined using heat-resistant DNL.
PCR amplified with A polymerase, purified using streptavidin beads, cut out using restriction enzyme MluI, ligated using DNA ligase, transformed into Escherichia coli, and containing ampicillin, ampicillin and streptomycin Accuracy of gene replication can be determined by inoculating two types of plates and calculating the ratio of colonies that appeared on each plate.

As a method for producing these modified enzymes, a conventionally known method can be used. For example, there is a method for producing a mutant DNA polymerase having a new function by a protein engineering technique by introducing a mutation into a gene encoding a wild-type DNA polymerase (J. B.
iol. Chem., 264 (11), 6447-6458 (1989)). The gene encoding the DNA polymerase into which the mutation is introduced is not particularly limited, and examples thereof include the gene described in Pyrococcus kodakaraensis KOD1 and shown in SEQ ID NO: 3. Similarly, Pyrococcus furiosus (Nucleic Acid Res., 21 (2), 259-265 (1993)) and Thermococcus litoralis (Proc. Natl. Acad. Sci. U.)
SA, 89, 5577-5581 (1992)). As a method for introducing a mutation into the wild-type DNA polymerase gene, any known method may be used. For example, a protein engineering technique such as a method of contacting a mutagenic agent with a wild-type DNA polymerase gene and a method of irradiating ultraviolet rays, and a method such as PCR or site-specific mutation can be used.

The QuickChange site-direc used in the present invention
The ted mutagenesis kit (Stratagene)
(1) denaturing the plasmid into which the gene of interest is inserted, annealing the mutant primer with the plasmid, and then performing an extension reaction using Pfu DNA polymerase; (2) repeating the cycle of (1) 15 times;
(3) For example, only the plasmid used as a template is selectively cleaved using the restriction enzyme DpnI. (4) Escherichia coli is transformed with the newly synthesized plasmid, and the plasmid having the desired mutation introduced therein is retained. This is a kit from which transformants can be obtained.

The above-mentioned modified DNA polymerase gene is transferred to an expression vector, if necessary, and Escherichia coli, for example, as a host is transformed using the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form a colony. Let it. Colonies are placed in a nutrient medium such as LB medium or 2 ×
After inoculating a YT medium and culturing at 37 ° C. for 12 to 20 hours, the cells are crushed to extract a crude enzyme solution. The vector is preferably one derived from pLED-MI or pBluescript. As a method for disrupting the cells, any known method may be used. For example, ultrasonic treatment, a physical disruption method such as French press or glass bead disruption, or a lytic enzyme such as lysozyme can be used. The crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived polymerase, and the DNA polymerase activity is measured. The 3'-5 'exonuclease activity is measured after the individual DNA polymerase activities are aligned, and the change in the 3'-5' exonuclease activity of each mutant can be measured by comparing with the wild-type DNA polymerase. .

Purified D from the strain selected by the above method
As a method for obtaining NA polymerase, any method may be used. For example, the following methods are available. After recovering the cells obtained by culturing in a nutrient medium, the cells are crushed and extracted by enzymatic or physical crushing to obtain a crude enzyme solution. The obtained crude enzyme extract is heat-treated, for example, treated at 80 ° C. for 30 minutes, and then a DNA polymerase fraction is recovered by ammonium sulfate precipitation. The crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, separation and purification can be performed by column chromatography such as Q Sepharose and Heparin Sepharose to obtain a purified enzyme preparation. The purified enzyme preparation was SDS-PA
It is refined by GE to show a substantially single band.

Furthermore, by performing PCR amplification using the obtained enzyme, the presence or absence or the strength of the amplification
R efficiency can be evaluated, and also the accuracy of DNA replication can be evaluated. The modified DNA polymerase of the present invention is excellent in gene amplification efficiency and amplification accuracy and can be used for PCR.

The nucleic acid amplification or extension method of the present invention uses the modified heat-resistant DNA polymerase of the present invention, using DNA as a template, at least one kind of primer, dNTP (ie, four kinds of deoxyribonucleotides). (3 phosphoric acid) to extend the primer to synthesize a DNA primer extension.
The method includes a method of extending a nucleic acid using one type of primer. Specifically, the method includes a primer extension method, a sequencing method, a method without performing a conventional temperature cycle, and a cycle sequence method. The method of the present invention also includes a method of amplifying a nucleic acid by a PCR method using two or more types of primers. Preferably, the primers are two oligonucleotides, one of each being complementary to the other DNA product. Further, it is preferable to repeat heating and cooling. Specifically, the gene amplification method based on the PCR method comprises three steps of denaturation, annealing, and extension in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and the thermostable DNA polymerase of the present invention. By repeating the cycle consisting of the following steps: amplifying the region of the target nucleic acid flanked by the pair of primers exponentially (Nature, 324 (6093), 13-19 (198
6)). That is, the nucleic acid of the sample is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded target nucleic acid complementary to each primer in the subsequent annealing step, and in the subsequent extension step, each primer is used as a starting point for DNA.
The DNA strand complementary to each single-stranded target nucleic acid serving as a template is extended by the action of the polymerase to obtain a double-stranded DNA.
By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified 2 ⁿ times. DNA of the present invention
The polymerase is required to maintain its activity by divalent ions such as, for example, magnesium ions, and one-valent ions such as, for example, ammonium ions and / or potassium ions.
It is preferable to coexist valence ions. In addition, PCR
The reaction solution contains a buffer and these ions,
BSA, a non-ionic detergent such as Triton X-100, and a buffer may be present. As a buffer,
Good buffers such as Tris and Hepes, and phosphate buffers are used. As a specific embodiment of PCR when using the thermostable DNA polymerase of the present invention,
Reaction buffer (120 mM Tris-HCl (pH 8.0), 10 mM KCl, 6 mM
(NH ₄ ) ₂ SO ₄ , 0.1% TritonX-100, 10 μg / ml BSA), each primer 0.4 pmol / μl, dNTPs 0.2 mM, template DNA 0.2 ng / μ
and a solution containing 0.05 u / μl of the DNA polymerase of the present invention are subjected to a temperature cycle of 94 ° C .; 15 seconds, 65 ° C .; 2 seconds, 74 ° C., 30 seconds about 25 times.

The nucleic acid amplification reagent of the present invention comprises two primers, one of which is complementary to the DNA extension product of the other primer, dNTP, and the heat-resistant DNA polymerase of the present invention as described above. More specifically, two primers comprising a divalent ion, a monovalent ion, and a buffer, wherein one primer is complementary to the other primer DNA extension product, dNTP and the above-mentioned thermostable DNA polymerase, magnesium. Ions, ammonium ions and / or potassium ions, BSA,
Includes a nonionic surfactant and buffer as described above.

In another embodiment of the nucleic acid amplification reagent of the present invention, two kinds of primers, dNT, in which one primer is complementary to the DNA extension product of the other primer,
P and the polymerase activity and / or 3'-5 'exonuclease activity of the thermostable DNA polymerase, divalent ions, monovalent ions, buffer, and, if necessary, the thermostable DNA polymerase of the present invention as described above. There is a nucleic acid amplification reagent containing an antibody having the activity of Examples of the antibody include a monoclonal antibody and a polyclonal antibody. The present nucleic acid amplification reagent is particularly effective in increasing the sensitivity of PCR and reducing non-specific amplification.

The reagent for gene mutation introduction of the present invention comprises a mutation-introducing primer in which one primer is mutually complementary to the DNA extension product of the other primer, dNTP, and the above-mentioned thermostable DNA polymerase of the present invention. Including. It may further contain a divalent ion, a monovalent ion, and a buffer as described above. The mutation-introducing primer of the present invention has a length of about 20 to 150 bases,
In both cases, the mutated portion is located near the center of the sequence, that is, a portion different from the DNA sequence serving as a template (for example, insertion, deletion,
Substitutions) and are complementary to each other. In the gene amplification reagent and the gene mutation reagent of the present invention, as the buffer, a good buffer such as Tris or Hepes and a phosphate buffer are used.
00 mM buffers (pH 7.5 to 9.0 (at 25
C))). In addition, the concentration of divalent ions such as magnesium ions and manganese ions is 0.5 to 2 mM at the reaction stage, and for example, ammonium ions and / or
Alternatively, the concentration of a monovalent ion such as potassium ion is preferably about 10 to 100 mM at the stage of the reaction. The DNA polymerase of the present invention has a capacity of 0.1 at the reaction stage.
It is preferable that the amount be about 01 to 0.1 unit / μl.
The concentration of the primer is about 0.2 to 2 pmol / μl.

Further, the thermostable DNA polymerase of the present invention attenuates or inactivates the polymerase activity of the thermostable DNA polymerase of the present invention as described above by using a chemical or genetic engineering technique, and various 3'-
It may have 5 'exonuclease activity and be used exclusively as 3'-5' exonuclease.

As an embodiment of the present invention, the heat-resistant DNA polymerase of the present invention as described above
There is a DNA polymerase composition characterized by a mixture of two or more species. Specifically, when a long-chain nucleic acid is amplified by mixing the above-mentioned thermostable DNA polymerase of the present invention with another DNA polymerase such as a DNA polymerase having a low 3′-5 ′ exonuclease activity ( For example, long PCR)
The composition useful for the above can be obtained. Actually, as a method for amplifying a long nucleic acid, a DNA polymerase composition obtained by mixing Taq polymerase lacking 3'-5 'exonuclease activity and Pfu polymerase or Ti polymerase having 3'-5' exonuclease activity or a mutant enzyme thereof is used. A method of performing PCR using a product has been reported (Barns, WM (1994) Proc. Natl. Acad. Sci. US
A 91, 2216-2220). In the present invention, the DN of the present invention
A combination of A polymerase and Taq polymerase, a combination of the DNA polymerase of the present invention and Tth polymerase, and a DNA polymerase of the present invention having low 3′-5 ′ exonuclease activity and a DNA polymerase of the present invention having high DNA polymerase activity For example, withdrawal from DNA polymerase can be exemplified. Examples of other components that can be added to the DNA polymerase composition of the present invention include antibodies that suppress the activity of the DNA polymerase.

[0035]

EXAMPLES The present invention will be described below more specifically with reference to examples.

Reference Example 1 Cloning of DNA Polymerase Gene Derived from Hyperthermophilic Archaeon KOD1 Strain The hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain isolated at Kohojima, Kagoshima was cultured at 95 ° C. The body was recovered. From the obtained cells, K
Chromosomal DNA of strain OD1 was prepared. Pyrococcus
Based on the amino acid sequence of the conserved region of the DNA polymerase derived from Juliosus (Pfu DNA polymerase), two primers (5′-GGATTAGTATAGTGCCAATGGSSGGCGA-3 ′ and
5′-GAGGGCAGAAGTTTATTCCGAGCTT-3 ′) (SEQ ID NOS: 26 and 27, “s” means a mixture of C and G) was synthesized. DN prepared using these two primers
PCR was performed using A as a template.

After determining the nucleotide sequence of the PCR-amplified fragment and determining the amino acid sequence, Southern hybridization is performed on the KOD1 strain chromosomal DNA restriction enzyme-treated product using the amplified DNA as a probe to obtain a DNA polymerase-encoding fragment. The size was determined (about 4-7Kb
p). Further, a DNA fragment of this size was recovered from an agarose gel, inserted into a plasmid pBluescript (manufactured by Stratagene), and Escherichia coli was isolated from the mixture.
(Escherichia coli) JM109 was transformed to prepare a library. Colony hybridization was carried out using the probe used for Southern hybridization, and a clone strain considered to contain a DNA polymerase gene derived from the KOD1 strain from the above library
(Escherichia coli JM109 / pBSKOD1) was obtained. A plasmid, pBSKOD1, was recovered from the obtained clone strain, and the nucleotide sequence was determined according to a standard method. Further, the amino acid sequence was deduced from the obtained base sequence. The DNA polymerase gene derived from the KOD1 strain was composed of 5010 bases and encoded 1670 amino acids (SEQ ID NO: 1).

To generate a complete polymerase gene, two intervening sequences (1374-2453 bp and 27
09-4316 bp) was removed by PCR fusion.
PCR was again performed using the two types of fragments thus obtained.
Is performed to remove the intervening sequence, and EcoRV, C
DNA derived from KOD1 strain having a BamHI site on the terminal side
A complete gene fragment encoding the polymerase was obtained (SEQ ID NO: 3). Further, the gene was subcloned using the NcoI / BamHI site of the expression vector pET-8c capable of inducing the gene with the T7 promoter and the restriction enzyme site created earlier to obtain a recombinant expression vector (pET-pol). Escherichia coli BL21 (DE3) / pET-pol has been deposited with the Biotechnology Institute (FERM BP-5513).

Example 1 To modify the thermostable DNA polymerase, the KOD polymerase gene was cut out from the plasmid pET-pol,
Bluescript was subcloned. That is, pE
T-pol was cut with restriction enzymes XbaI and BamHI (manufactured by Toyobo Co., Ltd.) to cut out a KOD DNA polymerase gene of about 2.3 kb. Next, this DNA fragment was subjected to XbaI and BamHI using a ligation kit (Ligation high, manufactured by Toyobo).
Ligated with the plasmid pBluescript SK (-) digested with, and competent cells (Toyobo's competent high JM109)
Was transformed. The cells were cultured on an LB agar medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar; Gibco) containing 100 μg / ml ampicillin for 16 hours at 35 ° C. A plasmid was prepared from the obtained colonies. Furthermore, the plasmid pKO containing the KOD DNA polymerase gene was confirmed by confirming the partial base sequence.
I got D1.

Example 2 Preparation of Modified Gene (HE) and Purification of Modified Thermostable DNA Polymerase Using the plasmid pKOD1 obtained in Example 1, KOD D
A plasmid having a modified thermostable DNA polymerase gene in which histidine at position 147 of NA polymerase was substituted with glutamic acid was prepared (pKOD HE). Preparation of the plasmid is performed by QuickChange site directed mutagenesis ki.
t (manufactured by Stratagene) was used. The method was performed according to the instruction manual. The primers described in SEQ ID NOs: 4 and 5 were used as mutation-producing primers. The confirmation of the mutant was performed by decoding the nucleotide sequence. Escherichia coli JM109 was transformed with the obtained plasmid to obtain Escherichia coli JM109 (pKOD HE).

The obtained cells were cultured as follows. First, a sterilized TB medium containing 100 μg / ml ampicillin (Molecular cloning 2nd editio) was used.
n, pA2) 6 L was dispensed into a 10 L jar fermenter. In this medium, 50 ml of LB medium (1% bactotryptone, 0.5%) containing 100 μg / ml ampicillin in advance.
% Yeast extract, 0.5% sodium chloride; Gibco) at 37 ° C. for 16 hours.
OD HE) (using 500ml Sakaguchi flask), 35 ℃
For 12 hours. The cells were recovered from the culture by centrifugation, and 400 ml of a disruption buffer (10 mM Tris-HCl
After suspending in a buffer solution (pH 8.0), 80 mM KCl, 5 mM 2-mercaptoethanol, 1 mM EDTA, the cells were disrupted by French press treatment to obtain a cell disrupted solution. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Further, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin Sepharose chromatography were performed, and finally a storage buffer (50 mM
Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1
mM dithiothreitol, 0.1% Tween20, 0.1%
Nonidet P40, 50% glycerin) to obtain a mutant thermostable DNA polymerase (HE). The measurement of the DNA polymerase activity in the above purification step was performed by the following operation. When the enzyme activity was high, the sample was diluted and measured.

(Reagent) Solution A: 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA Solution B: 2 μg / μl Activated calf thymus DNA solution C: 1.5 mM dNTP ( 250 cpm / pmol [3H] dT
TP) Solution D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) Solution E: 1 mg / ml salmon sperm DNA

(Method) Solution A 25 μl, Solution B 5 μl, Solution C 5
μl and 10 μl of sterile water were added to the microtube, and the mixture was stirred and mixed.
React for 0 minutes. Then, the mixture was cooled, 50 μl of solution E, 10 solutions of solution D
After adding 0 μl, the mixture is stirred and cooled on ice for further 10 minutes. This solution was filtered through a glass filter (GF / C filter manufactured by Whatman), washed thoroughly with solution D and ethanol, and the radioactivity of the filter was measured using a liquid scintillation counter (manufactured by Packard). Incorporation was measured. One unit of the enzymatic activity was the amount of the enzyme that incorporated 10 nmol of nucleotides into the acid-insoluble fraction per 30 minutes under these conditions.

Example 3 Preparation of Mutant (HD) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, heat resistance was obtained by substituting 147th histidine of KOD DNA polymerase with aspartic acid. DNA polymerase gene was prepared (pKOD H
D). The primers described in SEQ ID NOs: 6 and 7 were used as mutation primers. Furthermore, a modified thermostable DNA polymerase (HD) was obtained by the same purification method as in Example 2.

Example 4 Preparation of Mutant (HY) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, heat-resistant DNA in which histidine at position 147 of KOD DNA polymerase was replaced with tyrosine. A polymerase gene was made (pKOD HY).
The primers described in SEQ ID NOs: 8 and 9 were used as mutation primers. Further, a modified thermostable DNA polymerase (HY) was obtained by the same purification method as in Example 2.

Example 5 Preparation of Mutant (HA) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, heat-resistant DNA in which the 147th histidine of KOD DNA polymerase was substituted with alanine. A polymerase gene was made (pKOD HA).
The primers described in SEQ ID NOs: 10 and 11 were used as mutation primers. Furthermore, a modified thermostable DNA polymerase (HA) was obtained by the same purification method as in Example 2.

Example 6 Preparation of Mutant (HK) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, heat-resistant DNA in which histidine at position 147 of KOD DNA polymerase was replaced with lysine. A polymerase gene was made (pKODHK). The primers described in SEQ ID NOs: 12 and 13 were used as mutation primers. Further, a modified thermostable DNA polymerase (HK) was obtained by the same purification method as in Example 2.

Example 7 Preparation of Mutant (HR) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, heat-resistant DNA obtained by replacing the 147th histidine of KOD DNA polymerase with arginine. Polymerase gene was prepared (pKOD H
R). The primers described in SEQ ID NOs: 14 and 15 were used as mutation primers. Further, the modified thermostable DNA polymerase (H
R).

Example 8 Preparation of Mutant (HS) Gene and Purification of Modified Thermostable DNA Polymerase In the same manner as in Example 2, thermostable DNA in which the histidine at position 147 of KOD DNA polymerase was replaced with serine. A polymerase gene was made (pKODHS). The primers described in SEQ ID NOs: 16 and 17 were used as mutation primers. Further, a modified thermostable DNA polymerase (HS) was obtained by the same purification method as in Example 2.

Example 9 Preparation of Mutant (HQ) Gene and Purification of Modified Heat-Stable DNA Polymerase In the same manner as in Example 2, heat-resistant DNA in which histidine at position 147 of KOD DNA polymerase was replaced with glutamine. Polymerase gene was prepared (pKOD H
Q). The primers described in SEQ ID NOs: 18 and 19 were used as mutation primers. Further, the modified thermostable DNA polymerase (H
Q) was obtained.

Example 10 In addition to the modified thermostable DNA polymerase obtained in Examples 2 to 9, the exonuclease activities of IK and IQ were measured by the following method. Mutants IK and IQ are mutants obtained by substituting lysine and glutamine for the 142nd isoleucine of KOD DNA polymerase described in JP-A-10-42871.
It was prepared according to the method described in Japanese Patent No. 2871. As a control, wild-type KOD polymerase (manufactured by Toyobo) was used. 50 μl of the reaction solution (120 mM Tris-HCl buffer (pH
8.8, 25 ° C.), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl ₂ , 0.1% Triton X-1
And 0.001% BSA, 5 μg tritium-labeled Escherichia coli DNA) were dispensed into 1.5 ml microtubes, and DNA polymerase was added to each of 0.06 U, 0.
025U was added and reacted at 75 ° C. for 10 minutes. The reaction was stopped by ice cooling, and then 0.1% as a carrier.
Was added, and 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution were further added and mixed. Then, after leaving it on ice for 15 minutes, 12000
The precipitate was separated by centrifugation for 10 minutes by rotation,
μl of radioactivity was measured with a liquid scintillation counter (manufactured by Packard), and the amount of nucleotides released in the acid-soluble fraction was measured. Comparison of individual 3'-5 'exonuclease activities was performed based on the above radioactivity values when 0.06 and 0.025 polymerase Units were used. FIG. 2 shows the relative exonuclease activity of each DNA polymerase.

From this example, it has been proved that according to the present invention, a thermostable DNA polymerase having exonuclease activity of various strengths can be obtained. The 3′-5 ′ exonuclease activity of the mutant KOD polymerase obtained was determined by comparing the wild-type KOD DNA polymerase (100%).
In contrast, HD is about 6.25%, HE is about 25%, HY
About 90%, HA about 30%, HS about 50%, HQ about 50%, HK about 400%, HR about 300%, IK
Had an activity of about 6.25% and IQ had an activity of about 25%.

Example 11 Confirmation of Thermal Stability The thermal stability of the modified DNA polymerases obtained in Examples 2, 3 and 6 was measured by the following method. Five units of each DNA polymerase were added to 100 μl of buffer (20 mM Tris-HCl p
H8.8 at 25 ° C, 10 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 0.1% Triton X-100, 0.1
mg / ml BSA, 5mM 2-mercaptoethanol)
Pre-incubation was performed at 95 ° C. Samples were collected over time from this mixture and the polymerase activity was measured by the method described in Example 2. For comparison, the same operation was performed for natural KOD polymerase (manufactured by Toyobo). As shown in Table 1, any of the modified polymerases exhibited a residual activity of 60% or more after treatment at 95 ° C. for 6 hours, similarly to the natural KOD polymerase.

[0054]

[Table 1]

Example 12 Measurement of DNA Elongation Rate The DNA extension rate of the modified DNA polymerase obtained in Examples 2, 3, 6, and 7 was measured by the following method. One unit of the purified modified DNA polymerase was added to 10 μl of a reaction solution (20 mM Tris-HCl (pH 7.5), 8 mM magnesium chloride, 7.
5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNT
P, 0.2 μCi [α- ^32P ] dCTP) and 0.2 μg of SEQ ID NO: 1
M13mp181 annealed with 5 primers
The reaction was carried out at 75 ° C. for 20 seconds, 40 seconds, and 60 seconds with the single-stranded DNA. The reaction was stopped by adding an equal volume of a reaction stop solution (50 mM sodium hydroxide, 10 mM EDTA, 5% ficoll, 0.05% bromophenol blue). For comparison, the same operation was performed for Pfu polymerase (manufactured by Stratagene) and natural KOD polymerase (manufactured by Toyobo). After fractionating the DNA synthesized by the above reaction by alkaline agarose gel electrophoresis, the gel was dried and subjected to autoradiography. Labeled λ / HindIII was used as a DNA size marker. The DNA synthesis rate was determined by measuring the size of the synthesized DNA using the marker band as an index. Table 2 shows the results. Both modified polymerases use the native K
Like OD polymerase, it had a synthesis rate of about 120 bases / second. In contrast, Pfu polymerase had a synthesis rate of about 20 bases / second.

[0056]

[Table 2]

This example proves that the present invention provides a thermostable DNA polymerase having exonuclease activity of various strengths. The 3′-5 ′ exonuclease activity of the mutant KOD polymerase obtained was determined by comparing the wild-type KOD DNA polymerase (100%).
In contrast, HD is about 6.25%, HE is about 25%, HY
About 90%, HA about 65%, HS about 50%, HQ about 50%, HK about 400%, HR about 300%, IK
Had an activity of about 6.25% and IQ had an activity of about 25%.

Example 13 Example of PCR using modified DNA polymerase (1) Wild-type (WT) KOD polymerase and modified heat-resistant DNA polymerases HE, HD, HY, HA, HK, HR, I
PCR was performed using K and IQ. Mutant IK, IQ
Are K as described in JP-A-10-42871.
It is a mutant in which the 142nd isoleucine in OD DNA polymerase has been substituted with lysine and glutamine. HE and IQ, HD and IK are the same 3 '
It has been shown to exhibit -5 'exonuclease activity. PCR was performed in a 49 µl reaction solution (1 x KOD-Plus-buffer
(Manufactured by Toyobo) 1 mM MgSO4, 0.2 mM dNTP,
100 ng and 10 ng of K562 DNA (manufactured by Life Technology Co., Ltd.), 10 pmol of primers described in Sequence Listings 20 and 21, 1 μl of each enzyme (1 U / μl), and PC
R was performed. Thermal cycler is Perkin-Elmer P
It carried out as follows using CR system GeneAmp2400. That is, after performing the reaction at 94 ° C. for 2 minutes,
30 cycles of 15 seconds → 60 ° C., 30 seconds → 68 ° C., 3 minutes and 30 seconds were performed. After completion of the reaction, 10 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and an amplified DNA fragment of about 3.6 kb was confirmed under ultraviolet irradiation. FIG. 3 shows a photograph of agarose gel electrophoresis. These results demonstrate that mutants HE, HD, HY, and HA show better amplification than wild-type KOD polymerase, especially when amplifying from low copy number template DNA (10 ng). Was done.

Example 14 Example of PCR Using Modified DNA Polymerase (2) Amplification of Genes Larger in Size for Modified Thermostable DNA Polymerases HE, HD, HY and HA Which Obtained Good Results in Example 1 Tried. 49 μl of the reaction solution (1 × KO
D-Plus-buffer (Toyobo), 1 mM MgSO4, 0.
2 mM dNTPs, 100 ng and 50 ng K562 DNA
(Manufactured by Life Technology), 10 pmol of the primers described in SEQ ID NOs: 22 and 23) and
l plus PCR was performed. Perki Thermal Cycler
It carried out as follows using PCR system GeneAmp2400 made by n-Elmer. That is, after a reaction at 94 ° C. for 2 minutes, 9
30 cycles of 4 ° C., 15 seconds → 60 ° C., 30 seconds → 68 ° C., 6 minutes were performed. After the completion of the reaction, 10 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and an amplified fragment of about 6.2 kb was confirmed under ultraviolet irradiation. As a result, good amplification was confirmed particularly in HD and HE (FIG. 4). Although not shown here, amplification could not be confirmed at all with wild-type (WT) KOD DNA polymerase.

Example 15 Measurement of Accuracy of Modified KOD DNA Polymerase Accuracy of a wild-type KOD DNA polymerase and a modified heat-resistant DNA polymerase was measured by the following method. 49 μl of reaction solution (1 × KOD-Plus-buffer (manufactured by Toyobo), 1 mM MgSO ₄ , 0.2 mM dNTP, 2.5 ng)
Plasmid pMol 21 (Journal of Molecular Biology (19
99) 289, 835-850), 10 μmol of the primers described in SEQ ID NOS: 24 and 25), and 1 μl of the enzyme (1 U / μl).
A CR was performed. Here, among the mutants obtained this time, HD, HE, HY, HA, HK, HR, and
It carried out using IK and IQ of -42871 gazette. Thermal cycler is a Perkin-Elmer PCR system
It carried out as follows using GeneAmp2400. That is,
94 ° C, 2 minutes, 94 ° C, 15 seconds → 60 ° C, 30 minutes
Second cycle → 68 ° C., 4 minutes, 25 cycles. Also at the same time
An amplification reaction was also performed for rTaq DNA polymerase. As reaction conditions, 49 μl of a reaction solution (1 × rTaq bu
ffer (manufactured by Toyobo), 1.5 mM MgCl ₂ , 0.2 mM d
NTP, 2.5 ng plasmid pMol 21 (Journal of Molec
ular Biology (1999) 289, 835-850), 10 pmol of the primers described in SEQ ID NOS: 24 and 25) and each enzyme (5 U / μ).
l) was added thereto and reacted at 94 ° C for 2 minutes, and then 25 cycles of 94 ° C, 15 seconds → 60 ° C, 30 seconds → 68 ° C, 5 minutes were performed.

After completion of the PCR, the DNA was treated with phenol / chloroform, and then the DNA was precipitated by an ethanol precipitation method. The precipitate was dissolved in 100 μl of distilled water, 10 μl of avidin magnetic beads (manufactured by DYNAL) was added, and the mixture was mixed by inversion for 30 minutes. The magnetic beads were concentrated using a magnetic separation stand (Magical Trapper; manufactured by Toyobo Co., Ltd.). Subsequently, 100 μl of a washing solution A (10 mM Tris-HCl buffer (pH
8.0), 1 mM EDTA), 1M NaCl) to add 1
The mixture was stirred for 0 second, and concentrated again in the magnetic separation stand (washing). After repeating the above washing step once again, washing solution B (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDT)
The magnetic beads were washed using A)), and only the magnetic beads were collected using a magnetic separation stand. Next, 40 μl of distilled water, 5 μl of a restriction enzyme buffer and 50 U of restriction enzyme Mlu I (manufactured by Toyobo) were added to the collected magnetic beads, and 37
The mixture was treated for 3 hours while mixing by inversion at ℃. Thereafter, the magnetic beads were concentrated again using the magnetic separation stand, and only the supernatant was collected. The recovered DNA solution was desalted by an ethanol precipitation method, and 10 ng thereof was added with a ligation reagent (Ligation high (manufactured by Toyobo Co., Ltd.).
The ligation reaction was performed at 16 ° C. for 16 hours. next,
Molecular cloning 2nd edition Escherichia coli MF-101 strain (Jou
rnal of Molecular Biology (1999) 289, 835-850).

The transformed Escherichia coli solution was divided into two, and one of them was L-containing 200 μg / ml ampicillin.
B agar medium (0.6%) <A plate> and the other LB agar medium (0.6%) containing 200 μg / ml ampicillin and 400 μg / ml streptomycin (B)
The plate was cultured at 30 ° C. for 24 hours, the number of colonies that appeared was counted, the colonies that appeared on the B plate were divided by the colonies that appeared on the A plate, and the percentage value was taken as the mutation rate (Mutation frequency). The lower the mutation rate, the better the accuracy of DNA polymerase during DNA replication.

FIG. 5 shows the results. rTaq is 3'-5 '
Since it has no exonuclease (Proof readign) activity, it showed a high mutation rate of 7.91%, whereas
In the WT and the mutants obtained this time, the mutation rates were all 1% or less. Also, among them, 3 'to WT
HK, H with enhanced -5 'exonuclease activity
For R, the mutation rates are 0.12% and 0.17%, respectively.
And WT 0.47%.

[0064]

As described above, according to the present invention, it has become possible to prepare a thermostable DNA polymerase having various DNA amplification efficiencies, 3′-5 ′ exonuclease activity and accuracy. By using this method, a conventional heat-resistant DNA polymerase derived from a primordial bacterium can be used for various purposes,
That is, the modified heat-resistant DN is used so that it can be used for various applications such as amplification of a long region and amplification with higher accuracy.
It is possible to create A polymerase.

[0065]

[Sequence List] SEQUENCE LISTING <110> TOYO BOSEKI KABUSHIKI KAISHA <120> MODIFIED THERMOSTABLE DNA POLYMERASE <130> 01-0368 <141> 2001-05-11 <150> 2000-138796 <151> 2000-05-11 <160 > 27 <170> PatentIn Ver. 2.1 <210> 1 <211> 5342 <212> DNA <213> Pyrococcus kodakaraensis <220> <221> CDS <222> (156) .. (5165) <223> 1374-2453 intron, 2709-4316 intron <400> 1 gcttgagggc ctgcggttat gggacgttgc agtttgcgcc tactcaaaga tgccggtttt 60 ataacggaga aaaatgggga gctattacga tctctccgg atgtggggtt tacaata g gac tg gat gg tctacag gac tg 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 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 I le 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 ggg gca gtt att gac atc tac gag tac gac ata ccc ttc gcc aag 509 His Pro 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 gct gc cat g 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 G lu 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 Prop 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 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 atac 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 T hr 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 acc aca gcc tgg acc ggc gag aac ctt gag aga gtc gcc cgc 1085 Ile Thr Thr 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 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 gct gcc 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 g 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 gggt 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 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 520 525 530 530 ttg agg aaa gac gtt g tt g 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 a tg 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 635 640 645 645 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 650 655 655 660 ctt ggt atc cct cat caa acg tac acg tat cag tat cag gaa aat ggg 189 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 g 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 2 381 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 Ile 760 765 770 acc cac aac gtc tcg ccg gat acg ctc aac aga ga 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 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 810 815 815 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 ac ac ag ac ag ag 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 Glu Glu 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 875 880 885 885 gta aag aga gag gag gag gag 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 ga 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 9g 990 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 gag 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 Le u 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 10ag tcac 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 gat cc 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 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 ct gc tag cca gc tcg ct gc tcg tta gcac ct gc tcg tta gca tca 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 36 29 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 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 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 Ast 3821 G 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 t cg 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 40 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 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 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 Va l 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 gc tac aac g 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 Cy 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 ct aag tat 4541 Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Met Glu Phe Leu Lys 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 L eu 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 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 agg cag gg 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 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 g ga 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 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 16ac 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 gaagac ctg 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 Lys Gly Thr 1655 1660 1665 1670 tgacctttcc atttgttttc cagcggataa ccctttaact tccctttcaa aaactccctt 5225 tagggaaaga ccatgaagat agaaatccgg cggcgcccgg ttaaatacgc taggatagaa 5285 gtgaa gccag acggcagggt agtcgtcact gccccgaggg ttcaacgttg agaagtt 5342

<210> 2 <211> 774 <212> PRT <213> Pyrococcuskodakaraensis <400> 2 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 Pro 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 A rg 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 275 280 285 285 Lys Val Tyr Ala Glu Glu Ile Thr Thr 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 385 390 395 395 400 Val Tyr Leu A sp 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 Lys 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 A rg 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 625 630 635 635 640 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 755 760 765 765 Leu Lys Pro Lys Gly Thr 770

[0067] <210> 3 <211> 2325 <212> DNA <213> Pyrococcus kodakaraensis <400> 3 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 cgagagcatc cagcagttat tgacatctac gagtacgaca tacccttcgc caagcgctac 360 ctcatagaca agggattagt gccaatggaa ggcgacgagg agctgaaaat gctcgccttc 420 gacattgaaa 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 tatc ctgtga taagacggac gataaacctg cccacataca cgcttgaggc cgtttatgaa 840 gccgtcttcg gtcagccgaa ggagaaggtt tacgctgagg aaataaccac 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 tt tttgccac aatacctgga gccgatgctg aaaccgtcaa aaagaaggct 1680 atggagttcc tcaagtatat 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

<210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 gaaactctct acgaggaggg cgaggagttc gccg 34

<210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 cggcgaactc ctcgccctcc tcgtagagag tttc 34

<210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 gaaactctct acgacgaggg cgaggagttc gccg 34

<210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 cggcgaactc ctcgccctcg tcgtagagag tttc 34

<210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 gaaactctct actacgaggg cgaggagttc gccg 34

<210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 cggcgaactc ctcgccctcg tagtagagag tttc 34

<210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 gaaactctct acgccgaggg cgaggagttc gc 32

<210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 gcgaactcct cgccctcggc gtagagagtt tc 32

<210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 gaaactctct acaaggaggg cgaggagttc 30

<210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 13 gaactcctcg ccctccttgt agagagtttc 30

<210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 aaagctctct acagggaggg cgaggagttc gc 32

<210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 gcgaactcct cgccctccct gtagagagtt tc 32

<210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 16 gaaactctct actctgaggg cgaggagttc gccg 34

<210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 17 cggcgaactc ctcgccctca gagtagagag tttc 34

<210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 18 gaaactctct accaggaggg cgaggagttc gccg 34

<210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 cggcgaactc ctcgccctcc tggtagagag tttc 34

<210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 20 ggtgttccct tgatgtagca ca 22

<210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 21 acatgtattt gcatggaaaa caactc 26

<210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 22 agtgcttcgt gcccgatgac 20

<210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 tgccccttgg tgacatactc g 21

<210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 24 aaaaacgcgt caccagtcac agaaaagcat cttac 35

<210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 25 aaaaacgcgt caaccaagtc attctgagaa tagt 34

<210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 ggattagtat agtgccaatg gssggcga 28

<210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 27 gagggcagaa gtttattccg agctt 25

[Brief description of the drawings]

FIG. 1 shows the exo I regions (underlined) of various DNA polymerases and the amino acid sequences adjacent thereto.

FIG. 2 is a diagram showing the relative 3′-5 ′ exonuclease activity of each KOD DNA polymerase mutant (WT activity is defined as 100).

FIG. 3 is a diagram showing amplification of a β-globin gene (3.6 kb) by PCR using human genomic DNA of each KOD DNA polymerase mutant as a template. A: 100 ng of DNA derived from human cell line K562 was used for the reaction B: 10 ng of DNA derived from human cell line K562 was used for the reaction 1: WT 2: HD 3: HE 4: HY 5: HA 6: HK
7: HR 8: IK 9: IQ

FIG. 4. Myosinheavey chain gene using human genomic DNA of each KOD DNA polymerase mutant as a template
FIG. 4 is a diagram showing amplification by PCR of (6.2 kb). Using DNA extracted from 50ng of human cell line K562 1: HD, 2: HE, 3: HY, 4: HA

FIG. 5. PCR of each KOD DNA polymerase mutant
It is a figure which shows the mutation rate (Mutationfrequency) in amplification.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Shusuke Komatsubara 10-24 Toyocho, Tsuruga-shi, Fukui Prefecture Inside Toyobo Co., Ltd.Tsuruga Bio-Laboratory (72) Inventor Yoshiaki Nishiya 10-24 Toyocho, Tsuruga-shi, Fukui Prefecture Inside Toyobo Co., Ltd.Tsuruga Bio Research Laboratories (72) Inventor Fumiyoshi Kawakami 10-24 Toyocho, Tsuruga City, Fukui Prefecture Inside Toyobo Co., Ltd.Tsuruga Bio Research Laboratories (72) Inventor Yoshihisa Kawamura 10th Toyocho, Tsuruga City, Fukui Prefecture No. 24 Toyobo Co., Ltd. Tsuruga Bio Research Laboratory (72) Inventor Tadayuki Imanaka 2-28-11 Fujishirodai, Suita-shi, Osaka F-term (reference) 4B024 AA11 AA20 BA10 CA03 CA06 CA09 CA20 DA06 EA04 GA11 GA19 GA25 HA03 HA08 HA11 HA12 4B050 CC01 CC04 DD02 EE01 FF03E FF04E FF09E GG01 HH02 LL03 LL05 4B065 AA01Y AA26X

Claims (30)

[Claims] 1. A _{3'-5 'DX 1 EX 2} X 3 consisting of 4 amino acids long peptide adjacent to E of DXE sequence and the sequence of the exonuclease I (Exol) region of thermostable DNA polymerase having exonuclease activity A heat-resistant DNA polymerase in which histidine is replaced with another amino acid in the X ₄ H (D: aspartic acid, E: glutamic acid, H: histidine, X ₁ , X ₂ , X ₃ and X ₄ : any amino acid) sequence. 2. A DX ₁ EX ₂ X ₃ X ₄ histidine aspartic acid H sequence, glutamic acid, tyrosine, alanine, lysine, and claim 1 has been substituted with any amino acid selected from the group consisting of arginine Thermostable DNA polymerase. 3. The thermostable DNA polymerase according to claim 1, which has the following physicochemical properties. (1) DNA extension rate: at least 20 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has 10% or more DN as compared with the DNA polymerase before the heat treatment.
Retains A polymerase residual activity 4. The thermostable DNA polymerase according to claim 3, which has the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DNase of 40% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 5. The thermostable DNA polymerase according to claim 4, which has the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DN of 60% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 6. The heat-resistant DN according to claim 5, wherein in the amino acid sequence of SEQ ID NO: 2, histidine at position 147 is replaced with any one of aspartic acid, glutamic acid, tyrosine, alanine, lysine, and arginine.
A polymerase. 7. The heat-resistant DNA polymerase according to claim 6, wherein the histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with aspartic acid. 8. The heat-resistant DNA polymerase according to claim 6, wherein histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with glutamic acid. 9. The histidine at position 147 in the amino acid sequence of SEQ ID NO: 2 is substituted with tyrosine.
The thermostable DNA polymerase according to the above. 10. The amino acid sequence according to SEQ ID NO: 2,
The thermostable DNA polymerase according to claim 6, wherein the 147th histidine is substituted with alanine. 11. In the amino acid sequence of SEQ ID NO: 2,
The histidine at position 147 is replaced with lysine.
The thermostable DNA polymerase according to the above. 12. In the amino acid sequence of SEQ ID NO: 2,
7. The thermostable DNA polymerase according to claim 6, wherein histidine at position 147 is substituted with arginine. 13. Exo I (EXOI), a thermostable DNA polymerase having 3'-5 'exonuclease activity
Consisting of 4 amino acids long peptide adjacent to E of DXE sequence and the sequence of the region _{DX 1 EX 2 X 3 X 4} H (D: aspartic acid, E:
Glutamate, H: _{histidine, X 1, X 2, X} 3 and X _4: In any amino acid) sequence, gene histidine encoding the thermostable DNA polymerase has been substituted with another amino acid. 14. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 20 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has 10% or more DN as compared with the DNA polymerase before the heat treatment.
Retains A polymerase residual activity 15. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DNase of 40% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 16. The gene according to claim 13, which encodes a modified thermostable DNA polymerase having the following physicochemical properties. (1) DNA extension rate: at least 30 bases / second (2) Thermal stability: 9 at pH 8.8 (measured at 25 ° C.)
The DNA polymerase after the treatment at 5 ° C. for 6 hours has a DN of 60% or more as compared with the DNA polymerase before the heat treatment.
(3) amino acid sequence: DIETLYH located at amino acids 141 to 147 in the amino acid sequence of SEQ ID NO: 2
(D: aspartic acid, I: isoleucine, E: glutamic acid, T: threonine, L: leucine, Y: tyrosine, H: histidine) It has an amino acid sequence in which histidine is replaced with another amino acid. 17. A recombinant vector comprising the gene according to claim 13 inserted into an expression vector. 18. The vector may be pLED-MI or pBluescri.
The genetic recombinant vector according to claim 17, which is a pt-derived vector. 19. A transformant obtained by transforming a host cell using the recombinant vector according to claim 17 or 18. 20. The method according to claim 1, wherein the host cell is Escherichia coli.
20. The transformant according to claim 19, which is E. coli. 21. A method for producing a thermostable DNA polymerase, comprising culturing the transformant according to claim 20, and collecting a thermostable DNA polymerase from the culture. 22. A reaction between DNA as a template, at least one kind of primer, dNTP, and the thermostable DNA polymerase according to any one of claims 1 to 12, thereby extending the primer to obtain a DNA primer extension. A method for amplifying or extending a nucleic acid comprising synthesizing. 23. The method according to claim 22, wherein the primers are two kinds of oligonucleotides, one of which is complementary to the other DNA extension. 24. The method according to claim 22, wherein heating and cooling are repeated.
The nucleic acid amplification method according to the above. 25. A thermostable DNA polymerase according to any of claims 1 to 12, two primers wherein one primer is complementary to the DNA extension product of the other primer, dNTP, and a divalent ion. A nucleic acid amplification reagent comprising a monovalent ion and a buffer. 26. Two kinds of primers, one of which is complementary to the DNA extension product of the other primer, dNTP, the thermostable DNA polymerase according to any one of claims 1 to 12, magnesium ion, and ammonium. A nucleic acid amplification reagent comprising at least one selected from the group consisting of ions and potassium ions, BSA (bovine serum albumin), a nonionic surfactant, and a buffer. 27. Two kinds of primers, one of which is complementary to the DNA extension product of the other primer, dNTP, the thermostable DNA polymerase according to any one of claims 1 to 12, magnesium ion, and ammonium. At least one selected from the group consisting of ions and potassium ions, BSA, a nonionic surfactant, a buffer, and at least one of the polymerase activity and the 3′-5 ′ exonuclease activity of the thermostable DNA polymerase. A nucleic acid amplification reagent containing an antibody having an inhibitory activity. 28. A DNA polymerase composition comprising one or more heat-resistant DNA polymerases according to any one of claims 1 to 12. 29. A reaction between DNA as a template, a mutation-introducing primer, dNTP and the heat-resistant DNA polymerase according to any one of claims 1 to 12, thereby extending the primer to synthesize an extended DNA primer. A method for introducing a gene mutation comprising: 30. A reagent for introducing a gene mutation, comprising a mutation-introducing primer, dNTP, and the heat-resistant DNA polymerase according to any one of claims 1 to 12.