JPH05317058A - Dna fragment having heat-resistant dna polymerase gene information and its use - Google Patents

Abstract

PURPOSE:To provide a production process for a heat-resistant DNA polymerase of high productivity by collecting a heat-resistant DNA polymerase gene, analyzing the primary structure and applying the genetic engineering technique. CONSTITUTION:A process for producing heat-resistant DNA polymerase characterized by collecting a heat-resistant DNA polymerase originating from Thermus thermophilus, or a DNA encoding a substance functionally equal to the heat- resistant DNA polymerase, a recombinant vector containing the DNA or culturing a transformer prepared by transforming host cells with the recombinant vector to collect the heat-resistant DNA polymerase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding a thermostable DNA polymerase derived from Thermus thermophilus, a recombinant vector containing the DNA, and a transformant obtained by using the recombinant vector. And a method for producing a thermostable DNA polymerase obtained by expressing the genetic information of the DNA by the transformant.

[0002]

2. Description of the Related Art In recent years, the polymerase chain reaction method (PCR method, Saiki etc. Science230
Volume 1350-1354 (1985)) and the demand for thermostable DNA polymerases is increasing due to the analysis of DNA base sequences that are more difficult to analyze. However, the conventionally known thermostable DNA polymerase (Taq polymerase) derived from Thermus aquaticus does not have sufficient thermostability,
There was a problem that the activity decreased during the reaction. Therefore, we used a thermostable DN, which is more thermostable than Taq polymerase.
He found A polymerase and applied for Japanese Patent Application No. 63-213330.

[0003]

However, a thermostable DNA polymerase-producing bacterium found in Japanese Patent Application No. 63-213330, for example, Thermus thermophilus HB8.
(ATCC 27634) has low productivity of thermostable DNA polymerase, and since it also produces the restriction enzyme TthHB8, purification becomes complicated, and it is not always satisfactory for wide use as a research reagent.

[0004]

The present inventors have proposed the heat resistance D
As a result of diligent studies aimed at improving the productivity of NA polymerase, the thermostable DNA polymerase gene was successfully collected and the primary structure was analyzed, and by applying genetic engineering techniques, high productivity was obtained as described below. Established the manufacturing method.

That is, the gist of the present invention is the thermostable DN derived from Thermus thermophirus.
DNA encoding A polymerase or a functional equivalent to the thermostable DNA polymerase, a recombinant vector containing the DNA, a transformant obtained by transforming a host cell with the recombinant vector, and the transformant A method for producing a thermostable DNA polymerase is characterized in that the thermostable DNA polymerase is collected by culturing.

Examples of the above-mentioned DNA of the present invention include DNA having the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. The DNA of the present invention may be artificially mutated at a specific site of the basic DNA by gene recombination technology without changing the basic characteristics of the one encoded by the DNA or to improve the characteristics. It also includes those that are awakened.

The DNA of the present invention is obtained by separating the DNA of the microorganism from a microorganism that is a donor of the thermostable DNA polymerase gene producing thermostable DNA polymerase.
After purification, the digested DNA using ultrasonic waves, restriction enzymes, etc. and the linear expression vector are ligated and closed with a DNA ligase at the blunt or cohesive ends of both DNAs, and the recombinant DNA vector thus obtained is After transfer into a replicable host microorganism, the recombinant DNA vector carrying the screened microorganism is cultured using the marker of the vector and the activity of thermostable DNA polymerase as indicators, and the recombinant DNA is recovered from the cultured cells. The vector may be separated and purified, and then the DNA which is a thermostable DNA polymerase gene may be collected from the recombinant DNA vector.

The above sampling method will be described in more detail below. DNA derived from a microorganism that is a gene donor is collected as follows. That is, any of the above-mentioned bacteria that are donor microorganisms is cultivated by aeration and stirring in a liquid medium for about 1 to 3 days, the resulting culture is centrifuged to collect the cells, and then the cells are lysed to obtain a thermostable DNA.
A lysate containing the polymerase gene can be prepared. As a lysing method, for example, treatment with a cell wall lysing enzyme such as lysozyme is performed, if necessary, other enzymes such as protease and a surfactant such as sodium lauryl sulfate are used in combination, and further, lysing and thawing, which is a method of physically destroying the cell wall, The French press treatment may be performed in combination with the above-mentioned lysis method.

In order to separate and purify DNA from the lysate thus obtained, a method such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, and alcohol precipitation centrifugation is appropriately combined according to a conventional method. It can be carried out.

The method of cleaving the separated and purified microbial DNA can be carried out, for example, by ultrasonic treatment or restriction enzyme treatment. However, in order to facilitate the ligation of the obtained DNA fragment and the vector, a restriction enzyme is particularly specified. Acts on nucleotide sequences, eg EcoRI, HindII
Type II restriction enzymes such as I and BamHI are suitable.

Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. Examples of phages include Escherichia coli (Escheric)
hictoli) is used as a host microorganism, λgt
· 10, λgt · 11, etc. can be used.

As the plasmid, for example, when Escherichia coli is used as a host microorganism, pBR32
2, pBR325, pACYCI84, pUC12, p
UC13, pUC18, pUC19, pKK223-3
Etc. can be used. Such a vector is prepared by using the above-mentioned microorganism D which is a thermostable DNA polymerase gene donor.
It is desirable to obtain a vector fragment by cutting with the same restriction enzyme as that used for cutting NA.

The method for ligating the microbial DNA fragment and the vector fragment may be a method using a known DNA ligase. For example, after annealing the cohesive ends of the microbial DNA fragment and the vector fragment, a suitable DNA is prepared.
Recombinant DNA of microbial DNA fragment and vector fragment is prepared by using ligase. If necessary, it is also possible to transfer to a host microorganism after annealing and utilize in vivo DNA ligase to prepare a recombinant DNA.

The host microorganism may be any as long as the recombinant DNA can be stably and autonomously propagated and the trait of the foreign DNA can be expressed. For example, the host microorganism is Escherichia coli DH5, Escherichia coli HB.
101, Escherichia Collie W3110, Escherichia Collie C600, Escherichia Collie JM109
Etc. can be used.

As a method for transferring the recombinant DNA into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA is transferred using a commercially available competent cell, and then the microinjection method is used. May be. It was found that the thus obtained transformant microorganism can stably produce a large amount of thermostable DNA polymerase when cultured in a nutrient medium.

The selection as to whether or not the target recombinant DNA has been transferred into the host microorganism is carried out by a method of searching for a microorganism capable of simultaneously expressing a drug resistance marker of a vector carrying the target recombinant DNA and a thermostable DNA polymerase, and growing it in a selective medium. The colonies thus formed may be hybridized with a DNA probe synthesized on the basis of the amino acid sequence to perform a search and the like.

The base sequence of the thermostable DNA polymerase gene obtained by the above method is determined by Science (Scien).
ce), 214, 1205-1210 (1981), deciphering by the dideoxy method, and thermostable DNA
The entire amino acid sequence of the polymerase gene was deduced from the nucleotide sequence. The recombinant DNA carrying the thermostable DNA polymerase gene once selected in this manner can be easily taken out from the transformed microorganism and transferred to another host microorganism. In addition, a thermostable DNA polymerase gene D, which is a thermostable DNA polymerase gene, is cleaved from a recombinant DNA carrying the thermostable DNA polymerase gene by a restriction enzyme.
It is also easy to cut out NA, ligate it with a vector fragment obtained by cutting it by the same method, and transfer it to a host microorganism.

The culturing form of the host microorganism as a transformant may be selected by considering the culturing conditions in consideration of the nutritional physiological properties of the host. Usually, liquid culturing is usually carried out. Is advantageous. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose,
Maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract and the like are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, specific amino acids, specific vitamins, etc. are used as necessary.

The culturing temperature may be appropriately changed within the range where the bacterium grows and produces a thermostable DNA polymerase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Culture time varies slightly depending on the conditions, but heat-resistant DN
The culture may be completed at an appropriate time, considering the time when the maximum yield of A polymerase is reached, and usually about 12 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacterium grows and the thermostable DNA polymerase is produced, but the pH is particularly preferably about 6.0 to 8.0.

The thermostable DNA polymerase in the culture is
The culture is harvested by a means such as filtration or centrifugation, and then the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as EDTA and / or an interface. The thermostable DNA polymerase is solubilized by adding an activator and separated and collected as an aqueous solution.

The heat-resistant DNA polymerase-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or with a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the fractional precipitation method. Further, heat treatment and isoelectric point treatment are also effective refining means. A purified thermostable DNA polymerase is obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

The present invention will be described in detail below with reference to examples.

【Example】

Example 1 [Separation of chromosomal DNA] The chromosomal DNA of Thermus thermophilus HB8 was separated by the following method. 150 ml of 1.5% polypeptone, 1.5% yeast extract, 0.2% NaCl (pH 7.
After culturing with shaking in a liquid medium consisting of 5) at 70 ° C. overnight, the cells were collected by centrifugation (8000 rpm, 10 minutes). 10 mM
Tris-hydrochloric acid (pH 8.0), suspended in 24 ml of a solution containing 1 mM EDTA, and suspended in 3 ml of a lysozyme solution (2
0 mg / ml) and then 6% 10% SDS solution.
ml was added. 30 ml of phenol was added, and the mixture was gently mixed with stirring, centrifuged at 10,000 rpm for 15 minutes to separate an aqueous layer and a solvent layer, and the aqueous layers were combined. This operation was performed again, 2 volumes of ethanol was gently layered on this aqueous layer, and the DNA was sprinkled on the glass rod while gently stirring with the glass rod to separate the DNA. This was dissolved in a solution containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (hereinafter referred to as TE). Add an equal amount of chloroform
After treatment with phenol solution, separate the aqueous layer by centrifugation and
Add twice the amount of ethanol and repeat the above procedure using DN.
A was separated and dissolved with 2 ml TE. A commercially available competent cell of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.) was used and used as a host for preparing a library.

Example 2 [Preparation of a DNA fragment containing a gene encoding a thermostable DNA polymerase and a recombinant vector having the DNA fragment] 1 μg of the DNA obtained in Example 1 was used as a restriction enzyme HindIII.
(Toyobo) 5 units were allowed to react at 37 ° C. for 15 minutes, limited digestion was performed, and 0.5 μg of pUC19 completely digested with the restriction enzyme HindIII and 1 unit of T 4 -DNA ligase (Toyobo) were used at 16 ° C. The reaction was carried out for 12 hours to ligate the DNA. The ligated DNA is Escherichia
Transformation was performed using competent cells of Collie JM109. About 1 × 10 6 transformant colonies were obtained per 1 μg of the DNA used. 50 colonies obtained
L medium containing μg / ml ampicillin (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
The cells were cultivated in 0.25 mM IPTG) at 37 ° C. for 18 hours, cells were crushed, and the heat-resistant DNA polymerase activity was measured by the following method using the solution treated at 75 ° C. for 30 minutes.

(Reagent) A. 670 mM Tris-HCl (pH 8.8) 166 mM ammonium sulfate 67 mM magnesium chloride 10 mM 2-mercaptoethanol B.I. 1 μg / μl Activated Calf Thorax DNA C. 2 mM dNTP (250 cpm / pmol
“3H] dTTP) D. 20% trichloroacetic acid (2 mM pyrophosphate) E. 1 μg / μl carrier DNA

(Method) Solution A, solution B and solution C (5 μl each) and sterilized water (30 μl) were added to an Eppendorf tube and mixed by stirring, and then the cell disruption solution or the heat treatment solution (5 μl) was added and reacted at 75 ° C. for 30 minutes. To do. Then ice-cool and E-solution 50
Add μl and 100 ml of solution D, and cool with ice for 10 minutes.
This solution was filtered with a glass filter (Whatman GF / C filter), thoroughly washed with solution D and cold ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard) to incorporate nucleotides into the template DNA. It was measured. One unit of enzyme activity was the amount of enzyme that incorporated 10 nmol of nucleotide into the acid-insoluble fraction per 30 minutes under these conditions.

When the thermostable DNA polymerase activity of about 3,000 colonies was measured by the above method, 2
Colonies having thermostable DNA polymerase activity were obtained. These plasmids have Hind inside.
An insertion DNA fragment of about 6 kbp containing one III site was present, and this plasmid was designated as pUPLO.
Then from pUPL0, about 2.8kb in HindIII
The inserted DNA fragments of p and 3.2 kbp were excised and introduced into pUC19 to obtain pUPL01 and pUPL, respectively.
02 was built. Each of them was transformed into E. coli JM109, the transformants were cultured, and then heat-resistant DN was obtained by the above-mentioned method.
When the A polymerase activity was measured, no activity was observed. Next, pUPL02 is replaced with PstI and HindII
The 1.4 kbp fragment obtained by treating with I was introduced into pUC19 to construct pUPL03. Furthermore, a 3.2 kbp fragment obtained by treating pUPL01 with HindIII was introduced into the HindIII site of pUPL03 to obtain pUPL.
Built 1. The construction method of pUPL1 is shown in FIG. The productivity of the thermostable DNA polymerase of the strain transformed with Escherichia coli JM109 with pUPL1 was about twice that of Thermus thermophilus HB8.

[0027]

[Table 1]

Example 3 [Determination of nucleotide sequence] A HindIII fragment of pUPL01 of about 2.8 kbp,
And about 1.4 kbp HindIII-of pUPL03
For the PstI fragment, TAKARA
de using the sequence deletion kit
A production of a leakage mutant was performed. delete
Ion mutant is a single-stranded DNA according to a conventional method,
About the obtained single-stranded DNA, the Tth Sequencing kit (Tth Sequencing-Kit,
The base sequence was determined by using Toyobo. The determined nucleotide sequence and amino acid sequence are shown in Sequence 1.

[0029]

INDUSTRIAL APPLICABILITY The present invention clarified the base sequence of a thermostable DNA polymerase gene and the amino acid sequence of a thermostable DNA polymerase, and provided an efficient method for producing a thermostable DNA polymerase by a genetic engineering technique. Further, by using the thermostable DNA polymerase gene of the present invention and various genetic engineering techniques, a more efficient method for producing the thermostable DNA polymerase can be brought about.

[0030]

[Sequence list]

SEQ ID NO: 1 Sequence length: 3221 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Thermus thermophi
lus) strain name: HB8 (ATCC27634) TCTAGAGGAA GCATGAGCCT CACCCTGGCA GACAAGGTGG TCTACGAGGA GGAGATCCAG 60 AAAAGCCGCT TCATCGCCAA GGCGGCCCCC GTGGCCTCGG AGGAGGAGGC CTTGGCGTTT 120 TTGGCCGAGA ACCGGGAGCC TGAGGCCACC CACAACGGCC ACGCCTACAA GATCGGCCTC 180 CTCTACCGCT TCTCTGACGA CGGGGAGCCC TCGGGCACCG CAGGCAGGCC CATCCTCCAC 240 GCCATAGAGG CCCAGGGCCT GGACCGGGTG GCGGTCCTGG TGGTGCGCTA CTTCGGCGGG 300 GTGAAGCTCG GGGCCGGGGG GCTTGTGCGG GCCTACGGGG GGGTGGCGGC GGAGGCCTTA 360 AGGCGGGCGC CCAAGGTCCC CTTGGTGGAG CGGGTGGGGC TCGCCTTCCT CGTGCCCTTC 420 GCCGAGGTGG GCCGGGTCTA CGCCCTCCTG GAGGCCCGCG CCCCTGAAGG CCGAGGAGAC 480 CTACACCCCG GAGGCGTGCG CTTCGCCCTC CTCCTCCCCA AGCCCGAGCG GGAAGGTTTC 540 CTCAGGGCGC TCCTGGACGC CACCCGGGGA CAGGTGGCCC TGGAGTAGC ATG GAG 595 Met Glu 1 GCG ATG CTT CCG CTC TTT GAA CCC AAA GGC CGG GTC CTC CTG GTG GAC 643 Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val Asp 5 10 15 GGC CAC CAC CTG GCC TAC CGC ACC TTC TTC GCC CTG AAG GGC CTC ACC 691 Gly His His Leu Ala Tyr Arg Th r Phe Phe Ala Leu Lys Gly Leu Thr 20 25 30 ACG AGC CGG GGC GAA CCG GTG CAG GCG GTC TAC GGC TTC GCC AAG AGC 739 Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys Ser 35 40 45 50 CTC CTC AAG GCC CTG AAG GAG GAC GGG TAC AAG GCC GTC TTC GTG GTC 787 Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe Val Val 55 60 65 TTT GAC GCC AAG GCC CCC TCC TTC CGC CAC GAG GCC TAC GAG GCC TAC 835 Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr 70 75 80 AAG GCG GGG AGG GCC CCG ACC CCC GAG GAC TTC CCC CGG CAG CTC GCC 883 Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala 85 90 95 CTC ATC AAG GAG CTG GTG GAC CTC CTG GGG TTT ACC CGC CTC GAG GTC 931 Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu Glu Val 100 105 110 CCC GGC TAC GAG GCG GAC GAC GTT CTC GCC ACC CTG GCC AAG AAG GCG 979 Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Lys Ala 115 120 125 130 GAA AAG GAG GGG TAC GAG GTG CGC ATC CTC ACC GCC GAC CGC GAC CTC 1027 Glu Lys Glu Gly Tyr Glu Val A rg Ile Leu Thr Ala Asp Arg Asp Leu 135 140 145 TAC CAA CTC GTC TCC GAC CGC GTC GCC GTC CTC CAC CCC GAG GGC CAC 1075 Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly His 150 155 160 CTC ATC ACC CCG GAG TGG CTT TGG GAG AAG TAC GGC CTC AGG CCG GAG 1123 Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro Glu 165 170 175 CAG TGG GTG GAC TTC CGC GCC CTC GTG GGG GAC CCC TCC GAC AAC CTC 1171 Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp Asn Leu 180 185 190 CCC GGG GTC AAG GGC ATC GGG GAG AAG ACC GCC CTC AAG CTC CTC AAG 1219 Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu Leu Lys 195 200 205 210 GAG TGG GGA AGC CTG GAA AAC CTC CTC AAG AAC CTG GAC CGG GTA AAG 1267 Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val Lys 215 220 225 CCA GAA AAC GTC CGG GAG AAG ATC AAG GCC CAC CTG GAA GAC CTC AGG 1315 Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp Leu Arg 230 235 240 CTC TCC TTG GAG CTC TCC CGG GTG CGC ACC GAC CTC CCC CTG GAG GTG 1363 Leu Ser Le u Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 GAC CTC GCC CAG GGG CGG GAG CCC GAC CGG GAG GGG CTT AGG GCC TTC 1411 Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala Phe 260 265 270 CTG GAG AGG CTG GAG TTC GGC AGC CTC CTC CAC GAG TTC GGC CTC CTG 1459 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 290 GAG GCC CCC GCC CCC CTG GAG GAG GCC CCC TGG CCC CCG CCG GAA GGG 1507 Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 295 300 305 GCC TTC GTG GGC TTC GTC CTC TCC CGC CCC GAG CCC ATG TGG GCG GAG 1555 Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu 310 315 320 CTT AAA GCC CTG GCC GCC TGC AGG GAC GGC CGG GTG CAC CGG GCA GCA 1603 Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg Ala Ala 325 330 335 GAC CCC TTG GCG GGG CTA AAG GAC CTC AAG GAG GTC CGG GGC CTC CTC 1651 Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly Leu Leu 340 345 350 GCC AAG GAC CTC GCC GTC TTG GCC TCG AGG GAG GGG CTA GAC CTC GTG 1699 Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp Leu Val 355 360 365 370 CCC GGG GAC GAC CCC ATG CTC CTC GCC TAC CTC CTG GAC CCC TCC AAC 1747 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 375 380 385 ACC ACC CCC GAG GGG GTG GCG CGG CGC TAC GGG GGG GAG TGG ACG GAG 1795 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 390 395 400 GAC GCC GCC CAC CGG GCC CTC CTC TCG GAG AGG CTC CAT CGG AAC CTC 1843 Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu 405 410 415 CTT AAG CGC CTC GAG GGG GAG GAG AAG CTC CTT TGG CTC TAC CAC GAG 1891 Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu 420 425 430 GTG GAA AAG CCC CTC TCC CGG GTC CTG GCC CAC ATG GAG GCC ACC GGG 1939 Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 450 GTA CGG CGG GAC GTG GCC TAC CTT CAG GCC CTT TCC CTG GAG CTT GCG 1987 Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu Leu Ala 455 460 465 GAG GAG ATC CGC CGC CTC GAG GAG GAG GAG TC TTC CGC TTG GCG GGC CAC 2035 Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala Gly His 470 475 480 480 CCC TTC AAC CTC AAC TCC CGG GAC CAG CTG GAA AGG GTG CTC TTT GAC 2083 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 GAG CTT AGG CTT CCC GCC TTG GGG AAG ACG CAA AAG ACA GGC AAG CGC 2131 Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly Lys Arg 500 505 510 TCC ACC AGC GCC GCG GTG CTG GAG GCC CTA CGG GAG GCC CAC CCC ATC 2179 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 530 GTG GAG AAG ATC CTC CAG CAC CGG GAG CTC ACC AAG CTC AAG AAC ACC 2227 Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn Thr 535 540 545 TAC GTG GAC CCC CTC CCA AGC CTC GTC CAC CCG AGG ACG GGC CGC CTC 2275 Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg Leu 550 555 560 CAC ACC CGC TTC AAC CAG ACG GCC ACG GCC ACG GGG AGG CTT AGT AGC 2323 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 TCC GAC CCC AAC CT G CAG AAC ATC CCC GTC CGC ACC CCC TTG GGC CAG 2371 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 590 AGG ATC CGC CGG GCC TTC GTG GCC GAG GCG GGT TGG GCG TTG GTG GCC 2419 Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val Ala 595 600 605 610 CTG GAC TAT AGC CAG ATA GAG CTC CGC GTC CTC GCC CAC CTC TCC GGG 2467 615 620 625 GAC GAA AAC CTG ATC AGG GTC TTC CAG GAG GGG AAG GAC ATC CAC ACC 2515 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile His Thr 630 635 640 CAG ACC GCA AGC TGG ATG TTC GGC GTC CCC CCG GAG GCC GTG GAC CCC 2563 Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro 645 650 655 CTG ATG CGC CGG GCG GCC AAG ACG GTG AAC TTC GGC GTC CTC TAC GGC 2611 Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly 660 665 670 ATG TCC GCC CAT AGG CTC TCC CAG GAG CTT GCC ATC CCC TAC GAG GAG 2659 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 690 GCG GTG GCC TTT ATA GAG CGC TAC TTC CAA AGC TTC CCC AAG GTG CGG 2707 Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 695 700 705 GCC TGG ATA GAA AAG ACC CTG GAG GAG GGG AGG AAG CGG GGC TAC GTG 2755 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly Tyr Val 710 715 720 GAA ACC CTC TTC GGA AGA AGG CGC TAC GTG CCC GAC CTC AAC GCC CGG 2803 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala Arg 725 730 735 GTG AAG AGC GTC AGG GAG GCC GCG GAG CGC ATG GCC TTC AAC ATG CCC 2851 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG CTC GCC ATG GTG AAG CTC 2899 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 770 Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln Val His 775 780 785 GAC GAG CTC CTC CTG GAG GCC CCC CAA GCG CGG GCC GAG GAG GTG GCG 2995 Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu Val Ala 790 795 800 GCT TTG GCC AAG GAG GCC ATG GAG AAG GCC TAT CCC CTC GCC GTG CCC 3043 Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala Val Pro 805 810 815 CTG GAG GTG GAG GTG GGG ATG GGG GAG GAC TGG CTT TCC GCC AAG GGT 3091 Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala Lys Gly 820 825 830 TAGGGGGGCC CTGCTCTTCAGGAAGT CGCCTCCAGG 3151 GGAACGCCCT CTGGGCCTAC CAGGAGGCCT TTAGCCCCAA AGGTGCGGGT GAAGGCTTCC 3211 AGGCCCTGGG 3221

[Brief description of drawings]

FIG. 1 is a diagram showing a method for constructing pUPL1.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:01)

Claims (5)

[Claims] 1. Thermus th
ermophirus) -derived thermostable DNA polymerase or a DNA encoding a functional equivalent to the thermostable DNA polymerase. 2. The DNA according to claim 1, wherein the DNA encodes the base sequence shown in SEQ ID NO: 1 of the sequence listing or a base sequence equivalent thereto. 3. A recombinant vector containing the DNA of claim 1. 4. A transformant obtained by transforming a host cell with the recombinant vector according to claim 3. 5. The transformant according to claim 4 is cultured to produce a thermostable DNA polymerase, and the thermostable DN is then produced.
Thermostable DN characterized by collecting A polymerase
Method for producing A polymerase.