KR20110103750A - Thermostable dna polymerase derived from thermcoccus radiotolerans and its use - Google Patents

Abstract

The present invention is thermococcus ( thermococcus ) which is a hyperthermophilic archaea radiotolerans ) novel heat resistant DNA polymerase (hereinafter referred to as ' tra DNA polymerase'), a nucleic acid molecule encoding the same and its amino acid sequence, a vector comprising the nucleic acid sequence, Escherichia coli transformed with the vector coli ). The present invention also includes a method of manufacturing the Tra DNA polymerase and the DNA polymerase Tra PCR (polymerase chain reaction, hereinafter 'PCR'quot;) A kit comprising a. The present invention is a heat-resistant DNA polymerization which is the most essential element in PCR reactions used in various fields such as molecular biology research, diagnosis of genetic diseases, early diagnosis of viral and cancer genes, paternity identification, LMO differentiation, forensic research, etc. It is significant in that it can be provided as an enzyme. In particular, since the Tra DNA polymerase of the present invention has both DNA polymerization activity and proofreading activity, it can lower the error rate during PCR amplification, so it can be usefully used for PCR requiring accuracy. Have

Description

Heat-resistant DNA polymerase derived from Thermococcus radiotolerance strain and its use {THERMOSTABLE DNA POLYMERASE DERIVED FROM THERMCOCCUS RADIOTOLERANS AND ITS USE}

The present invention relates to a DNA polymerase derived from the strain of thermothermophilic archaeon Thermococcus radiotolerans and its use.

DNA polymerase (deoxyribonucleic acid polymerase, DNA polymerase, EC number 2.7.7.7) is an enzyme that synthesizes complementary DNA in the 5 '→ 3' direction depending on template DNA. It is known as an enzyme that plays the most important role in replication or DNA repair. DNA polymerase was first discovered in Escherichia coli by Kornberg et al in 1957 and is the current E. coli DNA polymerase I. The E. coli DNA polymerase Ⅰ (Escherichia coli DNA polymerase I and E. coli DNA polymerase I) include 5 '→ 3' nucleic acid terminal hydrolase, as well as 5 '→ 3' polymerase activity, DNA polymerization activity. It has a 3 '→ 5' nucleic acid terminal hydrolase activity (3 '→ 5' exonuclease activity) called activity (5 '→ 3' exonuclease activity) and proofreading activity. In the case of heat-resistant DNA polymerase, Thermor Aquaticus Witi-1 ( Thermus), aquaticus After their initial separation from YT-1), they have been separated from the Thermos flavus , Thermus thermophilus HB8, etc. belonging to the same genus, but their characteristics have been revealed. (Chien, A. et al. , 1976, J. Bacteriol. 127 , 1550-1557; Kaledin, AS et al ., 1981, Biokhimiya 46 , 1576-1584; Ruettimann, C. et. al . , 1985, Eur . J. Biochem . 149 , 41-46). However, in 1988, Saiki et al. Developed a polymerase chain reaction (PCR) technology using heat-resistant DNA polymerase, and heat-resistant DNA polymerase began to attract attention. littoralis less (Thermococcus litoralis), Thermo Lactococcus Mariners (Thermococcus marinus), Thermo Lactococcus guar forehead sensor system (Thermococcus guaymasensis), Rhodococcus furiosus (Pyrococcus furiosus), nanoah keum Iquique Tansu (Nanoarchaeum equitans), sseomeoseu knife filler Pyro Heat-resistant DNA polymerase has been competitively researched and developed from several high temperature and super high temperature archaea, such as Thermus caldophilus GK24, Thermus filiformis and Pyrococcus woesei . , H. et al ., 1993, The Journal of Biological Chemistry 168 (3) 1965-1975; Bae, H. et al ., 2009, Extremophiles 13 , 657-667; Lee , JI et al ., 2009, Enzyme and Microbial Technology 45 , 103-111; Lundberg, KS et al. , 1991, Gene 108 , 1-6; Choi, JJ et al ., 2008, Appl. Environ.Microbiol . 74 , 6563-6569; Park, JH et al. , 1993, Eur. J. Biochem. 214 , 135-140; Jung, SE et al. , 1997, Mol. Cells 7 , 769-776; Dabrowski, S. and Kur , J., 1998, Protein Expres. Purif. 14 , 131-138).

In particular, DNA polymerases derived from S. aureus and Pyrococcus strains have been attracting more attention because they are heat-resistant DNA polymerases with corrective activity, unlike DNA polymerases derived from S. aureus. It is used for.

PCR is a technique for mass amplification of extremely small amount of template DNA using DNA polymerase and primer, DNA denaturation (94 ° C), primer annealing (40-65 ° C) and DNA extension (DNA) elongation (72 ℃) is repeated 20-30 times in succession.

Since high temperature is required due to these reaction characteristics, development and development of heat-resistant DNA polymerase, which is the most important factor in PCR, must be made for the development and development of various PCR technologies.

Heat-resistant DNA polymerase is a very useful enzyme for identification and amplification of genes by PCR, DNA sequence determination, and clinical diagnosis. The enzyme is used in a wide range of fields, from molecular biology and genetic engineering research to early diagnosis of viral and cancer genes, genetic disease diagnosis and forensic research, and the demand is increasing day by day.

To date, there have been no studies on the nucleotide sequence of the DNA polymerase gene, the heat-resistant DNA polymerase encoded by the aforementioned gene, and the use of this enzyme.

Therefore, the present inventors have isolated the heat-resistant DNA polymerase gene from the ultra-high temperature bacterium Thermococcus radiotolerance, expressed and purified the enzyme encoded by the gene in Escherichia coli, and continued the research to identify the enzyme properties and use it for PCR. The present invention has been reached.

It is an object of the present invention to provide a heat-resistant Tra DNA polymerase derived from Thermococcus radiotolerans strain having excellent DNA polymerization activity and corrective activity, a nucleic acid molecule encoding the same, a vector comprising the nucleic acid molecule and a vector transformed with the vector. To provide a host cell.

Another object of the present invention is to provide a kit for nucleic acid amplification reaction using the Tra DNA polymerase.

Another object of the present invention is to provide a method for preparing Tra DNA polymerase.

Another object of the present invention is to provide a method of performing polymerase chain reaction (PCR) using Tra DNA polymerase.

In order to solve the above problems, the inventors of the present invention, the thermococcus radiotolerans DNA polymerase ( Tracoccus radiotolerans DNA polymerase, Tra DNA polymerase, hereinafter referred to as ' tra DNA polymerase') gene The nucleotide sequence of the gene and the amino acid sequence inferred therefrom are determined, and thus, the new heat resistance of Tra DNA polymerase has high amino acid sequence homology with the conventional heat-resistant B family DNA polymerases. It was confirmed that the B series DNA polymerase.

Then, by examining the various enzyme properties of the Tra DNA polymerase to confirm the optimum DNA polymerization activity (DNA polymerization activity) conditions, it was confirmed that it has a proofreading activity that improves the accuracy during DNA polymerization. Finally, by performing polymerase chain reaction (referred to as polymerase chain reaction, hereinafter 'PCR') using the Tra DNA polymerase, purified Tra It was confirmed that DNA polymerase can be used for PCR. It demonstrates in detail below.

The present invention provides a Tra DNA polymerase isolated from Thermococcus radiotolerans , which is a kind of ultra-high temperature archaea. Specifically, the present invention relates to a heat resistant Tra DNA polymerase isolated from a Thermococcus radiotolerance having the amino acid sequence of SEQ ID NO: 10.

The Tra DNA polymerase of the present invention is a heat-resistant B-type DNA polymerase, which is a DNA polymerization activity of 5 '→ 3' polymerase activity (5 '→ 3' polymerase activity). In addition, it is characterized by having a 3 '→ 5' nucleic acid terminal hydrolase activity (3 '→ 5' exonuclease activity) called proofreading activity.

Tra DNA polymerase of the present invention comprises a nucleic acid sequence of 2,328 bp, including a stop codon (SEQ ID NO: 9), and consists of 775 amino acids (SEQ ID NO: 10).

The present invention includes all of the protein variants having sequences different from those of the wild type Tra DNA polymerase of SEQ ID NO: 10-modifications by deletion, insertion, substitution, etc. of some sequences, and protein variants by combinations thereof.

In addition, the Tra DNA polymerase of the present invention also includes a form modified due to methylation, phosphorylation, glycosylation, acetylation and the like. Other physical or chemical properties may be modified due to physical factors such as temperature, pH, moisture, drying, pressure, reduced pressure, freezing, or chemical factors such as acids, alkalis, neutral salts, organic solvents, metal ions, oxidants, reducing agents, proteases, etc. Forms also fall within the scope of the present invention.

The variant may be a functional equivalent having substantially the same activity as the wild type protein, a form in which physicochemical properties such as increased or decreased enzymatic activity are changed, or a variant having increased structural stability to the external environment.

The present invention also provides information on the nucleotide sequence encoding the Tra DNA polymerase. Specifically, it relates to a nucleic acid molecule encoding the heat resistant Tra DNA polymerase. Tra DNA polymerase having an amino acid sequence of SEQ ID NO: 10 preferably has a nucleotide sequence of SEQ ID NO: 9.

The present invention also encompasses nucleic acid molecules encoding the above-mentioned Tra DNA polymerase variants.

The present invention also includes base sequences that encode polypeptides of the same sequence as the wild type DNA polymerase disclosed herein but different from the base sequences.

The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the heat resistant Tra DNA polymerase.

In the present invention, a "vector" is a recombinant vector capable of expressing a protein of interest in a host cell, and is a gene construct including essential regulatory elements operably linked to express a gene insert. In the present invention, "operably linked" refers to a functional link between an expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operative linkage with vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be facilitated using enzymes commonly known in the art. . Vectors of the present invention include all conventional vectors, including plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Preferably, it is a plasmid vector.

In the present invention, recombinant plasmid pTRAP was prepared using pET-22b (+), a kind of plasmid vector, as an expression vector (see FIG. 2).

Specifically, the present invention provides a vector which is pTRAP expressing Tra DNA polymerase having the amino acid sequence of SEQ ID NO: 10.

The present invention also provides a host cell transformed with the recombinant vector.

As used herein, "transformation" refers to a change in the genetic properties of an organism by DNA given from the outside. Host cells that can be transformed include, but are not limited to, prokaryotic cells, yeast cells, insect cells, plant cells, animal cells, and the like. It includes, for example, when the microorganism E. coli (Escherichia coli), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas) or the like. Most preferably, E. coli is used. In the present invention, Escherichia coli, Escherichia coli Rosetta (DE3) pLysS was used.

Specifically, the present invention provides Escherichia coli , Escherichia coli Rosetta (DE3) pLysS / pTRAP, which is a microorganism transformed with a pTRAP vector. E. coli, Escherichia coli Rosetta (DE3) pLysS / pTRAP, was deposited with the microbial accession number KACC95102P on February 23, 2010 to KACC (Korean Agricultural Culture Collection, 88-20 Seodun-dong, Gwonseon-gu, Suwon-si, Korea).

The present invention also relates to a kit comprising a heat resistant Tra DNA polymerase derived from Thermococcus radiotolerance having the amino acid sequence of SEQ ID NO: 10.

The Tra DNA polymerase of the present invention can be used as a kit component of a nucleic acid amplification reaction. Such nucleic acid amplification reactions include polymerase chain reation (PCR), ligand chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), strand displacement amplification (SDA), and branched DNA signal amplification. (branched DNA signal amplification), nested PCR (nested PCR), multiplex PCR (multiplex PCR) and the like.

The kit for nucleic acid amplification reaction of the present invention, in addition to the Tra DNA polymerase, includes one or more other components, solutions or devices suitable for the analysis method. For example, the kit of the present invention may include one or more components selected from a container containing a detection primer, an amplification tube or container, reaction buffer, dNTPs, RNAse, sterile water, and the like.

Kits comprising the Tra DNA polymerase of the present invention can be usefully used in various fields such as genetic engineering and molecular biology experiments, clinical diagnosis, forensic research.

The present invention also relates to a method for preparing Tra DNA polymerase.

The Tra DNA polymerase of the present invention can be prepared by a method of separating from a source of natural origin, by chemical synthesis, or by introducing a vector encoding a Tra DNA polymerase into a suitable host and expressing it thereafter.

Systems for preparing an expression vector capable of efficiently expressing Tra DNA polymerase and expressing it in a microorganism are well known to those skilled in the art, and can be implemented by various modifications / applications. One example of such an implementation is as follows.

Tra DNA polymerase comprises the steps of: (i) preparing a recombinant vector comprising a heat resistant Tra DNA polymerase gene isolated from a Thermococcus radiotolerance strain encoding the amino acid sequence of SEQ ID NO: 10; (ii) transforming the recombinant vector into a host cell; (iii) culturing the transformed host cell; And (iv) can be prepared by a process comprising the step of recovering the protein from the transformed host cell.

The type of vector in step (i), the method of transforming the vector in step (ii) and the type of sperm cell are as discussed above.

The culturing process of step (iii) is carried out according to methods well known for the type of cells or microorganisms used as transformants, and the conditions such as the media components, the culture temperature and the culture time can be appropriately adjusted. Specifically, the culture medium contains all the nutrients essential for the growth and survival of microorganisms such as carbon sources, nitrogen sources, trace element components and the like. The pH of the medium can be adjusted appropriately, and components such as antibiotics can be included.

Further, by processing the inducing agent (inducer) such as isopropyl Im Dwarf (referred to as isopropyl-β- _D -thiogalactopyranoside, hereinafter 'IPTG') Lactobacillus pyrano side can induce the expression of the protein. The type of inducer to be treated may be determined according to the vector system, and conditions such as the induction agent administration time and dosage may be appropriately controlled.

In step (iv) the protein is recovered and purified in a conventional manner. For example, the cells recovered by the centrifugation method are crushed using a French press, an ultrasonic crusher or the like. If protein is secreted into the culture, the culture supernatant is collected. When aggregated by overexpression, it can be obtained by dissolving, denaturing and refolding the protein in the appropriate solution. Oxidation and reduction systems of glutathione, dithiothreitol, β-mercaptoethanol, β-mercaptomethanol, cystine and cystamine are used, and refolding agents such as urea, guanidine, arginine and the like are used. Some of the salts may be used with the refolding agent.

At this time, a step of heat treatment for 10 to 60 minutes at 70 to 85 ℃ can be added.

Proteins obtained by culturing the transformant can be used without purification and are subjected to conventional biochemical separation techniques such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, chromatography Photography and the like can be used alone or in combination. Chromatography includes ion exchange chromatography, size exclusion chromatography, affinity chromatography, and the like.

The scale of the protein manufacturing process can be adjusted to suit the purpose. In addition, there may be additional and charging processes between each process.

Thus obtained protein can be converted into a salt by a well-known method when obtained as a free body, and when obtained into a salt, it can be converted into a free body or another salt by a well-known method.

Specifically, in the present invention, the heat-resistant Tra DNA polymerase preparation further comprises the step of purifying the separated DNA polymerase by heat treatment at 80 ° C. for 30 minutes and HisTrap ^TM HP column and HiTrap ^TM SP HP column chromatography. Provide a method. In the present invention, was expressed by cloning of recombinant plasmids pTRAP in E. coli Rosetta (DE3) pLysS, heat treatment and HisTrap ^TM HP column and HiTrap ^TM SP column chromatography of Step 2 using the HP column our Tra DNA through (column chromatography) process The polymerase can be purified.

In a specific embodiment of the present invention, the cells were recovered by centrifugation of the cultured cells, and then disrupted by ultrasonication and subjected to column chromatography using a HisTrap ^TM HP column (GE Healthcare) and a HiTrap ^TM SP HP column (GE Healthcare) after heat treatment. Obtained by the process performed.

Tra DNA polymerase produced according to the preparation method of the present invention is pH 7.5, temperature of 75-80 ℃, MgCl ₂ of 8 mM Concentration, 20 mM (NH ₄ ) ₂ SO ₄ Physical and chemical properties that exhibit optimal activity at concentrations and KCl concentrations of 90 mM.

The Tra DNA polymerase of the present invention can be used for PCR. Specifically, PCR was performed under a reaction buffer composed of 50 mM Tris-HCl (pH 8.6), 1 mM MgCl ₂ , 2 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, and 0.1-0.3% Triton X-100. As a result, it can be seen that it shows high accuracy with a considerably low error rate.

The present invention can be applied to PCR which is useful in modern biological experiments such as molecular biology, genetic engineering, etc. Through this, it can be used in various fields such as genetic disease diagnosis, early diagnosis of cancer genes and viral genes, paternity discrimination, forensic research, etc. Can be utilized. In particular, since the Tra DNA polymerase of the present invention has excellent DNA polymerization activity and high correction activity, it can be widely used in various nucleic acid polymerization reactions requiring accuracy.

Figure 1 shows the amino acid sequence of the Tra DNA polymerase of conventional heat-resistant B-based DNA polymerases (thermococcus sp 9 digrien-7, Thermococcus guaimasensis, Thermococcus codcarensis code 1 and Pyrococcus furio It is shown compared with the amino acid sequence of sus).
Figure 2 shows the construction of the recombinant plasmid pTRAP for the expression of Tra DNA polymerase gene.
Figure 3 shows the results of SDS denaturation gel electrophoresis according to the purification step (heat treatment and column chromatography of two steps) of the Tra DNA polymerase expressed in E. coli.
Figure 4 is a graph showing the relative degree of DNA polymerization activity according to the temperature (60-95 ℃) of Tra DNA polymerase.
5 is a graph showing the relative degree of DNA polymerization activity according to pH (pH 6.0-9.0) of Tra DNA polymerase.
6 is a graph showing the relative degree of DNA polymerization activity according to MgCl ₂ concentration (0-20 mM) of Tra DNA polymerase.
7 is a graph showing the relative degree of DNA polymerization activity according to (NH ₄ ) ₂ SO ₄ concentration (0-100 mM) of Tra DNA polymerase.
8 is a graph showing the relative degree of DNA polymerization activity according to KCl concentration (0-100 mM) of Tra DNA polymerase.
9 is a graph showing the relative thermal stability of Tra DNA polymerase at 94 ℃ and 99 ℃, respectively. 50% DNA polymerization activity was maintained at 94 ° C. for about 4 hours and at 99 ° C. for about 40 minutes.
FIG. 10 is a graph showing 3 '→ 5' nucleic acid terminal hydrolase activity of Tra DNA polymerase in the presence or absence of dNTP.
Figure 11 shows the results according to pH (pH 7.6-10.0) in the polymerase chain reaction (PCR) using Tra DNA polymerase.
Figure 12 shows the results according to the concentration of MgCl ₂ (0-3 mM) in the polymerase chain reaction using Tra DNA polymerase.
Figure 13 shows the results according to (NH ₄ ) ₂ SO ₄ concentration (0-10 mM) in the polymerase chain reaction using Tra DNA polymerase.
Figure 14 shows the results according to the KCl concentration (0 ~ 30 mM) in the polymerase chain reaction using Tra DNA polymerase.
Figure 15 shows the results according to the Triton X-100 concentration (0 ~ 0.03%) in the polymerase chain reaction using Tra DNA polymerase.
FIG. 16 shows polymerase chain reaction using lambda phage genomic DNA as a template with amplification time of 1 minute per kb using Tra DNA polymerase, Taq DNA polymerase and Pfu DNA polymerase under each optimal reaction buffer solution. The results are shown. When using Tra DNA polymerase, 2 and 5 kb of DNA were amplified.

Hereinafter, the present invention will be described in detail through non-limiting examples. However, the following examples are merely to illustrate the present invention for a more detailed description, the content of the present invention is not limited to the following examples.

Example 1: Cultivation of Thermococcus radiotolerance strain

Thermococcus radiotolerans strain (DSM 15228) was distributed by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and cultured in DSMZ 990 medium. DSMZ 990 medium (4.0 g peptone per liter, 2.0 g yeast extract, 35.0 g sea salt (Sigma, USA), 3.46 g PIPES, 5 g elemental sulphur, NH ₄ Cl 0.5 g, KH ₂ PO ₄ 0.35 g, CaCl ₂ 0.2 g, FeCl ₃ 6.7 mg, Na ₂ WO ₄ 2.9 mg, resazurin (resazurin_ 0.1 mg) were prepared and the pH was adjusted to 6.8 with 5 N sulfuric acid. When the peptone and yeast extract (prepared as 10% stock) and elemental sulfur were added separately without separately, the solution was heated overnight (8 hours) in an incubator at 100 ° C. to sterilize and remove oxygen from the solution at the same time. Some precipitates formed after heating were quickly filtered and then heated again for 3 hours in an incubator at 100 ° C. 0.5 g of elemental sulfur was added to a 120 ml serum bottle (Wheaton Co., USA) and heated. 100 ml of the medium solution is added, and the gas inside the serum bottle containing the medium solution is replaced with nitrogen gas for 30 minutes. After the film and then, the inlet of the serum bottle with a rubber bottle stopper and aluminum ring, were at 95 ℃ sterilization 24 hours thus prepared and then using a sterile syringe to the medium was added to the sterilized peptone and yeast extract, 5% Na ₂ A small amount of S · 9H ₂ O solution was injected, 1% inoculated Thermococcus radiotolerance strains, and then cultured at 80 ° C. for 24 hours.

Example 2: Isolation of Genomic DNA

The cultures incubated under anaerobic conditions were filtered to remove sulfur, the cells were recovered by centrifugation, and the cells were suspended in 10 mM Tris-HCl (pH 8.0) buffer solution containing 20% sucrose. The cell membrane was attenuated by repeating freezing and thawing twice, and then adding Triton X-100 (Triton X-100) to a final concentration of 0.3% for 1 hour at room temperature. To this was added SET (150 mM NaCl / 1 mM EDTA / 20 mM Tris-HCl (pH 8.0)) buffer solution, SDS and RNase were added and reacted at 55 ° C for 1 hour, followed by proteinase K ( proteinase K) was added and reacted at 37 ° C for 1 hour and at 55 ° C for 2 hours. Subsequently, an equal amount of a mixture of chloroform / isoamylalcohol (24: 1) was added to the reaction solution, followed by shaking overnight, followed by 2 times volume of 100% ethanol and 1/10 times volume. Genomic DNA was precipitated by addition of 3 M sodium acetate (pH 5.2), washed with 70% ethanol and dried in vacuo, TE (10 mM Tris-HCl (pH 7.6) / 1 mM EDTA) It was dissolved in buffer solution. Thermococcus radiotolerance genomic DNA isolated as described above was quantified by measuring the absorbance at 260 nm wavelength.

Example 3: Selection of DNA Polymerase Genes

To screen for Tra DNA polymerase genes from Thermococcus radiotolerance genomic DNA, Thermococcus radiotolerance genomic DNA was templated and previously reported Pyrococcus furiosus (Lundberg, KS et al . , 1991, Gene 108 (1), 1-6), Thermococcus sp. 9 ^o N-7 (Southworth, MW et al. , 1996, Proc. Natl. Acad. Sci. DNA polymerase genes of USA 93 (11), 5281-5285), Thermococcus guaymasensis (Lee, JI et al ., 2009, Enzyme Microb.Technol. 45 (2), 103-111) The primers of SEQ ID NOS: 1 and 2 were prepared with reference to two amino acid sequences of well-conserved sites. SEQ ID NO: 1 primer is the nucleotide sequence encoding the amino acid sequence EYDIPFA. SEQ ID NO: 2 primer is a complementary sequence of nucleotide sequence encoding amino acid sequence YYIENQV. PCR was performed using these primers as a template for the Thermococcus radiotolerance genomic DNA. The PCR conditions were carried out for 5 minutes at 94 ℃, 30 seconds at 94 ℃, 30 seconds at 50 ℃ and 30 minutes at 72 ℃ was repeated 30 times, the reaction product after extending the reaction at 72 ℃ 10 minutes 0.8 Electrophoresis on% agarose gel confirmed that the amplified DNA fragment of about 1.8 kb was amplified. The PCR reaction products were also purified on a 1% agarose gel using MEGAquick-spin ^™ PCR & Agarose Gel DNA Extraction Kit (iNtRON Biotechnology, Korea). The purified DNA was commissioned by Solgent Co., Ltd. to determine the nucleotide sequence, and compared with other DNA polymerase sequences of other known species, and showed high homology. Therefore, it was confirmed that the purified 1.7 kb DNA fragment was part of the Tra DNA polymerase gene of Thermococcus radiotolerance. This was used as the center for screening the Tra DNA polymerase gene of Thermococcus radiotolerance.

     SEQ ID NO: 1 Primer (T-111N): 5'-GARTACGACATACCCTTYGC-3 '

     SEQ ID NO: 2 Primer (T-CR): 5'-AACCTGGTTCTCRATKTAGTA-3 '

(Wherein R is an A or G base, Y is a C or T base, and K is a base of G or T.)

Example 4 Using Primer Walking Method Tra Search for unknown contiguous DNA sites (N and C termini) from the central region of the DNA polymerase gene

Four internal primers were prepared based on DNA sequences of 1.7 kb of Tra DNA polymerase gene already secured through Example 3 (SEQ ID NOs: 3, 4, 5, and 6 primers). The Walking SpeedUp ^™ Premix Kit (Seegene, Korea) was used to find unknown contiguous sequences. Tra DNA polymerase four kinds of random (random) primers and SEQ ID NO: 3 primer (Tra-N-1) in a Walking SpeedUp ^TM Premix Kit for cloning a gene region corresponding to the 5 'upstream (N-terminal region) of a gene and The first PCR was performed using Thermococcus radiotolerance genomic DNA. As a template, a second PCR was performed using SEQ ID No. 4 primer (Tra-N-2) as a template and a second PCR primer in the kit. As a result, a DNA fragment of about 1.5 kb was specifically amplified. The amplified DNA fragments were electrophoresed on agarose gel, and then extracted, purified using MEGAquick-spin ^TM PCR and Agarose Gel DNA Extraction Kit (iNtRON Biotechnology) and commissioned by Solgent. The base sequence was determined. As a result of determining the nucleotide sequence of these fragments, the nucleotide sequence in the 5 'upstream (N-terminal portion) including the start codon was obtained.

In addition, four random primers in the Walking SpeedUp ^TM Premix Kit and the known Tra DNA polymerase gene 1.7 are used to clone the gene region corresponding to the unknown 3 'downstream (C-terminal region) adjacent to the conserved nucleotide sequence. First PCR was performed using SEQ ID NO: 5 primer (Tra-C-1) based on kb DNA sequence and Thermococcus radiotolerance genomic DNA. The PCR product was specifically amplified about 1.2 kb of DNA fragment by using the SEQ ID No. 6 primer (Tra-C-2) as a template and the second PCR primer in the kit. Amplified DNA fragments were electrophoresed on agarose gel, then extracted, purified using MEGAquick-spin ^TM PCR and Agarose Gel DNA Extraction Kit (iNtRON Biotechnology) and commissioned by Solgent. The base sequence was determined. As a result of determining the nucleotide sequence of these fragments, a nucleotide sequence in the 3 'upstream (terminal C) portion containing the stop codon was obtained. Thus, the entire nucleotide sequence encoding the Tra DNA polymerase gene was determined (SEQ ID NO: 9).

    Internal primer sequence

   SEQ ID NO: 3 Primer (Tra-N-1): 5 '-ATCCTTCTCCTTCACGACCT-3'

   SEQ ID NO: 4 Primer (Tra-N-2): 5 '-TTTATCATCTCCTTCTCGGTGG-3'

   SEQ ID NO: 5 Primer (Tra-C-1): 5 '-GAGAAGCTGGTCATCCACG-3'

SEQ ID NO: 6 Primer (Tra-C-2): 5 '-GGGAGCTAAAGGATTACAAGG-3'

Tra DNA polymerase gene sequences were analyzed using DNASTAR (DNASTAR Inc., USA) program, Pyrococcus furiosus , Thermococus sp . 9 digry using NCBI BLAST program. Nucleotide sequence and similarity of N-7 ( Thermococcus sp. 9 ^o N-7), Thermococcus guaymasensis DNA polymerase gene were compared.

As a result, the entire nucleotide sequence of the DNA polymerase gene isolated from the Thermococcus radiotolerans strain consisted of a total of 2,328 bp including the start codon (ATG) and the stop codon (TGA) (SEQ ID NO: 9). The total amino acids consisted of 775 amino acids (SEQ ID NO: 10). From the amino acid sequence, the molecular weight of the DNA polymerase enzyme of the present invention was estimated to be about 89,791 Da. In addition, as a result of comparing the amino acid sequence of the DNA polymerase of the present invention isolated from the Thermococcus radiotolerans strain and the amino acid sequence of other DNA polymerases, Thermococcus sp. 9 digrien- 7 ( Thermococcus sp. 9 ^o N-7) (Southworth, MW et al. , 1996, Proc. Natl. Acad. Sci. USA 93 (11), 5281-5285) showed a 92% sequence homology with the DNA polymerase. Thermococcus guaymasensis (Lee, JI et al ., 2009, Enzyme Microb.Technol. 45 (2), 103-111) showed 89% sequence homology with the DNA polymerase. Sequence homology of 89% with DNA polymerase of Code 1 ( Thermococcus kodakaraensis KOD1) (Takagi M. et al ., 1997, Appl. Environ. Microbiol. , 4504-4510) was confirmed (see FIG. 1).

Example 5: Tra  Construction of Expression Vector pTRAP for Expression of DNA Polymerase Gene

A recombinant plasmid (pTRAP) incorporating the Tra DNA polymerase gene into the expression vector was constructed as follows (Fig. 2).

Tra first DNA polymerase SEQ encoding the N- terminal amino acid sequence from the nucleotide sequence of Tra DNA polymerase gene determined in Example 4 to obtain 7 gene primers (TraN) (5 'terminal primer) and the C- terminal amino acid sequence After preparing each SEQ ID No. 8 primer (TraC) (3 'terminal primer) complementary to the DNA base coding for, the Tra DNA polymerase gene was amplified by PCR. A primer of SEQ ID NO: 7 was synthesized to include a restriction enzyme Nde I cleavage site containing the start codon (ATG) of the Tra DNA polymerase gene, and SEQ ID NO: 8 primer to include a stop codon and a restriction enzyme Xho I cleavage site, respectively. Thermococcus radiotolerance genomic DNA was subjected to PCR using SEQ ID NOs: 7 and 8 primers. PCR was performed using a mixed composition consisting of 0.2 μg Thermococcus radiotolerance genomic DNA, 20 pmole of 5 'and 3' terminal primers, 200 μM dNTPs, 10 ㅧ PyroAce DNA polymerase buffer and 2.5 U Super PyroAce DNA polymerase. After reacting for 3 minutes at 95 ° C., it was repeated 30 times at 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 120 seconds, and then reacted for 10 minutes at 72 ° C. Electrophoresis on 0.8% agarose gel with DNA size marker confirmed the presence of a band at about 2.3 kb. After the PCR reaction mixture was electrophoresed on a 1% agarose gel, approximately 2.3 kb of DNA product amplified by PCR was obtained from MEGAquick-spin ^TM PCR and Agarose Gel DNA Extraction Kit (iNtRON Biotechnology). Purified and recovered using. The recovered DNA was digested with restriction enzymes Nde I and Xho I and then electrophoresed again on a 1% agarose gel to purify the gel fragment corresponding to the 2.3 kb position as a Tra DNA polymerase gene fragment using the kit. Recovered. The purified Tra DNA polymerase gene fragment was inserted into the expression vector pET-22b (+) (Novagen, USA) digested with the same restriction enzyme and linked using T ₄ DNA ligase and overnight ligation at 16 ° C. E. coli Escherichia coli Rosetta (DE3) pLysS (Stratagene, USA) was then transformed (Sambrook, J. et al ., 1989, Molecular cloning, a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press). Plasmid DNA was isolated from the transformants by alkaline lysis method, digested with restriction enzymes Nde I and Xho I, and then electrophoresed with DNA size marker on 0.8% agarose gel to express Tra DNA polymerase gene. It was again confirmed that it was correctly inserted in the vector. The expression vector constructed for the expression of the Tra DNA polymerase gene constructed as above was named "pTRAP" (FIG. 2). The Tra DNA polymerase of E. coli containing the expression vector and the expression vector containing the gene and the gene was to the National Academy of Agricultural Sciences Agricultural Genetic Resources Center, deposited on February 23, 2010, the deposit genetically name Escherichia coli Rosetta (DE3) pLysS / pTRAP The accession number is KACC95102P.

SEQ ID NO: 7 Primer (TraN):

5 '-NNNNNN CATATG ATCCTCGATACCGACTACATCACA-3'

Nde I

SEQ ID NO: 8 Primer (TraC):

5 '-NNNNNN CTCGAG CTTCTTCCCCTTCGGCTTAAGC-3'

Xho I

Example 6: Expression and Purification of Recombinant DNA Polymerase Gene

The recombinant protein transformed into Escherichia coli by the method of Example 5 was expressed as Tra DNA polymerase protein from Escherichia coli Rosetta (DE3) pLysS / pTRAP, and the expressed protein was purified as follows.

E. coli Rosetta (DE3) pLysS containing the recombinant plasmid of Example 5 was inoculated in LB liquid medium to which 100 ug / ul ampicillin and 34 ug / ul chloramphenicol were added, and then incubated at 37 ° C. overnight, followed by 5 ml of culture. Escherichia coli was inoculated in 500 ml of the same medium and cultured at 37 ° C. When the absorbance at 600 nm was about 0.6, IPTG (isopropyl β- _D -galactopyranoside) was added to a final concentration of 0.1 mM and incubated overnight. Cells (2.1 g / wet weight) were recovered by centrifugation at 6,000 rpm for 15 minutes and then 25 ml of sonication buffer containing 20 mM Tris-HCl (pH 7.4), 0.5 mM NaCl containing 1 mM PMSF. Suspension) was disrupted by ultrasonication and centrifuged at 18,000 rpm for 15 minutes to remove E. coli cell debris. Next, after heat treatment at 80 ° C. for 30 minutes, most of the proteins derived from E. coli were removed by centrifugation to recover the supernatant. The supernatant was finally purified by column chromatography using HisTrap ^™ HP column (GE Healthcare) and HiTrap ^™ SP HP column (GE Healthcare). The Tra DNA polymerase purified as described above was stored in a storage buffer (20 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 0.1% Tween 20, 0.5% Nonidet P40, 100 mM KCl, 50% Glycerol) After dialysis at, it was used to investigate the enzymatic properties of Tra DNA polymerase while storing at -20 ℃ and to perform PCR using Tra DNA polymerase. In purifying heat-resistant Tra DNA polymerase from E. coli Rosetta (DE3) pLysS having a recombinant plasmid pTRAP, the results of the purification step are shown in Table 1 below.

DNA polymerization activity was measured using Choi et al. (Choi, JJ et al. , 1999, Biotechnol. Appl. Biochem. 30 , 19-25). Reaction mixture ( Tra DNA polymerase purified in Example 6, 1.25 μg active calf thymus DNA, 50 mM Tris-HCl, pH 7.5), 10 mM MgCl ₂ , 40 mM KCl, 1 mM β-mercaptoethanol, 100 μM dATP, dCTP and dGTP, 10 μM dTTP, 0.5 μCi [ methyl - ³ H] thymidine 5′-triphosphate) were reacted at 75 ° C. for 10 minutes and then quenched on ice. The reaction solution was added dropwise to a DE-81 filter paper disk (23 mm, Whatman Co., USA) and dried at 65 ° C., followed by a 0.5 M sodium phosphate (pH 7.0) buffer solution. After washing for 10 minutes in 70% ethanol in order for 5 minutes, it was dried again at 65 ℃. Dry DE-81 filter paper discs were used to measure the amount of DNA incorporation of [ ³ H] TTP using a Tri-Carb Liquid Scintillation Analyzer (PerkinElmer, USA). At this time, 1 unit (U) was defined as the amount of DNA polymerase required to synthesize 10 nmol of dNTP synthesized for 30 minutes at 75 ° C to synthesize an acid insoluble DNA product.

The amount of protein in the purification steps was determined using a Lowry assay ( J. Biol. Chem. 193, 261 (1951)). In addition, denatured gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) was performed to confirm the degree of purification (Fig. 3). Purification degree according to the purification step of the Tra DNA polymerase derived from E. coli Rosetta (DE3) pLysS / pTRAP is shown in Figure 3 by SDS-PAGE electrophoresis picture. Lane 1 is an ultrasonic crushed sample of E. coli Rosetta (DE3) pLysS / pTRAP cells cultured without IPTG induction, and lane 2 is an ultrasound of E. coli Rosetta (DE3) pLysS / pTRAP cells cultured with IPTG induction The crushed sample, lane 3 is a sample after heat treatment at 80 ° C. for 30 minutes, lane 4 is a sample after HisTrap ^™ HP column HisTrap ^™ HP column chromatography, and lane 5 is a sample after HiTrap ^™ SP HP column chromatography. Purification confirmed that the Tra DNA polymerase protein had a molecular weight of about 90,000 Da, which was similar to 89,788.9 Da, which was estimated from the amino acid sequence of Example 4. Lane M represents a protein low molecular weight marker.

Example 7: Tra  Investigation of enzyme properties in DNA polymerization activity of DNA polymerase

Enzymatic properties of the DNA polymerization activity of the Tra DNA polymerase were investigated based on the method for measuring the DNA polymerization activity mentioned in Example 6 above. When examining the enzyme properties in the DNA polymerization activity, it was carried out in the above method by changing the pH of the reaction mixture or the concentration of each component or the reaction temperature.

The effect of temperature on the DNA polymerization activity of the Tra DNA polymerase purified in Example 6 was investigated at 60-95 ° C., showing the maximum DNA polymerization activity at 75-80 ° C. (FIG. 4). 4 is a graph showing the relative degree of DNA polymerization activity according to the temperature of Tra DNA polymerase.

In order to observe the effect of pH on the enzymatic activity of the Tra DNA polymerase purified in Example 6, 50 mM MOPS buffer was used in the pH 6.0-7.0 range, and 50 mM Tris-HCl buffer was used in the pH 7.0-9.0 range. The enzyme activity was examined. As a result, Tra DNA polymerase showed the maximum DNA polymerization activity at pH 7.5 (FIG. 5). Figure 5 is a graph showing the degree of DNA polymerization activity relative to the pH of the Tra DNA polymerase, in Figure 5 ● shows the results using 50 mM MOPS buffer, ○ ○ results using 50 mM Tris-HCl buffer Indicates.

Figure 6 is a graph showing the relative degree of DNA polymerization activity according to MgCl ₂ concentration of Tra DNA polymerase. As a result of investigating the effect of Mg ²⁺ , a divalent cation, on the DNA polymerization activity of Tra DNA polymerase, the optimum MgCl ₂ concentration was 8 mM.

Figure 7 (NH _{4) 2} SO and ₄ concentration graph showing the degree of DNA polymerization activity relatively according to, Figure 8 is a graph showing the degree of DNA polymerase activity according to the KCl concentration of Tra DNA polymerase with relatively Tra DNA polymerase to be. Investigation of the effects of monovalent cations on the DNA polymerization activity of Tra DNA polymerase showed that the optimal (NH ₄ ) ₂ SO ₄ concentration was 20 mM (FIG. 7) and the optimal KCl concentration was 90 mM (FIG. 8).

After a while the Tra DNA polymerase stored respectively in 94 ℃ and 99 ℃ for 8 hours sampling every hour, examination, the thermal stability by measuring DNA polymerization activity as described above, Tra DNA polymerase is 94 ℃ 50% of the DNA polymerization activity was maintained for about 4 hours (Fig. 9). FIG. 9 is a graph showing the thermostability of the Thermococcus radiotolerance DNA polymerase. In FIG. 9, ● and ○ indicate the results of using Tra DNA polymerase incubated at 99 ° C. and 94 ° C., respectively.

Example 8: Tra  Measurement of 3 '→ 5' Nucleic Acid Terminal Hydrolase Activity of DNA Polymerase

The 3 '→ 5' nucleic acid terminal hydrolase activity of Tra DNA polymerase (3 '→ 5' exonuclease activity) was measured as follows. First, as a substrate for measuring 3 '→ 5' nucleic acid terminal hydrolase activity, pBluescript SK-vector was digested with restriction enzyme Not I, followed by radioisotope [α- ³² P] dCTP and Klenow fragment (Klenow). 3 'end of the pBluescript SK-vector digested with the restriction enzyme was used. Next, the reaction mixture ( Tra DNA polymerase, radioisotope labeled substrate at the 3 ′ end, 50 mM Tris-HCl (pH 7.5), 14 mM MgCl ₂ , 80 mM KCl, 0.01% BSA) was respectively present in dNTP. Alternatively, samples were taken at 5 minutes intervals for 5 minutes at 75 ° C in the absence, quenched on ice, centrifuged with 10% trichloroacetic acid solution, and the supernatant was recovered. The nucleic acid terminal hydrolase activity was measured using a -Carb Liquid Scintillation Analyzer. As a result, it was confirmed that the Tra DNA polymerase had 3 '→ 5' nucleic acid terminal hydrolase activity (Fig. 10). In particular, it can be seen that the enzyme activity is higher in the absence of dNTP. The 3 '→ 5' nucleic acid terminal hydrolase activity of Tra DNA polymerase was estimated in the amino acid sequence comparison and was consistent with the experimental results. This 3 '→ 5' nucleic acid terminal hydrolase activity is also called corrective activity and plays a role of enhancing the accuracy during DNA polymerization. FIG. 10 is a graph showing 3 '→ 5' nucleic acid terminal hydrolase activity of Thermococcus radiotolerance DNA polymerase. In FIG. 10, 3 is a 3 '→ 5' nucleic acid terminal hydrolase in the presence of dNTP and ○ The results of the activity measurements are shown respectively.

Example 9: Tra  Determination of Optimum Conditions for PCR Using DNA Polymerase

In order to examine the possibility of using Tra DNA polymerase in PCR where heat-resistant DNA polymerase is most importantly used, and to determine the optimal reaction buffer composition in PCR using Tra DNA polymerase, PCR was performed as follows. Was performed. 23 ng of lambda phage genomic DNA as a template, respectively, consisting of 1 pmole 5 'and 3' terminal primers, 250 μM dNTPs, based on optimal conditions determined by characterization of the Tra DNA polymerase in Example 8 1 × Tra DNA polymerase reaction buffer and 0.6 U (unit) of purified Tra DNA polymerase (see Example 6 above) were used as the reaction mixture, followed by reaction at 94 ° C. for 3 minutes, followed by 94 at 30 seconds, 55 The procedure was repeated 30 times at 30 ° C. and 2 minutes at 72 ° C. for 30 minutes, followed by reaction at 72 ° C. for 10 minutes, and PCR results were confirmed by 0.8% agarose gel electrophoresis. When determining the optimal composition of the reaction buffer in PCR using Tra DNA polymerase, the pH of the reaction buffer or the concentration of each component were varied.

As a result of examining the effect of pH on PCR using Tra DNA polymerase, the optimum pH of the PCR reaction buffer solution was 8.6 (Fig. 11). Figure 11 shows the results according to the pH in the polymerase chain reaction using Tra DNA polymerase, in Figure 11 M represents a 1 kb DNA ladder (New England BioLabs), each lane represents a pH. PCR amplification size is 2 kb.

As a result of examining the effect of divalent cation Mg ²⁺ on PCR using Tra DNA polymerase, the optimal concentration was 1 mM (FIG. 12). 12 shows the results according to MgCl ₂ concentration in the polymerase chain reaction using Tra DNA polymerase. In FIG. 12, M represents a 1 kb DNA ladder (New England BioLabs), and each lane represents the MgCl ₂ concentration. It is shown.

As a result of investigating the effect of (NH ₄ ) ₂ SO ₄ on PCR using Tra DNA polymerase, the optimal concentration was 2 mM (FIG. 13). Figure 13 shows the results according to (NH ₄ ) ₂ SO ₄ concentration in the polymerase chain reaction using Tra DNA polymerase, in Figure 13 M represents a 1 kb DNA ladder (New England BioLabs), each lane Shows the (NH ₄ ) ₂ SO ₄ concentration.

As a result of examining the effect of KCl on PCR using Tra DNA polymerase, the optimal concentration was 10 mM (FIG. 14). Figure 14 shows the results according to the KCl concentration in the polymerase chain reaction using the Tra DNA polymerase, in Figure 14 M represents the 1 kb DNA ladder (New England BioLabs), each lane represents the KCl concentration. .

As a result of examining the effect of Triton X-100 on PCR using Tra DNA polymerase, the polymerase chain reaction occurred well regardless of the amount when added (Fig. 15). Figure 15 shows the results according to the Triton X-100 concentration in the polymerase chain reaction using Tra DNA polymerase, in Figure 15 M represents a 1 kb DNA ladder (New England BioLabs), each lane is Triton X -100 concentration.

The results show that Tra DNA polymerase can be used for PCR. The optimal reaction buffer composition for PCR using Tra DNA polymerase is 50 mM Tris-HCl (pH 8.6), 1 mM MgCl ₂ , 10 It was determined to add Triton X-100 to mM KCl and 2 mM (NH ₄ ) ₂ SO ₄ .

Example 10: Tra  Determination of PCR Amplifiable Size Using DNA Polymerase

23 ng of lambda phage genomic DNA as a template, respectively, 1 pmole of 5 'and 3' terminal primers, 250 μM dNTPs, 1 × Tra DNA polymerase optimal reaction buffer determined in Example 9 and in Example 6 above According to the length of the target amplification product expected to investigate the amplifiable length of PCR using the purified Tra DNA polymerase, PCR was performed as follows. First, the reaction was performed at 94 ° C. for 3 minutes, and then 30 times at 94 ° C., 30 seconds at 55 ° C., and 30 times at 1 min / kb depending on the expected size of the DNA fragment at 72 ° C. After that, the reaction was finally performed at 72 ° C. for 10 minutes. After completion of the PCR reaction, 0.8% agarose gel electrophoresis confirmed the PCR results. As a result, Tra DNA polymerase can be synthesized up to 5 kb by PCR using the genomic DNA of lambda phage under the optimal reaction buffer solution. It could be confirmed (Fig. 16). FIG. 16 shows the results of polymerase chain reaction using Tra DNA polymerase using lambda phage genomic DNA as a reaction mixture under an optimal reaction buffer solution. In FIG. 16, each lane represents an amplification size. , M represents the 1 kb DNA ladder (New England BioLabs).

Example 12: Tra  Investigation of accuracy in PCR using DNA polymerase

PCR fidelity (PCR fidelity) in PCR using Tra DNA polymerase was performed by slightly modifying the Lundberg method (Lundberg et al ., 1991, Gene 108 (1), 1-6). ). In fact, the experiment was measured by the same method as Lee et al ., The same expression vector pJR2- lacZ (5.7 Kb) and primers were used (Lee, JI et al ., 2009, Enzyme Microb.Technol. 45 (2) , 103-111). First, the 835 bp fragment of the 5 'portion of the expression vector pJR2- lacZ into which the lacZ gene was inserted was amplified by PCR using Tra DNA polymerase. At this time, PCR using a DNA polymerase Tra later by performing the same PCR using DNA polymerase already advanced to the goods in place of Tra DNA polymerase was to compare the results. Next, the PCR amplification products were digested with restriction enzymes Bam HI and Cla I, respectively, and then reinserted into the expression vectors digested with the same restriction enzymes, and then transformed into E. coli by heat shock gene transfer. Antibiotic ampicillin and IPTG and X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) was evenly plated in agar plate medium and incubated for 16 hours at 37 ℃. Subsequently, the agar plate was stored at 4 ° C. for 2 hours, and then the number of blue colonies and white colonies was counted, and mutation frequencies and error rates were calculated based on the numbers of blue colonies and white colonies. Was the result is the same as Table 2 below, Tra PCR accuracy of the PCR using a DNA polymerase is Pfu DNA slightly tteoleojina compared with the polymerase, more than does the Taq DNA polymerase which is mainly used in a conventional PCR higher Tra It was confirmed that DNA polymerase can be used for PCR requiring a high degree of accuracy.

Mutation frequency is expressed as the ratio of white colony number to total colony number (blue + white). The number of template doublings ( d ) is equation 2 ^d = (Amplification amount of PCR product) / (starting target amount added to PCR reaction). The error rate is the equation ER = mf / ( bp X d ), where mf is the mutation frequency, bp is the lacZ target size (835), and d is the template doubling (Lundberg et al ., 1991, Gene 108 (1)). , 1-6)

The heat resistant polymerase of the present invention can be applied to a wide range of fields from genetic engineering research, early diagnosis of viral and cancer genes, genetic disease diagnosis, GMO and forensic research.

Institute of Agricultural Biotechnology KACC95102 20100223

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> THERMOSTABLE DNA POLYMERASE DERIVED FROM THERMCOCCUS          RADIOTOLERANS AND ITS USE <130> P10-0062 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T-111N Primer <400> 1 gartacgaca tacccttygc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> T-CR Primer <400> 2 aacctggttc tcratktagt a 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tra-N-1 Primer <400> 3 atccttctcc ttcacgacct 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tra-N-2 Primer <400> 4 tttatcatct ccttctcggt gg 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tra-C-1 Primer <400> 5 gagaagctgg tcatccacg 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tra-C-2 Primer <400> 6 gggagctaaa ggattacaag g 21 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TraN Primer <400> 7 nnnnnncata tgatcctcga taccgactac atcaca 36 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TraC Primer <400> 8 nnnnnnctcg agcttcttcc ccttcggctt aagc 34 <210> 9 <211> 2328 <212> DNA <213> Thermococcus radiotolerans <400> 9 atgatcctcg ataccgacta catcacagag gatggaaagc ccgtcataag gatattcaag 60 aaggaaaacg gcgagttcaa gatagaatat gacaggaact ttgagcctta catctacgct 120 ctcctgaagg acgattctgc catagaggac gtgaagaaga taaccgccga gcgccacggc 180 aaggtcgtta aggtgaagcg cgccgagaag attaagaaga agttcctcgg caggcccctg 240 gaggtctggg ttctctactt cacccatccc caggacgtcc cggcgataag ggacaggata 300 cgcgcccatc ctgccgtcgt tgacatctac gagtacgaca tacccttcgc caagcgctac 360 ctcataggca agggtctgat tccgatgaaa ggcgacgagg agctcaagct actctccttc 420 gacatcgaga ctctctacca cgagggcgag gagttcggaa ccgggccgat tctgatgatt 480 agctacgccg acgaggacga ggcgagggtc ataacgtgga agaaagtcga cctcccctac 540 gttgacgtcg tctccaccga gaaggagatg ataaagcgct tcctcaaggt cgtgaaggag 600 aaggatccgg atgtcctcat aacctacaac ggcgacaact tcgacttcgc ctacctcaag 660 aagcggtgcg agaagctcgg gataaagttc acccttggga gagatgggag cgagcccaaa 720 atccagcgca tgggagacag gtttgcagtc gaggtgaagg gcagaataca cttcgacctc 780 taccccgtca taaggaggac gataaacctt ccaacctaca cccttgaggc ggtttacgag 840 gcagtctttg gaaagccgaa ggagaaggtc taccccgagg agataaccac cgcctgggag 900 agcggcgagg ggcttgagag ggtcgcgcac tactccatgg aggacgcgaa ggtcaccttt 960 gaactcggca gggagttctt cccgatggag gcccagctct caaggctgat aggccagagc 1020 ctctgggacg tctcccgctc aagcaccggc aacctcgtgg agtggttcct cctgaggaag 1080 gcctacgaga ggaacgagct cgccccaaac aagcccgacg agcgggaact tgcgagacgg 1140 cgcgggggct acgcgggtgg atacgttaag gagccggagc ggggactgtg ggacaacata 1200 gtctacctgg acttccgcag cctgtatccc tcgatcatca tcacccacaa cgtctcgccg 1260 gacactctaa accgcgaggg ctgtaaagag tacgacaccg ccccacaggt cggccacagg 1320 ttctgtaagg acgtcccggg cttcatcccg agccttttgg gtgctcttct cgacgagagg 1380 cagaagataa agaagaggat gaaggccagc atagacccgc tggagaagaa actcctcgat 1440 tacaggcaga aggccattaa aatcctcgcc aacagcttct acggctacta cggctacgcc 1500 cggtcccgct ggtactgcaa ggagtgcgcc gagagcgtta cggcatgggg cagggactac 1560 atcgagatgg tgatccgcga gctggaggag aagtttggct tcaaggtgct ctatgctgac 1620 accgacggtc tccatgccac tattcctgga gcggacgccg agacggtcaa gaagaaggcg 1680 aaggagttct taaaatacat caaccccaaa ctcccgggac ttctcgaact cgaatacgag 1740 ggcttctacg tcagggggtt cttcgtgacg aagaagaagt acgcggtcat agacgaggag 1800 ggcaagataa ccacgcgggg gcttgagatc gtccggcgcg actggagcga gatagccaag 1860 gagacccagg cgagggtttt ggaggccata ctgaggcacg gtgacgtcga ggaggccgtc 1920 aggatagtca aggacgttac cgagaagctg agcaggtacg aggttccgcc ggagaagctg 1980 gtcatccacg agcagattac cagggagcta aaggattaca aggccaccgg cccgcacgtg 2040 gccatagcga agcgcctcgc ggcgagggga ataaaggtac gccccggcac ggtgataagc 2100 tacatcgtcc tcaagggctc cggaaggata ggcgacaggg cggttccctt cgacgagttc 2160 gacccgacga agcacaagta cgacgcggaa tactacatcg agaaccaggt cctgccggcc 2220 gtggagagga ttctgaaggc cttcggctac aagaaggagg agctcaggta tcagaaaacg 2280 aggcaggtcg gcctcggggc gtggcttaag ccgaagggga agaagtga 2328 <210> 10 <211> 775 <212> PRT <213> Thermococcus radiotolerans <400> 10 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 Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile          35 40 45 Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Lys      50 55 60 Val Lys Arg Ala Glu Lys Ile Lys Lys Lys Phe Leu Gly Arg Pro Leu  65 70 75 80 Glu Val Trp Val Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile                  85 90 95 Arg Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr             100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Gly Lys Gly Leu Ile Pro         115 120 125 Met Lys Gly Asp Glu Glu Leu Lys Leu Leu Ser Phe Asp Ile Glu Thr     130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asp Glu Ala Arg Val Ile Thr Trp Lys Lys Val                 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys             180 185 190 Arg Phe Leu Lys 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 Lys Phe Thr 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 Lys Pro Lys Glu         275 280 285 Lys Val Tyr Pro Glu Glu Ile Thr Thr Ala Trp Glu Ser Gly Glu Gly     290 295 300 Leu Glu Arg Val Ala His Tyr Ser Met Glu Asp Ala Lys Val Thr Phe 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe 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 Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr     370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His                 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp             420 425 430 Thr Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Val Pro Gly Phe         435 440 445 Ile Pro Ser Leu Leu Gly Ala Leu Leu Asp Glu Arg Gln Lys Ile Lys     450 455 460 Lys Arg Met Lys Ala Ser Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Lys Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr                 485 490 495 Tyr Gly Tyr Ala Arg Ser Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser             500 505 510 Val Thr Ala Trp Gly Arg Asp Tyr Ile Glu Met Val Ile Arg Glu Leu         515 520 525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu     530 535 540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu                 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys             580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu         595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala     610 615 620 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Asp Val Thr Glu Lys Leu Ser Arg Tyr Glu Val Pro                 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp             660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala         675 680 685 Arg Gly Ile Lys Val Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu     690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Val 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 Lys Ala Phe Gly Tyr Lys Lys             740 745 750 Glu Glu Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp         755 760 765 Leu Lys Pro Lys Gly Lys Lys     770 775

Claims (12)

A heat resistant Tra DNA polymerase derived from Thermococcus radiotolerance having the amino acid sequence of SEQ ID NO: 10.
The method of claim 1, wherein the Tra DNA polymerase is pH 7.5, a temperature of 75 ~ 80 ℃, MgCl ₂ of 8 mM Concentration, 20 mM (NH ₄ ) ₂ SO ₄ Tra DNA polymerase characterized in that it has the optimal activity at the concentration and KCl concentration of 90 mM.
A nucleic acid molecule encoding Tra DNA polymerase of claim 1.
The nucleic acid molecule of claim 3, wherein the nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO. 9.
A recombinant vector comprising a nucleic acid molecule encoding the heat resistant Tra DNA polymerase of claim 1.
6. The vector of claim 5, wherein said vector is pTRAP.
A host cell transformed with the vector of claim 5.
The host cell according to claim 7, wherein said host cell is Escherichia coli Rosetta (DE3) pLysS / pTRAP (accession number KACC95102P) transformed with a vector which is pTRAP.
A kit for nucleic acid amplification reaction comprising the Tra DNA polymerase of claim 1.
(i) preparing a vector expressing the Tra DNA polymerase of claim 1;
(ii) transforming the vector into a host cell;
(iii) culturing the transformed host cell; And
(iv) recovering the protein from the transformed host cell;
Including, Tra DNA polymerase production method.
Method of performing polymerase chain reaction (PCR) using the Tra DNA polymerase of claim 1.
The reaction of claim 11, wherein the PCR consists of 50 mM Tris-HCl (pH 8.6), 1 mM MgCl ₂ , 10 mM KCl, 2 mM (NH ₄ ) ₂ SO _4, and 0.1-0.3% of Triton X-100 Method for performing polymerase chain reaction (PCR), characterized in that the buffer solution.

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