CA2267101C - Thermostable nucleic acid polymerase from thermococcus gorgonarius - Google Patents

Abstract

A purified thermostable enzyme is derived from the thermophilic archaebacterium Thermococcus gorgonarius. The enzyme can be native or recombinant, retains approximately 90 % of its activity after incubation for two hours at 95 .degree.C in the presence of stabilizing agen ts and possesses 3'-5' proofreading exonuclease activity. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR) .

Description

Thermostable nucleic acid polymerase from Tlrermococcus gorgonarius The present invention relates to an extremely thermostable enzyme. More specifically, it relates to a thermostable DNA polymerase obtainable from Thermococcus gorgonarius.

DNA polymerases are a family of enzymes which are in particular involved in DNA replication and repair. Extensive research has been conducted on the isolation of DNA
polymerases from mesophilic microorganisms such as E.coli (see, for example, Bessman et al.
(1957) J. Biol.
Chem. 223:171-177, and Buttin and Kornberg, (1966)J. Biol. Chem. 241:5419-5427).

Research has also been conducted on the isolation and purification of DNA
polymerases from thermophiles, such as Thermus aquaticus (Chien, A., (1976) et al. J.
Bacteriol. 127:1550-1557) Further, the isolation and purification of a DNA polymerase with a temperature opti-mum of 80 C from Thermus aquaticus YT1 strain has been described (EP 0 258 017 and US
4, 889, 819).

Research has indicated that while the Taq DNA polymerase has a 5'-3' polymerase-dependent exonuclease function, the Taq DNA polymerase does not possess a 3'-5' proofreading exonuclease function (Lawyer, F.C. et al., (1989) J. Biol. Chem., 264:6427-6437. Bernad A., et al. (1989) Ce1159:219). As a result, Taq DNA polymerase is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on where in the PCR cycle that error occurs (e.g., , in an early replication cycle), the entire DNA amplified could contain the erroneously incor-porated base, thus, giving rise to a mutated gene product. Furthermore, research has indicated that Taq DNA polymerase has a thermal stability of not more than several minutes at 100 C.

The 3'-5' exonuclease activity is generally considered to be desirable, because misincorporated or unmatched bases of the synthesized nucleic acid sequence are eliminated by this activity.
Therefore, the fidelity of PCR utilizing a polymerase with 3'-5' exonuclease activity is increased. Such an enzyme is, e.g. the DNA polymerase from Pyrococcus furiosus (Lundberg et al., (1991) Gene., 108; p. 1-6).

Other more recent investigation focusses on the isolation and purification of DNA polymerases from archaebacteria such as Thermococcus sp. (EP 0 455 430), in particular a purified DNA
polymerase obtainable from Thermococcus litoralis is described. Also the recombinant lo preparation and the gene encoding for this enzyme is known in the art (EP 0 547 920).

In EP 0 455 430 is also described a DNA polymerase from Pyrococcus sp. and the gene there-of which also contains introns to be removed for expression of the functional enzyme in E.coli.
In EP 0 701 000 A and in Proc. Natl. Acad. Sci. USA, Vol. 93, No. 11, (1996) pg. 5281-5285 a thermostable DNA polymerase 9 N7 is described which exhibits a very strong 3'-5'-exonuclease activity. However, it has been observed that the 9 N7 polymerase exhibits a tendency to degrade single stranded DNA (primer). Therefore, the exonuclease activity has been modulated and a mutant 9 Nm polymerase has been obtained which is more useful for a number of applications as the native enzyme. However, when using a 9 Nm polymerase for PCR (see figure 6) a primer-template independent DNA-synthesis seems to occur (as can be deducted from the observed highmolecular smear in the gel (figure 6)) instead of the occurence of defined PCR products when using e.g. Taq-Polymerase. Therefore, neither the native nor the exonuclease modulated 9 N-7 polymerase can be succesfully used in PCR.

In WO 92/03556 a thermostable DNA polymerase obtainable from the eubacterium Thermotoga maritima is described which also exhibits proofreading activity.
However, in comparison to other DNA PolYmerases e.g. Pfu polymerase or Tgo polymerase, the Tma polymermase exhibits a relatively low fidelity (Flaman, J.M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C., Ishioka, C., Friend, S.J. and Iggo, R. (1994) Nucl. Acids. Res. 22 3259-3260; Cline, I., Braman, J C. and Hogrefe, H.H. (1996) Nucl. Acids. Res.
24, 3546-' 3551).

The DNA polymerase obtainable from Pyrococcus furiosis (Pfu) is described in and exhibits a relatively high fidelity.

Accordingly, there is a desire in the art to obtain and produce a purified, highly thermostable DNA polymerase with 3'-5' proofreading exonuclease activity which exhibits a high fidelity and is suitable to improve the PCR process.

The present invention meets this need by providing a DNA polymerase from Thermococcus 1o gorgonarius (Tgo) together with the related DNA and amino acid sequence information, re-combinant expression vector and a purification protocol for said DNA
polymerase. The DNA
polymerase according to the present invention exhibits more than a two fold greater replication fidelity than known DNA polymerases, e.g. obtainable from Pyrococcusfuriosus.
A further advantage is that the 3'-5'exonuclease activity found in T. gorgonarius polymerase can also decrease non-specific background amplification in PCR by degrading defrayed ends of primers bound to unspecific sequences thereby destabilizing the binding of the primer because of decreasing the length of the helix. Tgo polymerase is thus unexpectedly superior to known DNA polymerases in amplification protocols requiring high fidelity DNA
synthesis (see figure 8-10). Another advantageous property of the DNA polymerase of Thermococcus gorgonarius is the fact, that the gene does not contain intervening sequences which would have to be removed to accomplish expression in E. coli.

The thermostable DNA polymerase enzyme obtainable from T. gorgonarius catalyzes the template directed polymerization of DNA, has an apparent molecular weight of about 92,000-96,000 daltons and retains 90 % of its activity after incubation for two hours at 95 C in the presence of a stabilizer like a non-ionic detergent as, e. g., 0.01 % ThesitTM
(Dodecylpoly(ethylenglycolether)õ) or 0.01 % Nonidet P40TM
(Ethylphenolpoly(ethylenglycolether).).

Moreover, DNA encoding the 92,000-96,000 daltons thermostable DNA polymerase obtain-able from Tyiermococcus gorgonarius has been isolated and which allows to obtain the ther-mostable enzyme of the present invention by expression in E. coli. The DNA
sequence of the DNA polymerase obtainable from Thermococcus gorgonarius is shown in SEQ IDNo.
6. The recombinant Thermococcus gorgonarius DNA polymerase also possesses 3'-5' exonuclease (proofreading) activity. Furthermore the gene encoding DNA polymerase from Thermococcus gorgorarius does not contain intervening sequences.

Thermococcus gorgonarius was isolated from E. A. Bonch-Osmolovskaya and V. A.
Svet-lichny, Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia. Thermo-coccus gorgonarius is a new strain, isolated from a thermal vent in New Zealand. This strain does not show DNA-DNA homology with T. celer, T. litoralis or T. stetteri.

The preferred thermostable enzyme herein is a DNA polymerase obtainable from Thermococ-cus gorgonarius DSM 8976 (deposited on the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig). This organism is an extremely thermophilic, sulfur metabolizing, archaebacterium, with a growth range between 55 C and 98 C.

A preferred method for isolation and purification of the enzyme is accomplished - affter all growth - using the multi-step process as follows:

First, the frozen cells are thawed, suspended in a suitable buffer such as buffer A (40 mM Tris-HCI buffer, pH 7.4; 0.1 mM EDTA, 7 mM 2-mercaptoethanol; 1 mM Pefabloc SCTm (4-(2-Aminoethyl)-benzolsulfonylfluorid)), disnipted by high pressure at 1.200 bar.
KCI was added to the extract to a final concentration of 400 mM and the solution cleared by centrifugation.
The supernatant is then passed through a Heparin Sepharose C16B column (Pharmacia), which has a strong affinity for nucleic acid binding proteins. The nucleic acids present in the supernatant solution of Thermococcus gorgonarius and many of the other proteins pass through the column and are removed by washing the column with two column volumes of buffer A. After washing, the enzyme is eluted with a linear gradient from 0 to I M NaCI in 3o buffer A. The peak DNA polymerase activity is dialyzed and applied to a DEAE Sephacer column (Phannacia). The column is washed with buffer A and the enzyme activity eluted with a linear gradient from 0 to 1 M NaCI in buffer A. The peak DNA polymerase activity is *Trade-mark dialyzed and applied to a Cellulose Phosphate column (Whatman). The enzyme is again eluted with a linear gradient such as 0 to I M NaCI in buffer A. The enzyme is about 40 % pure at this stage.

The apparent molecular weight of the DNA polymerase obtainable from Thermococcus gor-gonarius is between about 92,000 to 96,000 daltons when compared with DNA
polymerases of known molecular weight, such as E.coli DNA polymerase I and Thermus thermophilus DNA polymerase. It should be understood, however, that as a protein from an extreme ther-mophile, Thermococcus gorgonarius DNA polymerase may migrate during electrophoresis at lo an aberrant relative molecular weight due to failure to completely denature or other intrinsic properties. The exact molecular weight of the thermostable enzyme of the present invention may be determined from the coding sequence of the Thermococcus gorgonarius DNA
poly-merase gene. The molecular weight of the DNA polymerase may be determined by any tech-nique , for example, by in situ analysis after separation by SDS-polyacrylamide gel electro-t5 phoresis (SDS-PAGE) as described in Spanos, A. and Hiibscher,U., (1983) Methods in En-zymology 91:263-277.

Polymerase activity is either measured by the incorporation of radioactively labeled deoxy-nucleotides into DNAse-treated, or activated DNA, following subsequent separation of the 20 unincorporated deoxynucleotides from the DNA substrate. Polymerase activity is proportional to the amount of radioactivity in the acid-insoluble fraction comprising the DNA, as described by Lehman, I.R., et al. (1958) J. Biol. Chem. 233:163, or by incorporation of digoxigenin-labeled dUTP and determination of incorporated Digoxigenin-dUTP using chemolununescence according to the method described in HtSltke, H.-J.; Sagner, G; Kessler, C.;
and Schmitz, G., 25 (1992)Biotechniques 12:104 -113.

The DNA polymerase of the present invention has a very high thermal stability at 95 C. It retains approximately 90 percent of its activity after incubation at 95 C for 120 minutes in the presence of stabilizer. The thermal stability is determined by preincubating the enzyme at the 30 temperature of interest in the presence of all assay components (buffer, MgClz, deoxynucleo-tides, activated DNA and a stabilizer like 0.01 % ThesitTm and 0.01 % Nonidet P40Tm) except the single radioactively-labeled deoxynucleotide. At predetermined time intervals, ranging from *Trade-mark 1-120 minutes, small aliquots are removed, and assayed for polymerase activity using one of the methods described above.

The thermostable enzyme of this invention may also be produced by recombinant DNA tech-niques, as the gene encoding this enzyme has been cloned from Thermococcus gorgonarius genomic DNA. The complete coding sequence for the Thermococcus gorgonarius DNA
poly-merase can be derived from the plasmid pBTac2Tgo on an approximately 2.3 kB
EcoRI/PstI
restriction fragment.

The production of a recombinant form of Thermococcus gorgonarius DNA
polymerase gen-erally includes the following steps: DNA is isolated which codes for the active form of the po-lymerase. This can be accomplished e.g. by screening of a DNA library derived from the ge-nomic DNA of T. gorgonarius using the DNA sequence described in SEQ ID No.: 1 as a probe. Clones containing DNA fragments of T. gorgonarius hybridizing to the probe are iso-lated and the nucleotide sequence of the plasmid inserts determined. Complete isolation of the coding region and the flanking sequences of the DNA polymerase gene can be performed by restriction fragmentation of the T. gorgonarius DNA with another restriction enzyme as in the first round of screening and by inverse PCR (Innis et al., (1990) PCR
Protocols; Academic Press, Inc., p. 219-227). This can be accomplished with synthesized oligonucleotide primers binding at the outer DNA sequences of the gene part but in opposite orientation e.g. with the SEQ ID Nos. 2 and 3. As template T. gorgonarius DNA is used which is cleaved by restriction digestion and circularized by contacting with T4 DNA ligase. To isolate the coding region of the whole polymerase gene, another PCR is performed using primers as shown in SEQ ID
Nos. 4 and 5 to amplify the complete DNA polymerase gene directly from genomic DNA and introducing ends compatible with the linearized expression vector.
SEQ ID NO. 1:
5'-ATG ATH YTN GAY ACN GAY TAY ATH AC-3' SEQIDNO.2:
5'-GGC CTA CGA GAG GAA CGA ACT GGC-3' SEQ ID NO. 3 :
5'-GGC GTA GAT GTA GGG CTC-3' SEQID NO.4:
5'-GAG CTG GTC GAA TTC ATG ATC CTG GAC GCT GAC TAC ATC ACC -3' SEQ ID NO. 5:
5'- AGC CTG CAG TCA TGT CTT AGG TTT TAG CCA CGC-3' The gene is operably linked to appropriate control sequences for expression in either pro-karyotic or eucaryotic host/vector systems. The vector preferably encodes all functions re-quired for transformation and maintenance in a suitable host, and may encode selectable markers and/or control sequences for polymerase expression. Active recombinant thermostable polymerase can be produced by transformed host cultures either continuously or after induction of expression. Active thermostable polymerase can be recovered either from host cells or from the culture media if the protein is secreted through the ceil membrane.

It is also preferable that Thermococcus gorgonarius thermostable polymerase expression is tightly controlled in E.coli during cloning and expression. Vectors useful in practising the present invention should provide varying degrees of controlled expression of Thermococcus gorgonarius polymerase by providing some or all of the following control features: (1) pro-moters or sites of initiation of transcription, either directly adjacent to the start of the poly-merase gene or as fusion proteins, (2) operators which could be used to turn gene expression on or off, (3) ribosome binding sites for improved translation, and (4) transcription or trans-lation termination sites for improved stability. Appropriate vectors used in cloning and ex-pression of Thermococcus gorgonarius polymerase include, for example, phage and plasmids.
Example of phage include lambda gtl l(Promega), lambda Dash (Stratagene) lambda ZapII
(Stratagene). Examples of plasmids include pBR322, pBTac2 (Boehringer Mannheim), pBluescript (Stratagene), pSP73 (Promega), pET3A (Rosenberg, A.H. et al., (1987) Gene 56:125-135) and pET11C (Studier-J. W. et al. (1990) Methods in Enzymology, 185:60-89).
According to the present invention the use of a plasmid has shown to be advantageously, particularly pBTac2. The Plasmid pBTac2 carrying the Thermococcus gorgonarius DNA
polymerase gene is then designated pBTac2Tgo.

Standard protocols exist for transformation, phage infection and cell culture (Maniatis, et al.
(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press).
Of the numerous E.coli strains which can be used for plasmid transformation, the preferred strains include JM110 (ATCC 47013), LE392 pUBS 520 (Maniatis et al. supra;
Brinkmann et al., (1989) Gene 85:109-114;), JM101 (ATCC No. 33876), XLI (Stratagene), and RRl (ATCC no. 31343), and BL21 (DE3) plysS (Studier, F. W. et al., (1990)Methods inEnzymo-lo logy, supra). According to the present invention the use of the E. coli strain LE392 pUBS 520 has shown to be advantageously. The E. coli strain LE392 pUBS 520 transformed with the plasmid pBTac2Tgo is then designated E. coli pBtac2Tgo (DSM No. 11328). E.coli strain XL1 Blue (Stratagene) is among the strains that can be used for lambda phage, and Y1089 can be used for lambda gt 11 lysogeny. The transformed cells are preferably grown at 37 C and expression of the cloned gene is induced with IPTG (Isopropyl-B-D-thiogalactopyranosid).
Isolation of the recombinant DNA polymerase can be performed by standard techniques. Se-paration and purification of the DNA polymerase from the E. coli extract can be performed by standard methods. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific interaction such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography and methods utilizing a difference in isoelectric point such as isoelectric focussing electrophoresis.

One preferred method for isolating and purification of the recombinant enzyme is accomplished using the multi-stage process as follows.

The frozen cells are thawed and suspended in a suitable buffer such as buffer A (40 mM Tris-HCI, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol) in the presence of Pefabloc SC in a final concentration of 1 mM, lysed by the addition of lysozyme (200 g/ml) under stirring for 30 min. at 4 C. Sodium deoxycholate is added to a final concentration of 0.05 %. After an incubation for another 30 min. KCI is added to a final concentration of 0.75 M. The suspension is incubated at 72 C for 15 min. and centrifuged. The supernatant is adjusted to 25 %
saturation with (Nfi4)2SO4 and then applied to a hydrophobic interaction chromatography column such as TSK Butyl Toyopearl 650C (TosoHaas). Most of the nucleic acids and unspe-cific proteins are in the flow through and wash of the column while the polymerase is eluting at the end of a decreasing gradient from 30 % to 0 % saturation of (NH4)2SO4 in buffer A (with additional 10 % glycerol). The polymerase-active fractions are pooled, dialyzed against buffer A containing 10 % glycerol, adjusted to 10 mM MgC12 and applied to a high affinity column for nucleotide-binding enzymes such as Fractogel TSK AF-Blue column (Merck).
The column is washed with buffer A containing 10 % glycerol and the polymerase protein is eluted with a linear gradient of 0 to 3 M NaCI in buffer A (with additional 10 % glycerol).
The polymerase fractions are pooled and dialyzed against the storage buffer B (20 mM Tris-HCI, pH 8.0;
0.1 mM EDTA; 10 mM 2-mercaptoethanol; 50 mM (NH4)2SO4,; 50 % glycerol) and stored at -20 C.

The Thermococcus gorgonarius DNA polymerase of the present invention may be used for any purpose in which such an enzyme is necessary or desirable. For example, in recombinant DNA
technology including, second-strand cDNA synthesis in cDNA cloning and DNA
sequencing.
See Maniatis, et al., supra.

The Thermococcus gorgonarius DNA polymerase of the present invention may be modified chemically or genetically - site directed or random - to inactivate the 3'-5' exonuclease func-tion and used for any purpose in which such a modified enzyme is desirable, e.g., DNA
sequencing or DNA labelling.

In addition, the Thermococcus gorgonarius DNA polymerase of the present invention may also be used to amplify DNA, e.g., by the procedure disclosed in EP 0 200 362, EP 0 201 184 and EP 0 693 078.

*Trade-mark The following examples are given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that the examples are illustrative, and that the invention is not be considered as restricted except as indicated in the appended claims.

5 Brief description of the drawings Figure 1:
SDS polyacrylamide gel analysis of partially purified and purified recombinant DNA poly-merase from T. gorgonarius.

10 Lane 1: 1 l of crude extrat.
Lane 2: 5 l of polymerase fraction obtained after the first chromatography step (TSK Butyl Toyopearl 650C) Lane 3: 5 fcl of fraction obtained after second chromatography step (Fractogel Blue).
Lane 4: 10 l of fraction obtained after second chromatography step (Fractogel Blue).
Lane 5: 10 units of DNA polymerase from Thermococcus gorgonarius.
Lane 6: Molecular weight markers Lane 7: 10 units of DNA polymerase from Pyrococcus woesei.
Lane 8: Molecular weight markers Figure 2:
In situ activity analysis of native and recombinant Thermococcus gorgonarius DNA poly-merase in comparison to Klenow fragment, Pol I of E.coli and Thermus thermophilus DNA
polymerase as described in Example I. Native and recombinant Thermococcus gorgonarius DNA polymerase have the same electrophoretic mobility.

Figure. 3:
DNA sequence (SEQ ID NO. 6) and the deduced amino acid sequence (SEQ ID NO. 7) of the gene encoding the DNA polymerase from Thermococcus gorgonarius.

Figure. 4:
-Determination of heat stability of T.gorgonarius polymerase as described in Example V.

Figure. 5:
Analysis of 3'-5' exonuclease activity as described in Example VI.
Various amounts (units are indicated in the figure) of Tgorgonarius DNA
polymerase were incubated with DNA fragments in the absence (- dNTPs) and presence (+dNTPs) of desoxy-nucleotide triphosphates. ctrll and 2: Control reactions without DNA
polymerase.

The 3'-5'exonuclease activity is dependent on the presence or absence of dNTPs.
Figure 6:
Comparison of various thermostabil DNA polymerases (Vent exo-, 9 Nm, Taq) with respect to the incorporation of Cy5-dUTP. The reaction mixtures contained 2mM MgC12, 30nM
of each primer, ing DNA and 200 M deoxynucleotide. Buffer conditions were used as rcomrnended by the supplier of the enzymes. Plasmid DNA has been used in which the O-Actin-gene of the mouse has been inserted. TTP has been partly replaced by Cy5-dUTP. The reaction mixture contained Cy5-dUTP:TTP in the following ratios: 65:35 (lane 1), 50:50 (lane 2), 35:65 (lane 3), 15:85 (lane 4). As a control the above described reaction has been performed without modified nucleosidetriphosphates (lane 5).

Figure 7:
Use of Tgo-polymerase in PCR applying different amounts of polymerase as well as different MgC12-concentrations.

Figure 8:
Use of Tgo-polymerase in PCR applying different amounts of TgO polymerase;
comparison of TgO and Pfu polymerase.

Figure 9:

Amplification of A.-DNA; Comparison of TgO and Pfu polymerase.
Figure 10:
Comparison of TgO and Pfu polymerase; investigation of the influence of the concentration on the PCR; 2,5 U polymerase has been used in every assay.

Example I

Purification of a thermostable DNA polymerase from Thermococcus gorgonarius Thermococcus gorgonarius (DSM 8976) was grown in the medium which was prepared as follows: A mineral solution containing KCI, 325 mg/l; MgC12=2 H20, 2.75 mg/1;
MgS04=7 H20, 3.45 mg/l; NHaCI, 0.25 mg/1; CaC12-2 H20, 0.15 mg/I; KH2PO4, 0.15 mg/1;
NaCI, 18 g/1; NaHCO3, 1 g/l; trace elements, 4 ml/1(Balch et al., (1979) Microbiol. Rev.
43:260), vitamins, 4 ml/1(Balch supra,); Rezazurin, 1 mg/l; 0.4 ml/1 of a 0.2 % solution of Fe(NH2)2(SO4)2=7 H20 was boiled and cooled. The following components were added to the final concentrations as indicated: Peptone, 5 g/l; yeast extract, 1 g/1; Na2S-9 H20, 250 mg/i and cystein-HCI, 250 mg/l, the pH was adjusted to 6.2 - 6.4. The incubation temperature was 88 C. The cells were cooled to room temperature, collected by centrifugation and stored at -70 C. 6 g of cells were suspended in 12 ml of buffer A (40 mM Tris-HCI, pH
7.5; 0.1 mM
EDTA; 7 mM 2-mercaptoethanol) containing 1 mM Pefabloc SCTM and disrupted by pressure at 1200 bar. KCI was added to a final concentration of 400 mM, dissolved and the solution was centrifugated at 48,200 x g for 30 minutes at 4 C. The supernatant was passed through a 31 ml Heparin Sepharose Cl 6B column (Pharmacia). The column was then washed with 62 ml of buffer B (buffer A containing 10 % glycerol). The column was eluted with a 310 ml linear gradient from 0 to 1.0 M NaCI in buffer B. The DNA polymerase eluted between 30 and 45 mS/cm. The fractions containing DNA polymerase activity were pooled and dialyzed twice against 600 ml buffer B respectively and applied to a 18 ml DEAE Sephacel column (Pharmacia). The column was washed with two column volumes of buffer B, and eluted with a 160 ml linear gradient of 0 to 0.9 M NaC1 in buffer B. The polymerase activity eluted between 4 and 14 mS/cm. Fractions were pooled, dialyzed twice against buffer B (200 ml each time) and applied to a 4 ml Cellulose Phosphate P 11 column (Whatman). The column was washed with 8 ml of buffer B and the activity eluted with a 40 ml linear gradient of 0 to 1 M NaCl. The active fractions which eluted between 13 and 32 mS/cm were pooled, dialyzed against buffer B
containing (NH4)ZSO4 to 25% saturation and applied to a 4 ml TSK Butyl Toyopearl 650C
column (TosoHaas). The column was washed with 8 m125% (NH4)ZSO4-saturated buffer B
and eluted with 40 mi of a decreasing gradient of 25 % to 0%(NH4)2SO4-saturated buffer B.

W0 98/14590 CA 022 67io i 2 0 0 7- 05- o i PCT/EP97/05393 The polymerase eluted between 74 and 31 mS/cm, the pool was dialyzed against buffer B and applied to a 4 ml FractogetTSK AF-Orange column (Merck). The column was washed with 8 ml of buffer B and eluted with a 80 ml linear gradient of 0 to 2.0 M NaCI.
The active fractions (between' 76 and 104 mS/cm) were pooled and dialyzed against storage buffer C
(20 mM Tris-HCI, pH 8.0; 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 50 mM
(NH4)2S04;
50 % glycerol) and stored at -20 C. At this step the DNA polymerase was approximately 40 %
pure.

The molecular weight of the isolated DNA polymerase was determined by "activity gel analy-lo sis" according to a modified version of the method described by Spanos,A.
and Hubscher, U, supra. The DNA polymerase sample was separated on a SDS polyacrylamide gel containing activated calf thymus DNA. The polymerase was renaturated in the gel in 50 mM
Tris-HCI, pH 8.8; 1 mM EDTA; 3 mM 2-mercaptoethanol; 50 mM KCI; 5 % glycerol. Labeling of the DNA with Dig-dUTP (Boehringer Mannheim) was performed in 10 ml of the following buffer:
15. 50 mM Tris-HCI, pH 8.8; 7 mM MgCI2; 3 mM 2-mercaptoethanol; 100 mM KCI; 12 M
dGTP; 12 M dCTP; 12 M dATP; 6 pM dTTP; 6 M Dig-dUTP. The gel was first incubated under shaking at room temperature (30 min.) and then slowly warmed up to 72 C by temperature increments of 5 C. At each temperature interval DNA synthesis is allowed to proceed for 30 min., in order to detect also polymerase activity of inesophile control poly-20 merases. Then the gel was washed and the DNA was blotted on a nylon membrane (Boehringer Mannheim), UV crosslinked. The digoxygenin labeled DNA was detected using the protocol described in the "Boehringer Mannheim's Dig System User's Guide for Filter Hybridization". As molecular weight markers Eco1i DNA polymerase I, Thermus thermo-philus DNA polymerase and Kienow fragment were analyzed on the same gel. The DNA
25 polymerase isolated from Thermococcus gorgonarius has an apparent molecular weight in the range of 92,000 to 96,000 daltons as shown in figure 2.

*Trade-mark Example 2 Cloning of the T. gorgonarius DNA polymerase 1. DNA from T. gorgonarius was isolated and purified by the method described in Lawyer, F. C. et al. (1989) J. Biol. Chem. 264:6427-6437.

2. The DNA was restricted with BamHl, separated on an low melting point agarose gel, denatured and blotted onto a nylon membrane. The blot was probed with a Digoxigenin labeled oligonucleotide of the sequence shown in SEQ ID No. 1. A signal could be detected and the region corresponding to the hybridization signal was cut out of the gel.
The gel piece was melted and the DNA isolated by ethanol precipitation.

3. The DNA fragments isolated were ligated into a plasmid vector, hybridized with SEQ ID.
No. 1. The plasmid DNA from positive clones were isolated and the nucleic acid se-quences of the insert determined. The DNA sequences obtained were then compared with sequences of DNA polymerase genes published in Braithwaite, D. K. and Ito J.(1993), Nucl. Acids Res. 21:787-802.

4. From the sequence of one of the cloned fragments which showed a high degree of homo-logy to the B type DNA polymerases described in the publication of Braithwaite et al., supra, the primers SEQ ID No. 2 and 3 were designed. These primers bind close to the ends of the cloned DNA fragment in opposit orientations to allow amplification of the flanking genomic sequences in circularized template DNA.
5. With these primers "inverse PCR" was performed according of the method of Innis, M.
A., supra, with the DNA from step 1 which was cleaved with EcoRI and circularized with T4 DNA ligase. With this technique two fragments were generated and the sequences determined. An open reading frame could be identified. The deduced aminoacid sequence showed strong homologies to known DNA polymerases of the pol B type.

6. From the sequence of the DNA fragment identified in step 5 new primers were designed, the sequences are shown in SEQ ID No. 4 and 5 which were complementary to the start and the end of the reading frame. The primers contained additional non complementary 5' sequences with restriction sites to introduce clonable ends into the PCR
product in such an 5 orientation that the product would be under transcriptional and translational control of the promoter.

7. The PCR product was cleaved with EcoRI and Pstl, purified and ligated into the vector pBTac2. This clone, expressing the DNA polymerase from Thermococcus gorgonarius 10 was designated pBTac2Tgo.

SEQID NO. 1:
5'-ATG ATH YTN GAY ACN GAY TAY ATH AC-3' SEQ ID NO. 2:
5'-GGC CTA CGA GAG GAA CGA ACT GGC-3' SEQ ID NO. 3:
2o 5'-GGC GTA GAT GTA GGG CTC-3' SEQ ID NO. 4:
5'-GAG CTG GTC GAA TTC ATG ATC CTG GAC GCT GAC TAC ATC ACC -3' SEQ ID NO. 5:
5'-AGC CTG CAG TCA TGT CTT AGG TTT TAG CCA CGC-3' Example 3 Expression of recombinant T. gorgonarius DNA

The vector from example 2 was transformed into E.coli strain LE 392 pUBS 520, cultivated in a fermentor in a rich medium containing the appropriate antibiotic. Induction was performed at an optical density of 1.25 A540 with 0.5 mM IPTG. The DNA polymerase from T.
gorgonarius may also be cloned and expressed by other methods.

Cells are harvested at an optical density of 5.4 A540 by centrifugation and frozen until needed or lyzed by treatment with lysozyme to produce a crude cell extract containing the T. gorgonarius DNA polymerase activity.

The crude extract containing the T gorgonarius DNA polymerase activity is purified by the method described in Example 1, or by other purification techniques such as affinity-chroma-tography, ion-exchange-chromatography or hydrophobic-interaction-chromatography.

Example 4 Purification of recombinant T. gorgonarius DNA Polymerase E.coli (LE392 pUBS520) pBtac2Tgo (DSM No. 11328) was grown in a 10 1 fermentor in me-dia containing 20 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCI
and 100 mg/liter ampicillin at 37 C and induced with 0.5 mM IPTG at midexponential growth phase and in-cubated an additional 4 hours. About 45 g of cells were harvested by centrifugation and stored at -70 C.

2 g of cells were thawed and suspended at room temperature in 4 ml of Buffer A
(40 mM Tris-HCI, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 1 mM Pefabloc SC). 1.2 mg of lysozyme were added and the cells were lyzed under stirring for 30 minutes at 4 C. 4.56 mg sodium deoxycholate were added and the suspension incubated for 10 minutes at room tem-perature followed by 20 minutes at 0 C. The crude extract was adjusted to 750 mM KCI, heated for 15 minutes at 72 C and centrifuged for removal of denatured protein.

The supernatant was adjusted to 25 % saturation with (NH4)2SO4 and applied to a TSK Butyl Toyopearl 650C column (1.5 x 10 cm; 17.7 ml bed volume) equilibrated with buffer B
(buffer A containing 10 % glycerol) and 30 %(NH4)2SO4-saturation. The column was washed with 70 ml of buffer B and the polymerase was eluted with a 177 ml linear gradient of buffer B
containing 30 % to 0%(NH4)2SO4 saturation and 0 to 0.2 % ThesitTM (v/v).

The column fractions were assayed for DNA polymerase activity. DNA polymerase activity was measured by incorporation of digoxigenin labeled dUTP into the newly synthesized DNA
and detection and quantification of the incorporated digoxigenin essentially as described below.
The reaction is performed in a reaction volume of 50 l containing 50 mM Tris-HCI, pH 8.5;
mM (NHa)2SO4; 7 mM MgC12; 10 mM 2-mercaptoethanol; 100 M of dATP, dGTP, 15 dCTP, dTTP, respectively; 200 g/ml BSA; 12 g of DNAse activated DNA from calf thymus and 0.036 M digoxigenin-dUTP and 1 or 2 l of diluted (0.05 U to 0.01 U) DNA
polymerase from T. gorgonarius. The samples are incubated for 30 min. at 72 C, the reaction is stopped by addition of 2 pl of 0.5 M EDTA, and the tubes placed on ice. After addition of 8 l of 5 M
NaCI and 150 l of Ethanol (precooled to -20 C) the DNA is precipitated by incubation for 15 min. on ice and pelleted by centrifugation for 10 min. at 13,000 rpm and 4 C. The pellet is washed with 100 l of 70% Ethanol (precooled to -20 C) and 0.2 M NaCI, centrifuged again and dried under vacuum. The pellets are dissolved in 50 l Tris/EDTA (10 mM/0.1 mM;
pH 7.5). 5 l of the sample are spotted into a well of a nylon membrane bottomed white microwell plate (Pall Filtrationstechnik GmbH, Dreieich, FRG, product no:
SM045BWP). The DNA is fixed to the membrane by baking for 10 min. at 70 C. The DNA loaded wells are filled with 100 l of 0.45 m filtrated 1% blocking solution (maleic acid, 100 mM;
NaCI, 150 mM;
casein, 1%(w/v); pH 7.5). All following incubation steps are done at room temperature. After incubation for 2 min. the solution is sucked through the membrane with a situable vacuum manifold at -0.4 bar. After repeating the washing step once the wells are filled with 100 l of a 1:10,000-dilution of Anti-digoxigenin-AP Fab fragments (Boehringer Mannheim, FRG, No: 1 093 274) diluted in the blocking solution described above. After incubation for 2 min. and sucking the solution through the membrane, this step is repeated once. The wells are washed ~ WO 98/14590 PCT/EP97/05393 twice under vacuum with 200 l washing-buffer I (maleic-acid, 100 mM; NaCI, 150 mM;
TweenTM 20, 0.3 % (v/v); pH 7.5) After washing for another two times under vacuum with 200 1 washing-buffer 2 (Tris-HCI, 10 mM; NaCI, 100 mM; MgCIZ, 50 mM; pH 9.5) the wells are incubated for 5 min. with 50 l of CSPD7"1 (Boehringer Mannheim, No: 1 655 884), diluted 1:100 in washing buffer 2 which serves as a chemiluminescent substrate for the subsequent alkaline phosphatase reaction.

The solution is sucked through the membrane and after 10 tnin. incubation the RLU/s (Relative Light Unit per second) are detected in a Luminometer e.g. MicroLumat LB 96 P
(EG&G
Berthold, Wilbad, FRG).

In order to correlate the relative light units to the polymerase units as defined commonly, a standard curve was prepared using a serial dilution of Taq DNA polymerase as a standard en-zyme. The Taq polymerase was assayed in the buffer recommended by the supplier. The linear range of the standard curve was used to detenmine the relative activity of the T. gorgonarius DNA polymerase preparations.

The active fractions were pooled, dialyzed twice against 500 ml Buffer B and applied to a FractogetTSK AF-Blue column (1x10; 7.8 m1 bed volume) equilibrated with buffer B. After washing with 15 ml buffer B the column was eluted with a linear gradient of 156 ml from 0 to 3 M NaCI in buffer B supplemented with 0.05 % Thesit. The active fractions were pooled and dialyzed against the storage buffer C (20 mM Tris-HCI, pH 8.2; 10 mM 2-mercaptoethanol;
0.1 mM EDTA; 50 mM (NH4)2SO4, 50 % glycerol). After adding of 0.5 % of NonidetTM P 40 (v/v) and 0.5 % of ThesitT"' (v/v) the preparation was stored at -20 C.
Characterisation of the recombinant DNA Polymerase from Thermococcus gorgonarius Recombinant and native T. gorgonarius DNA polymerase had the same apparent molecular weight when electrophoresed in 8 - 25 % SDS-PAGE gradient gels. Recombinant T.
gor-gonarius polymerase maintains the heat stability of the native enzyme.
Recombinant T gor-gonarius polymerase has the same 3'-5'exonuclease activity as native T.
gorgonarius poly-merase, which is also sensitive to inhibition by an excess of dNTPs.

*Trade-mark Example 5 Thermostability of T. gorgonarius DNA Polymerase The thermostability of the DNA polymerase from T. gorgonarius purified as described in Example I was determinated as follows: 5 units purified T. gorgonarius polymerase were incubated at 95 C in 100 l of the following buffer: 50 mM Tris-HCI, pH 8.8 (at 25 C);
1o 15 mM (NH4)2S04; 7 mM MgC12; 10 mM 2-mercaptoethanol; 200 M each of dATP, dGTP, dCTP and dTTP; 0.1 % Nonidet P40, 0.1 % Thesit; 25 g DNAse treated calf thymus DNA.
l samples were taken at 0, 5, 10, 15, 30, 45, 60 and 120 minutes. The remaining poly-merase activity was measured as described in example IV by determining incorporation of labeled 3H-TTP into DNA in a 50 l volume of the incubation mixture described above con-15 taining in addition 150 nCi of 3H-TTP. After incubation at 72 C for 30 minutes the reactions were stopped by addition of 300 l 10 % TCA, and after 10 minutes at 0 C the mixtures were applied onto 3MM filters (Whatman). The filters were washed three times with approximately 10 ml 5 % TCA each time, dried for 10 minutes at 75 C and the DNA bound radioactivity of each filter was measured in 5 mi scintillation liquid in a scintillation vial in LKB rack beta 1217/1218 (Pharmacia).

As shown in figure No. 4 the T. gorgonarius DNA polymerase retained almost 90 % of its initial activity after incubation for 120 minutes at 95 C, Pwo polymerase has a similar stability, while Taq DNA polymerase has a remaining activity of approximately 16 % only.

_ _ _ _ ......_.... _.__...~_ Example 6 Determination of 3'-5' proofreading activity 5 A series of units of T. gorgonarius DNA polymerase (see figure 5) were incubated for 4 hours at 72 C with 1 g DNA molecular weight marker VI (Boehringer Mannheim) in the presence and absence of dNTP's, 1 mM each, in 50 l of the following incubation buffer:
50 mM
Tris-HCI, pH 7.8; 10 mM MgCl2; 7 mM 2-mercaptoethanol with Paraffin overlay.
After addition of 10 l stop solution the DNA fragments were separated on a 1 %
agarose gel. In the lo absence of dNTP's a smear of DNA fragments or no DNA could be detected while in presence of dNTP's the DNA fragments remained undegraded.

Example 7 Fidelity of T. gorgonarius DNA polymerase in the PCR process The fidelity of T. gorgonarius DNA polymerase in the PCR process was determined in an assay based on the amplification, circularisation and transformation of the pUC19 derivate pUCIQ17, containing a functional lac I4 allele (Frey, B. and Suppmann B.
(1995) Biochemica 2:34-35). PCR-derived mutations in lac I are resulting in a derepression of the expression of lac Za and subsequent formation of a functional B-galactosidase enzyme which can be easily detected on X-Gal indicator plates . The error rates determined with this lac I-based PCR
fidelity assay were in the range of 3.4 to 2.2 - 10-6.

The plasmid pUCIQ 17 was linearized by digestion with DraII to serve as a substrate for PCR
amplification with DNA polymerase of T. gorgonarius. Both of the primers used have C1aI
sites at their 5 prime ends:

SEQ ID NO. 8 Primer 1: 5'-AGCTTATCGATGGCACTTTTCGGGGAAATGTGCG-3' SEQ ID NO. 9 Primer 2: 5'-AGCTTATCGATAAGCGGATGCCGGGAGCAGACAAGC-3' The length of the resulting PCR product is 3493 pb.

The PCR was performed in a final volume of 50 l in the presence of 1.5 mM
MgCIZ, 50 mM
Tris-HCI, pH 8.5 (25 C), 12.5 mM (NHa)2SO4, 35 mM KCI, 200 M dNTPs and 2.5 units of T. gorgonarius DNA polymerase. Conditions of the amplification reaction using T.gorgonarius DNA polymerase are lo The cycle conditions were as follows:

1 x denaturation of template for 2 min. at 95 C
denaturation at 95 C for 10 sec.
8 x annealing at 57 C for 30 sec.
L elongation at 72 C for 4 min.
denaturation at 95 C for 10 sec.

16 x annealing at 57 C for 30 sec.
elongation at 72 C for 4 niin.

+ cycle elongation of 20 sec. for each cycle After PCR, the PCR products were PEG-precipitated (Barnes, W. M. (1992) Gene 112:229) the DNA restricted with Clal and purified by agarose gel electrophoresis. The isolated DNA
was ligated using the Rapid DNA Ligation Kit (Boehringer Mannheim GmbH) and the ligation products transformed in E.coli DH5a, plated on TN Amp X-Gal plates. The a-complementing E.coli strain DH5a transformed with the resulting plasmid pUCIQ17 (3632 bp), shows white (lacI+) colonies on TN plates (1.5 % Bacto Tryptone, 1% NaCI, 1.5 % Agar) containing ampicillin (100 g/ml) and X-Gal (0.004 % w/v). Mutations result in blue colonies.

After incubation overnight at 37 C, blue and white colonies were counted. The error rate (f) per bp was calculated with a rearranged equation as published by Keohavong and Thilly (Keohavong, P. and Thilly, W. (1989) PNAS USA 86:9253):

f= -InF/dgbbp where F is the fraction of white colonies:

F = white (lacI+) colonies / total colony number;
d is the number of DNA duplications:
2d = output DNA / input DNA;

and b is the effective target size of the (1080bp) lac I gene, which is 349 bp according to Provost et al. (Provost et al. (1993) Mut. Res. 288:133).

Example 8 Fidelity assay Determination of the misincorporation rates of DNA polymerases from Pyrococcus furiosus and Thermococcus gorgonarius under PCR conditions.

Error rates of many DNA polymerases are published. For example for the DNA
polymerase of Pyrococcus furiosus various error rates were measured (Lit. 1-5). They may vary with the conditions used e.g. nucleotide triphosphate concentrations, enzyme preparation, buffer con-ditions and of course with the method used, the determination of the number of duplications and the way to calculate the misincorporation rate.

Therefore, the DNA poiymerases Pfu (Stratagene) and Tgo (Boehringer Mannheim GmbH) were analyzed in parallel in the same system (Protocol: Frey, B. and Suppman, B. Boehringer Mannheim Biochemica Information, Nr. 96-1995, 21-23).

Table 1:

Fidelity of Pfu and Tgo DNA polymerases in PCR fidelity assay DNA Polymerase Plaques scored Mutation Error rate (a) Error rate (b) Total Mutant frequency RLU
1. sample 3082 76 2.47 1,56 x 10-5 8,2 x 10-6 2. sample 2693 68 2.52 1,6 x 10"S 8,4 x 10-6 rg-o 1. sample 1904 12 0.63 3,5 x 10-6 1,8 x 10-6 2. sample 2003 20 1 5,6 x 10-6 2,9 x 10-6 (a) Error rate calculated according to the equation used by Stratagene (Lundberg K.S. et al.
(1991) Gene 108, 1-6).

ER=mf/bpxd ER = error rate mf = mutation frequency in % minus background frequency of 0.0017 %
bp is the number of detectable sites in the lac I gene sequence (182) d is the number of duplications. In this particular experiment the number of duplications was determined/estimate for Pfu to be 8,64 and for Tgo to be 9,64 (b) Error rate calculated per bp with a rearranged equation published by Keohavong P. and Thilly W. (1989) PNAS USA 86, 9253.

~ WO 98/14590 24 PCT/EP97/05393 ER=-1nF/dxbbp F = fraction of white colonies (white colonies / total number of colonies) d = the number of duplications. 2d = output DNA / input DNA
b is the effective target size of the (1080 bp) lac I gene, which is 349 bp according to Provost, G.S., Kretz, P.L., Hammer, R.T., Matthews, C.D., Rogers, B.J., Lundberg K., S., Dycaico, M.J. and Short, J.M. (1993) Mut. Res. 288, 133 f Result:
These data show that the mutation frequency of Tgo DNA polymerase is lower than that of Pfu, and the fidelity (calculated in errors per base pair) is higher no matter which way of calculation was used.

References describing error rates for Pfu:

1. Lundberg, K.S., Shoemaker, D.D., Adams M.W.W:, Short, J.M., Sorge, J.A. and Mathur, E.J. (1991) Gene 108, 1-6. (1.6 x 10-6 errors/base) 2. Flaman, J.-M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C., Ishioka, C., Friend, S.H. and Iggo, R. (1994) NAR 22, 3259-3260. (2 x 10'6 errors/base) For Tli (Vent) Polymerase: (Variations in error rate depending on assay) 3. Cariello, N.F., Swenberg, J.A. and Skopek, T.R. (1991) NAR 19, 4193-4198.
(2.4 x 10'5 errors/base) 4. Ling, L.L., Keohavong, P., Dias, C. and Thilly, W.G. (1991) PCR Methods Appl. 1, 63-69. (4.5 x 10'5 errors/base) 5. Matilla, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991)NAR 19, 4967-4973. (5.7 x 10'5 errors/base) SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: Roche Diagnostics GmbH
(B) STREET: Sandhoferstr. 116 (C) CITY: Mannheim (E) COUNTRY: DE
(F) POSTAL CODE (ZIP): 68298 (G) TELEPHONE: 06217595482 (H) TELEFAX: 06217594457 (ii) TITLE OF INVENTION: Thermostable nucleic acid polymerase from Thermococcus gorgonarius (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SWABEY OGILVY RENAULT
(B) STREET: 1981 McGill College, Suite 1600 (C) CITY: Montreal (D) STATE: Quebec (E) COUNTRY: Canada (F) ZIP: H3A 2Y3 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: not known (B) FILING DATE: October 1, 1997 (C) CLASSIFICATION

(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBERS: EP 96 115 874.8 EP 97 100 584.8 (B) FILING DATES: October 3, 1996 January 16, 1997 (vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kevin P. Murphy (B) REGISTRATION NUMBER: 3302 (C) REFERENCE/DOCKET NUMBER: 3580-774 KPM/CC/LM
(viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 845-7126 (B) TELEFAX: (514) 288-8389 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

- ----- ---------(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2322 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..2322 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Asp Tyr Asp Arg Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Pro Ile Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr Val Arg Val Val Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Lys Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Ile Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Glu Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Gly Glu Gly Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Glu Glu Tyr Asp Val Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Val Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr Tyr Gly Tyr Thr Lys Ala Arg Trp Tyr Tyr Lys Glu Cys Ala Glu Ser Val Thr Gly Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Lys Glu Phe Leu Asp Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Ala Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp Leu Lys Pro Lys Thr (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 773 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Asp Tyr Asp Arg Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Pro Ile Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr Val Arg Val Val Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Lys Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Ile Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Glu Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Gly Glu Gly Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Glu Glu Tyr Asp Val Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Val Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr Tyr Gly Tyr Thr Lys Ala Arg Trp Tyr Tyr Lys Glu Cys Ala Glu Ser Val Thr Gly Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Lys Glu Phe Leu Asp Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Ala Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp Leu Lys Pro Lys Thr (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Claims (40)

1. A purified thermostable DNA polymerase obtained from Thermococcus gorgonarius which catalyses the template directed polymerisation of DNA, possesses 3'-5' exonuclease activity and is characterized by at least a two-fold greater replication fidelity than DNA polymerase obtained from Pyrococcus furiosus, and wherein said polymerase has an apparent molecular weight between 92 000 to 96 000 daltons.

2. A purified thermostable DNA polymerase according to claim 1 which retains % of its activity after incubation for two hours at 95°C in the presence of a stabilizer.

3. A polymerase obtained from E. coli transformed with a vector comprising a nucleotide sequence encoding the polymerase as claimed in claim 1 or 2.

4. A stabilized composition consisting of a polymerase as claimed in any one of claims 1-3 and a stabilizer.

5. The composition according to claim 4, wherein said stabilizer is a non-ionic detergent.

6. The composition according to claim 4 or 5, wherein Thesit or Nonidet P 40 serve as stabilizer.

7. An isolated DNA sequence coding for the polymerase according to any one of claims 1-3 obtained from Thermococcus gorgonarius and represented by the formula shown in SEQ ID NO: 6.

8. An isolated DNA sequence coding for the polymerase as claimed in claim 7 contained within the plasmid pBTac2.

9. An isolated DNA sequence of claim 8 contained within a 2.3 kB EcoRI/Pst1 restriction fragment of plasmid pBTac2.

10. A vector containing the isolated DNA sequence as claimed in claim 7.

11. The vector of claim 10, wherein such vector is plasmid pBTac2.

12. The vector according to claim 10 or 11 providing some or all of the following features:
(1) promotors or sites of initiation of transcription;
(2) operators which are used to turn gene expression on or off;
(3) ribosome binding sites for improved translation; or (4) transcription or translation termination sites.

13. A microbial host transformed with the vector of claims 10-12.

14. A microbial host according to claim 13 wherein said host is E. coli LE 392 pUBS
520 (DSM No. 11328).

15. A process for the preparation of DNA polymerase according to any one of claims 1-3 or a composition according to any one of claims 4-6, comprising the steps:

(a) culturing cells of the natural strain Thermococcus gorgonarius;
(b) suspending the cells of the natural cells in buffer;
(c) disrupting the cells; and (d) purifying the DNA polymerase by chromatographic steps.

16. A process for the preparation of DNA polymerase according to any one of claims 1-3 or a composition according to any one of claims 4-6, comprising growing a microbial host strain according to claim 13 or 14 and purifying the DNA
polymerase there from.

17. Use of a thermostable DNA polymerase as claimed in any one of claims 1-3 or a composition as claimed in any one of claims 4-6, for amplifying DNA.

18. Use of a thermostable DNA polymerase as claimed in any one of claims 1-3 or a composition as claimed in any one of claims 4-6, for cloning a second-strand cDNA.

19. Use of a thermostable DNA polymerase as claimed in any one of claims 1-3 or a composition as claimed in any one of claims 4-6, for DNA sequencing.

20. A purified thermostable DNA polymerase encoded by SEQ ID NO: 6.

21. The polymerase as claimed 20, obtained from Thermococcus gorgonarius.

22. The polymerase as claimed in claim 20 or 21, wherein said polymerase has an apparent molecular weight between 92 000 to 96 000 daltons.

23. The polymerase as claimed in any one of claims 20-22, which retains 90% of its activity after incubation for two hours at 95°C in the presence of a stabilizer.

24. A polymerase obtained from E. coli transformed with a vector comprising the nucleotide sequence encoding the polymerase as claimed in any one of claims 20-23.

25. A stabilized composition consisting of a polymerase as claimed in any one of claims 20-24 and a stabilizer.

26. The composition according to claim 25, wherein said stabilizer is a non-ionic detergent.

27. The composition according to claim 25 or 26, wherein Thesit or Nonidet P

serve as stabilizer.

28. An isolated DNA sequence coding for the polymerase according to any one of claims 20-24 obtained from Thermococcus gorgonarius.

29. An isolated DNA sequence coding for the polymerase as claimed in claim 28 contained within the plasmid pBTac2.

30. An isolated DNA sequence of claim 29 contained within a 2.3 kB EcoRI/Pst1 restriction fragment of plasmid pBTac2.

31. A vector containing the isolated DNA sequence as claimed in claim 28.

32. The vector of claim 31, wherein such vector is plasmid pBTac2.

33. The vector according to claim 31 or 32 providing some or all of the following features:
(1) promotors or sites of initiation of transcription;
(2) operators which are used to turn gene expression on or off;
(3) ribosome binding sites for improved translation; or (4) transcription or translation termination sites.

34. A microbial host transformed with the vector of claims 31-33.

35. A microbial host according to claim 34 wherein said host is E. coli LE 392 pUBS
520 (DSM No. 11328).

36. A process for the preparation of DNA polymerase according to any one of claims 20-24 comprising the steps of:
(a) culturing cells of the natural strain Thermococcus gorgonarius;
(b) suspending the cells of the natural cells in buffer;
(c) disrupting the cells; and (d) purifying the DNA polymerase by chromatographic steps.

37. A process for the preparation of DNA polymerase according to any one of claims 20-24 comprising growing a microbial host strain according to claim 34 or 35 and purifying the DNA polymerase there from.

38. Use of a thermostable DNA polymerase according to any one of claims 20-24 or of a composition according to any one of claims 25-27 for amplifying DNA.

39. Use of a thermostable DNA polymerase according to any one of claims 20-24 or of a composition according to any one of claims 25-27 for cloning a second-strand cDNA.

40. Use of a thermostable DNA polymerase according to any one of claims 20-24 or of a composition according to any one of claims 25-27 for DNA sequencing.