WO1995014770A1 - Improved polymerase - Google Patents

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WO1995014770A1
WO1995014770A1 PCT/NZ1994/000135 NZ9400135W WO9514770A1 WO 1995014770 A1 WO1995014770 A1 WO 1995014770A1 NZ 9400135 W NZ9400135 W NZ 9400135W WO 9514770 A1 WO9514770 A1 WO 9514770A1
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dna
polymerase
dna polymerase
activity
recombinant
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PCT/NZ1994/000135
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French (fr)
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Peter Leonard Bergquist
Darren John Day
Moreland David Gibbs
Rosalind Alison Reeves
David James Saul
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Pacific Enzymes (1993) Limited
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241Nucleotidyltransferases (2.7.7)
    • C12N9/1252DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention comprises a thermophilic enzyme, more specifically a heat-stable DNA polymerase, isolated from the New Zealand Thermus species Thermus filiformis. The invention also includes recombinant plasmids and transformed host cells capable of producing the enzyme. The enzyme is classified into class EC 2.7.7.7; a DNA nucleotidyltransferase DNA-directed type.

Description

IMPROVED POLYMERASE

FIELD OF INVENTION

The invention comprises a thermophilic enzyme, more specifically a heat-stable DNA polymerase, isolated from the New Zealand Thermus species Thermus filiformis. The invention also includes recombinant plasmids and transformed host cells capable of producing the enzyme. The enzyme is classified into class EC 2.7.7.7; a DNA nucleotidyltransferase DNA-directed type. •**,

DEFINITIONS

In referring to a peptide chain as being comprised of a series of amino acids "substantially or effectively" in accordance with a list offering no alternatives within itself , we include within that reference any versions of the peptide chain bearing substitutions made to one or more amino acids by similar amino acids in such a way that the overall structure and the overall function of the protein composed of that peptide chain is substantially the same as - or undetectably different to - that of the unsubstituted version. For example it is generally possible to exchange alanine and valine without greatly changing the properties of the protein, especially if the changed site or sites are at positions not critical to the morphology of the folded protein.

The term "thermostable" as applied to an enzyme means that the enzyme is relatively unaffected by heat. Normally such enzymes are used in aqueous solutions and the upper limits of the temperature range are determined by the boiling point of water at the relevant environmental pressures. Preferably a thermostable enzyme remains active for a long period at a high temperature and preferably it also has an enhanced Km at a high temperature.

Other definitions are used in a manner consistent with the art.

BACKGROUND OF THE INVENTION

Heat stable DNA polymerases (EC 2.7.7.7, DNA nucleotidyltransferase DNA-directed) have been isolated from numerous thermophilic organisms (for example: Kaledin et al. 1980. Biokimiya 44, 644-651; Kaledin et al. 1981. Biokimiya 46, 1247-1254 ; Kaledin et al. 1982. Biolimiya 47, 1515-1521; Ruttimann, et al. 1985. Eur. J. Biochem 149, 41-46; Neuner et al. 1990. Arch. Microbiol. 153, 205-207). For some organisms, the polymerase gene has been cloned and expressed (Lawy r et al. 1989. J. Biol. Chem. 264, 6427-6437; Engelke et al. 1990. Anal. Biochem. 191, 396-400; Lundberg et al. 1991. Gene 108, 1-6; Perler et al. 1992. Proc. Natl. Acad. Sci. USA 89, 5577-5581). (References to cited literature are provided at the end of the description of the preferred embodiments.)

Thermophilic DNA polymerases are increasingly becoming important tools for use in molecular biology and there is growing interest in finding new polymerases which have more suitable properties and activities for use in diagnostic detection, cloning and DNA sequencing. For example, the use of thermostable enzymes in the PCR reaction, which is used to amplify existing nucleic acid sequences by a very large ratio. At present, three polymerases have become available from Thermus species. Taq polymerase from T. aquaticus (Yellowstone Park, USA, Brock et al. 1969. J. Bacteriol. 98, 289-297); Tth polymerase from "T. thermophilus" (Japan, Oshima and hnahori. 1974. J. Syst. Bacteriol. 24, 104-112) and Tfl polymerase from "T. flavus" (Japan, Saiki et al. 1972. Agric. Biol. Chem. 34, 2357-2366). The present patent concerns a fourth DNA polymerase from T. filiformis, a species isolated from Waimangu Hot Springs, New Zealand (Hudson et al. 1987. Int. J. Syst. Bacteriol. 37, 431-436). Neither "T. thermophilus" nor "T.fiavus" are yet accepted as validly named species, whereas T. filiformis and T. aquaticus are accepted as distinct, valid species. Phylogenetic analysis of the Thermus genus (as described later in this document) indicates that the New Zealand Thermus isolates (including T. filiformis) are evolutionarily well separated from the American and Japanese isolates (Saul et al. 1993. Int. J. Syst. Bacteriol. 43, 754-760).

Currently, Taq polymerase is the preferred enzyme for DNA sequencing using automated thermal sequencing machines such as the Applied Biosystems 373A DNA Sequencer. The advantages of using a thermophilic enzyme are that less template is required due to the linear amplification that occurs with thermal cycling and also the elevated temperatures help to melt secondary structures in the DNA that may be problematic with conventional sequencing techniques. Taq polymerase is currently the only enzyme used for the polymerase chain reaction (PCR). For performing a PCR on RNA rather than DNA templates (RT PCR), a polymerase must be capable of first producing a DNA copy of an RNA template before normal PCR can commence. This activity, known as reverse transcription, has been noted to varying degrees with Tth and Taq DNA polymerases (Jones, et al. 1989. Nucl. Acids Res. 17, 8387- 8388; Meyers and Gelfand 1991. Biochem. 30, 7661-7666). Reverse transcription by thermophilic DNA polymerases has advantages over mesophilic viral reverse transcriptases such as Moloney murine leukemia virus reverse transcriptase (RT) and avian myeloblastosis virus RT which are commonly used for cDNA synthesis, because the higher reaction temperatures possible with thermophilic polymerases helps to destabilise RNA secondary structures which can pose significant problems for the mesophilic viral RTs.

The wide range of uses for DNA polymerases means that it is advantageous to have available a variety of enzymes. For instance, the reverse transcriptase activity of Taq polymerase is considerably lower than that of Tfil or Tth polymerases and is consequently less suitable for RT PCR. In automated DNA sequencing, an enzyme is required which can incorporate fluorescently-labelled dideoxynucleosides efficiently. Taq polymerase performs this task best at reduced temperatures and with high concentrations of the base analogs. This is disadvantageous for templates which tend to form secondary structures which require high temperatures for denaturation.

Accordingly there is a desire in the art to produce alternative DNA polymerases to the most frequently used Taq polymerase, in order to improve both the cost-effectiveness and the quality of the results obtained. It is well known in the art that use of an improved enzyme capable of operating at a higher temperature improves the specificity and selectivity of the primer-directed extension process within the PCR reaction.

OBJECT

It is an object of this invention to provide a polymerase enzyme (EC 2.7.7.7) and/or cloned DNA and/or vectors and or transformed host cells capable of producing this enzyme, or at least to provide the public with a useful choice.

STATEMENT OF THE INVENTION

In one aspect the invention provides a purified heat-stable DNA polymerase enzyme (EC 2.7.7.7, a DNA nucleotidyltransferase DNA-directed enzyme), characterised in that it has reverse transcriptase activity in the presence of magnesium ions.

In another aspect the invention comprises a recombinant DNA sequence that encodes DNA polymerase activity of the microorganism Thermus filiformis.

In a related aspect, the DNA sequence is that shown in Fig 1. In a second related aspect the invention comprises a recombinant DNA sequence that encodes amino acid residues 1 to 833 as also shown in Fig 1.

In a further aspect the invention comprises recombinant DNA plasmids that comprise the DNA sequence of the invention inserted into plasmid vectors and which can be used to drive the expression of the thermostable DNA polymerase of Thermus filiformis in a host cell transformed with the plasmid.

In a related aspect the invention includes a recombinant plasmid comprising the vector pUC18 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2300.

Also in a related aspect the invention includes a recombinant plasmid comprising the vector pT7-7 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2303. In a further related state the invention comprises a host cell transformed with the recombinant DNA of the invention.

As one option, the invention comprises such a host cell, being Escherischia cø/z'/pNZ2300, the strain being designated PB5900.

As another option, the invention comprises such a host cell, being Escherischia coli/pNZ2303, the strain being designated PB5904.

In a further aspect the invention comprises a DNA polymerase having a molecular mass of about 93.4 KDa.

In a further aspect the invention comprises a DNA polymerase having a pH-dependent activity as shown in Fig 3. In a yet further aspect the invention comprises a DNA polymerase having a dependency on magnesium ion concentration, as shown in Fig 4.

In yet another aspect the invention comprises a DNA polymerase having Reverse Transcriptase activity in the substantial absence of manganese ions.

In a still further aspect the invention comprises a DNA polymerase having an activity, referenced to Taq polymerase, as illustrated in Fig 5.

Preferably the invention provides a thermostable DNA polymerase which has been purified from a recombinant Escherichia coli strain containing the gene encoding for this enzyme, which has been isolated from Thermus filiformis.

The coding region for the polymerase was isolated by PCR amplification from T. filiformis genomic DNA. The PCR product was cloned in the vector pUC18 and active thermophilic polymerase was expressed as a fusion protein with the amino-terminus of β-galactosidase. Higher expression was achieved by cloning the gene into the expression vector pT7-7 where the polymerase was expressed as a fusion with the T7 gene 10 protein. Analysis of the sequence of the polymerase showed that it was clearly distinguishable from similar enzymes from Thermus aquaticus, "Thermus flavus" and "Thermus thermophilus" .

The DNA polymerase has been purified to homogeneity by a simple three-step procedure involving heat precipitation of E. coli proteins followed by Q-sepharose and heparin sepharose chromatography. The purified protein has a relative molecular mass of 94 kDa as determined by SDS polyacrylamide gel electrophoresis (PAGE) using 12% and 7.5% polyacrylamide gels, whereas the predicted molecular mass, from the polypeptide sequence of Figure 1, is 93.2 kDa. The purified enzyme exhibits no endonuclease, exonuclease or ribonuclease activity, shows maximal activity in low salt buffers at alkaline pHs and has an absolute requirement for Mg2+ ions (Km 1.5 mM). The DNA polymerase shows reverse transcriptase activity on primed RNA templates. The polymerase is highly processive and has an extension rate of approximately lOOObases/min.

DRAWINGS

These and other aspects of this invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying drawings, in which:

FIG. 1 (3 sheets, fig 1-1, fig 1-2 and fig 1-3) shows the DNA sequence of the polymerase gene of Thermus filiformis and the derived peptide sequence for Tfil polymerase. The one- letter abbreviations for the amino acids are shown here for convenience.

F = Phenylalanine T = Threonine D = Aspartic acid

L = Leucine A = Alanine E = Glutamic acid

1 = Isoleucine Y = Tyrosine C = Cysteine

M = Methionine H = Histidine W = Tryptophan

V = Valine Q = Glutamine R = Arginine

S = Serine N = Asparagine G = Glycine

P = Proline κ= Lysine

FIG. 2 Alignment of the deduced amino acid sequences for Tfil , Taq and Tfl DNA polymerases. Only the residues differing from the Tfil sequence are shown for Taq and Tfl.

FIG. 3. Relative yield of PCR product at differing pHs. PCR was performed using primers P4 and P5 and a pUC18 plasmid containing the sheep myόD gene as described in the General Methods section. The relative yield at each pH value was estimated by electrophoresis of 10 ml of reaction product on a 2% agarose gel which was stained with ethidium bromide and photographed (panel A). The intensity of the bands was estimated by densitometer scanning of the negative (negatives used in figure) and the values plotted (panel B) relative to the darkest and lightest area of the photograph.

FIG. 4 The Km of Tfil polymerase for Mg2+ ions. The Km of Tfil polymerase for MgCl2 in the solid phase assay was determined by varying the amount of MgCl2 from the standard 5 mM, and measuring the incorporation of radio-nucleotide after a lh reaction at 70°C as in the standard solid phase assay. The plot shows the number of incorporated counts at each MgCl2 concentration tested and the inset presents the data as an Eadie-Hofstee plot.

FIG. 5 The activity of Tfil polymerase and Taq polymerase at different KC1 concentrations. The activity of 0.05 units of each polymerase was determined in the solid phase assay described below except that various amounts of KC1 were included in the reaction at the indicated concentrations.

FIG. 6. PCR amplification using Tfil DNA polymerase with different buffers and templates. Panel (A) shows the PCR amplification of the 16S rRNA genes from Thermus flavus using genomic DNA and primers P6 and P7, and panel (B) shows amplification of a 280 bp fragment of myoD from a plasmid template using primers P4 and P5. The amplifications were performed as described in the General Methods section with differing concentrations of MgCl2, Tween 20 and (NH4)2SO4 as indicated. For amplification of the rRNA genes the PCR buffer was 50 M Tris-HCl pH 8.8 containing 1.5 mM MgCl2, 400 mM dNTPs and either 0%, 0.01%, 0.02% or 0.04% Tween 20 (lanes 2 to 6 respectively). Taq polymerase was used in the same buffer with 0.01% Tween 20. Amplifications from plasmid DNA were also as described in the General Methods section. The PCR buffer was 50 mM Tris-HCl pH 8.8 with the following additives: lanes 8, 9, 10 and 11 each contained, 15 mM (NH4)2SO4, 0.01% Tween 20 and 1, 1.5, 2.0, or 2.5 mM MgCl2 respectively; lane 12 contained 1.5 mM MgCl2, 0.01% Tween 20; lane 13 contained 1.5 mM MgCl2, 25 mM (NH4)2SO4, 0.01% Tween 20; lane 14 contained 1.5 mM MgCl2 and 15 mM (NH )2SO4. The reaction products from both PCR reactions were analysed by electrophoresis of 5 ml of the 50 ml reaction on an 0.8% (panel A) or 2% (panel B) agarose gel stained with ethidium bromide. The markers are the BRL 1 kb ladder.

FIG. 7. Sensitivity of RT/PCR amplifications. The sensitivity of RT/PCR was determined by serially diluting an RNA transcript of α-lactalbumin and using the dilutions as template for the RT/PCR reaction. RT/PCR amplification of α-lactalbumin was performed by using both primers in the RT reaction and 2 units of Tfil DNA polymerase as described in the General Methods section. Lanes 1 to 5 correspond to 32 pg, 160 pg, 800 pg, 4 ng, 20 ng and 100 ng of RNA template respectively. The reaction products were analysed by electrophoresis of 5 ml of product on a 0.8% agarose gel which was stained with ethidium bromide. Lane 6 is the BRL 1 kb ladder. The gels are illustrated in negative form, for clarity.

FIG. 8. RT/PCR amplification from total cellular RNA. Panel A shows the amplification of a 200 bp region of topoisomerase Ha from total cell RNA isolated from Jurkat cell lines that were amsacrine-resistant, adriamycin-resistant, or normally sensitive (lanes 1, 2 and 3 respectively). Approximately 400 ng of RNA was used for each amplification and 5 units of polymerase. The sense primer was added with the PCR buffer after the RT as described in methods. Panel B shows the amplification of α-lactalbumin (lane 4) from 500 ng of total cell RNA as described in Experimental Procedure. Reaction products were analysed by electrophoresis of 10 ml on a 2% (panel A) or a 0.8% (panel B) agarose gel and ethidium bromide-staining. Molecular weight markers are the BRL 1 kb ladder.

FIG. 9. Phylogenetic tree of Thermus isolates ex Saul et al. (1993. Int. J. System. Bacteriol". 43, 754-760). The tree is based on 16S rRNA sequence data and generated by the method of maximum parsimony. The scale bar represents an expected nucleotide substitution rate of 0.01 per site. The values on the branches indicate the levels of support from 100 bootstrapped trees. The shaded boxes delineate clades supported by more than 80% of the bootstrapped trees. Also included are the geographic origins of the isolates.

PREFERRED EMBODIMENTS

GENERAL METHODS USED

Assay for polymerase activity. Thermophilic DNA polymerase activity was measured using a solid phase assay as described by Day et al. 1993 Anal Biochem, 211: 174-176.

Polyacrylamide gel electrophoresis. SDS PAGE was performed essentially as described by Laemmli (1970) Nαtwre 227, 680-685; using 12.5% and 7.5% SDS polyacrylamide gels and a Pharmacia "Phastgel" system. Gels were silver stained as described in the Pharmacia "PhastSystem" Users Manual.

Detection of endonuclease, exonuclease and ribonuclease activities. The purified polymerase was tested for the presence of endonuclease and exonuclease activities by incubating enzyme with linearised and supercoiled plasmid or an RΝA transcript. Ten units of DΝA polymerase were incubated with 200 ng of linearised plasmid or uncut plasmid, or 500 ng of RΝA transcript, in 20 mM Tris-HCl buffer pH 8.0 or pH 8.8 containing 5 mM Mg -1" for 3 hr at 70°C or 37°C. Degradation of RΝA or plasmid DΝA, or relaxation of the plasmid, was determined by agarose gel electrophoresis and staining with ethidium bromide.

Dye-labelled primer sequencing. Sequencing using the ABI dye-labelled Ml 3 forward sequencing primer was performed on a single stranded M13 template that contained a G:C rich insert from a 16S RΝA gene from a thermophilic organism (pΝZ2201). The sequencing reactions were performed exactly as described in the Applied Biosystems Taq Dye Primer Cycle Sequencing Kit Manual except differing buffer conditions for use with the Tfil polymerase.

Dye-labelled dideoxynucleotide terminator sequencing. The same template as for dye- labelled primer sequencing was used. The protocol suggested by ABI was followed with some modifications. Buffers containing varying amounts of MgCl2 and salts were tried in place of the recommended "TACS" buffer. The ratio of deoxynucleotide to dye labelled terminator was also varied by adding increasing amounts of the ABI supplied nucleotide mix (750 mM dITP, 150 mM dATP, 150 mM dTTP and 150 mM dCTP). The standard sequencing cycle of 25 cycles of 96°C for 30s, 50°C for 15s, 60°C for 4 min, was varied by increasing the extension temperature from 60°C to either 65°C or 70°C for 4 min.

RNA preparation. Total cellular RNA was isolated from cell lines derived from MA 104 cells (α-lactalbumin cell lines) or Jurkat cells (topoisomerase Ila cell lines) as described by Sambrook et al. 1989 in "Molecular Cloning: A Laboratory Manual". Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. RNA run-off transcripts of the cloned a- lactalbumin gene have been described elsewhere (L'Hullier et al. 1992. EMBO J. 11, 4411- 4418).

Oligodeoxynucleotide primers used.

PI 5'-CGGATCCCAAATCAGGCTTTTATTCGG-3'

P2 5'-ATTTTCCATGATCTGCTTATGAG-3'

P3 5'-TATTCCCAAACTGGATGATGCTAAT-3'

P4 5'-GCGGGCCCTTAGGCTACTACGG-3'

P5 5'-GCTCGATATCCCGGCGTGGGGC-3'

P6 5'-AAAGGAGGTGATCCAGCCGCACCTTC-3' P7 5'-TTGTTGGAGAGTTTGATCCTG-3' T7 5'-AATACGACTCACTATAG-3'

PCR and RT/PCR coupled amplifications. All PCR amplifications (50 ml) were performed in 50 mM Tris-HCl buffer pH 8.8 containing 1.5 mM MgCl2, 0.01% Tween 20, 400 mM each of dATP, dCTP, dGTP and dTTP (dNTPs), 10 pmoles each of forward and reverse primer, 2 units of DNA polymerase and the indicated amount of template unless otherwise stated. Reactions were overlaid with 50 ml of mineral oil prior to amplification. RT/PCR amplifications had the initial reverse transcription performed in a reaction volume of 25 ml and contained 50 mM Tris-HCl buffer pH 8.8, 2 mM MgCl2, 0.05% Tween 20, 0.05% Nonidet P40, 400 mM of each dNTP, and 100 ng of reverse primer PI or p2. The amount of enzyme used was varied between 1 and 5 units, and often, the forward primer T7 or P3, which are necessary for the PCR amplification step, was also included in the reverse transcription mixture. Reverse transcriptions were performed at 60°C (under mineral oil) with the enzyme added after the 60°C reaction temperature had been attained. Reverse transcription was allowed to proceed for 5 min after which the reaction temperature was raised to 94°C and the reaction diluted with 75 ml of PCR buffer (50 mM Tris-HCl buffer pH 8.8 containing 1.5 mM MgCl2, 0.01% Tween 20 and 400 mM dNTPs) that had been preheated to 94°C. The forward primer was added at this point if it had been omitted from the initial reverse transcription reaction. The reaction was overlaid with more mineral oil and then amplified in a thermal cycler using 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min for both α- lactalbumin and topoisomerase Ha templates. This 'hot start' procedure helped prevent mis- priming during the reverse transcription and subsequent PCR amplification. Optimisation of PCR buffer conditions. The buffer conditions that gave maximum yield of PCR product were determined using primers P4 and P5, and 10 ng of a plasmid template containing the sheep myoD gene (Huynen, et al. 1991. Nucl. Acids Res. 20, 374) and also with primers P6 and P7 (16S rRNA gene primers), with 50 ng of T. flavus genomic DNA as template. The PCR buffer was systematically varied by altering the amount of MgCl2, (NH4)2SO4 and Tween 20 added to the 50 mM Tris-HCl buffer pH 8.8. Each dNTP was present at 400 mM and 1 unit of polymerase was used per reaction. The PCR cycle used was 25 cycles of 94°C for 1 min., 60°C for 1 min. and 72°C for 1 min. for the plasmid amplification and 35 cycles of 94°C for 1 min., 53°C for 1 min. and 72° for 2 min., with an initial denaturing step of 4 mins at 94°C.

Determination of the pH optimum for PCR. The above primers were used (100 ng each) to determine the yield of PCR product obtained with different buffers. The reactions all contained 400 mM of each dNTP, 1.5 mM MgCl2, 0.01 % Tween 20, 10 ng of template DNA, 1 unit of polymerase and 50 mM of either Tris, Bis-Tris propane (l,3bis[tris(Hydroxymethyl)methlyamino]propane) or EPPS (N-[2-Hydroxyethyl]-piperazine- N'-[3-propanesulphonic acid]) buffer in the pH range 7.4 to 9.5. The reactions were thermally cycled for 25 cycles of 94°C for 1 min., 60°C for 1 min. and 72°C for 1 min. The yield of PCR product was determined by electrophoresis of a 10 ml portion of the reaction product followed by densitometer scanning of a photographic negative of the ethidium bromide stained agarose gel.

GENE CLONING OF THE THERMUS FILIFO.RMIS DNA POLYMERASE GENE. Gene isolation. The gene encoding the DNA polymerase was isolated from Thermus filiformis genomic DNA using the technique of the Polymerase Chain Reaction (PCR). Two oligonucleotide primers were used for the amplification:

Primer A, S'-CACGAATTCGGGGATGCTGCCCCTCTTTGAGCCCAAG-S' Primer B, 5'-GTGGGArCCATCACTCCTTGGCGGAGAGCCAGT-3'. The primers contained an EcoRI site and a BamH site at their 5' ends respectively for A and B and are derived from the primers used by Εngelke et al. (1990) Anal. Biochem. 191: 396- 400; to clone the Taq polymerase gene from Thermus aquaticus. The PCR amplification was performed in 50 ml of buffer containing 10 mM Tris-HCl pH 8.8, 2.5 mM MgCl2, 50 mM KC1, 400 μM dNTPs, 10 pmoles of each primer and 2.5 units of Taq polymerase (AmpliTaq; Cetus Corp). The target sequence was amplified by first denaturing at 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 4 mins. Thermal cycling was performed in a Perkin Elmer Cetus thermal cycler.

Cloning and expression in pUC18 The PCR product was purified by electrophoresis of 100 ml of the PCR mixture on a 0.8% Tris-Acetate agarose gel. The 2.6 kb band of the polymerase coding region was purified from the agarose by binding and elution from glass powder (Geneclean, Bio 101, San Diego, CA). The product was prepared for cloning by treating with 1 unit each of T4 polynucleotide kinase, T4 DNA polymerase and Klenow fragment in 66 mM Tris-HCl pH 7.6 containing 6 mM MgCl2 and 50 μM dNTPs for 15 mins at 37°C. The DNA was then extracted with phenol/chloroform and then chloroform prior to precipitation with ethanol. The pellet was resuspended and digested with EcoRI to give one cohesive end for directional cloning. The DNA was ligated into pUC18 that had been digested with EcøRI and Smαl, and the ligated products were transformed into E. coli strain DH. Transformants were grown on L-agar containing 100 μg/ml ampicillin and IPTG/Xgal (5 mM and 33 μg ml respectively) to allow selection of recombinants. White colonies, in which the LacZ gene had been insertionally inactivated, were picked and grown in L-broth containing 100 μg/ml ampicillin, and plasmid DNAs were prepared by alkaline lysis. The plasmids were checked for insertions by digestion with PVMII. Those recombinants containing inserts were grown in L- broth containing ampicillin and tested for the expression of thermophilic DNA polymerase by induction of exponentially growing culture with 0.5 mM IPTG and assaying the heat-treated extracts for DNA polymerase activity. A recombinant expressing polymerase was obtained. The strain was designated PB5900 and the plasmid pNZ2300.

Cloning and expression in pT7-7 The Tfil polymerase coding region was excised from pNZ2300 by digestion at the unique EcoRI and HindHI sites of the multiple cloning site of the vector. The EcoRI - Hindiπ restriction fragment was purified and ligated into the expression vector pT7-7 that had been similarly digested with EcoRI and HindHI. The ligated DNA was transformed into E. coli strain JMlOl and transformants were selected by plating on L-agar containing ampicillin (60 μg/ml). Colonies were grown and plasmid prepared by alkaline lysis. Colonies containing insertions were identified by digestion with EcoRI. The new recombinant strain was designated PB5902 and the new plasmid pNZ2302.

The DNA polymerase coding sequence in the plasmid pNZ2302 was out of frame with respect to the phage T7 gene 10 product. The coding region for the DNA polymerase was placed in frame with the ATG of the gene 10 protein by digestion of pNZ2302 with EcoRI, followed back-filling of the EcoRI site with Klenow fragment and religation. This new construct was designated pNZ2303.

Plasmid pNZ2303 was used to transform competent E. coli K12 containing plasmid pGPl-2. Transformants were selected by plating on L-agar containing ampicillin and kanamycin (60 μ g/ml each) and were grown at 32°C. Colonies were picked and grown in L-broth containing ampicillin and kanamycin (60 μg/ml each) at 32°C, then induced and tested for the expression of active thermophilic DNA polymerase in heat treated induced extracts, as described above. A recombinant strain expressing active thermostable DNA polymerase (PB5905) was obtained. PURIFICATION OF Tfil POLYMERASE

Growth of the recombinant E.coli strain PB5905 for the production of Tfil DNA polymerase. Ten litres of medium (L-broth containing 60 μg/ml of ampicillin) were inoculated with a 1/40 volume stationary phase culture of PB5905 and grown in batch culture. The cells were grown at 32°C to an absorbance at 600 nm of 0.5, then induced by rapidly raising the growth temperature to 42°C, and maintained at this temperature for a further 45 min. The temperature was then reduced to 37°C and the cells grown for a further 2 h before harvesting and washing in 10 mM Tris-HCl buffer pH 8.0 containing 1 mM EDTA (TE buffer). Approximately 20 g wet weight of cells were obtained and may be frozen prior to use.

Purification of Tfil DNA polymerase. The cells were thawed and resuspended in 100 ml of TE buffer. The protease inhibitor phenylmethylsulphonylfluoride was added to a final concentration of 0.5 mM and the cells broken by one passage through a french pressure cell. The broken cells were incubated in a 70°C water bath in a preheated 1 litre flask for 30 min after which they were cooled on ice. Precipitated protein and cell debris were removed by centrifugation at 32,600x g for 15 min at 4°C. The supernatant was decanted and dialysed for 12 h against 3 changes of 5 litres of TE buffer and filtered through a 0.22 μm filter to remove paniculate matter.

A Pharmacia "Q-Hiload 26/10" was washed with 1M KC1 then equilibrated with buffer A (20 mM Tris-HCl pH 8.0 containing 1 mM EDTA and 10% v/v glycerol) at 4°C. The heat-treated extract (85 ml) was applied to the column using a peristaltic pump (Pharmacia P-l) at a flow rate of 2 ml min. Unbound material was washed from the column with 50 ml of buffer A. The column was then attached to a Pharmacia FPLC system and bound protein eluted using a linear KC1 gradient in buffer A (0 - 400 mM KC1 in 300 ml) at a flow rate of 5 ml/min. The elution of protein was monitored at 280 nm and 7.5 ml fractions collected and placed immediately on ice. Fractions containing DNA polymerase activity were pooled and dialysed against 3 changes of 5 litres of 20 mM potassium phosphate pH 7.5 (buffer B). A 5 ml heparin sepharose column (Pharmacia HiTrap heparin, 5 ml) is equilibrated with buffer B at 4°C and all of the dialysed extract applied in two identical runs, each using half of the extract (70 ml). The sample was applied at a flow rate of 2 ml min, and the column washed with 10 ml of buffer B. Bound polymerase was eluted with a linear gradient of KC1 in buffer B (0 - 500 mM KC1 in 30 ml). Pure DNA polymerase eluted as a single peak at about 350 mM KC1. Active fractions were pooled and diluted with an equal volume of glycerol and stored at -20°C. Approximately 20 mg of purified polymerase can be recovered by this purification scheme.

CHARACTERISTICS OF Tfil POLYMERASE

Analysis of the sequence of the Tfil polymerase gene. The sequence of the DNA polymerase gene was determined by inserting and sequencing restriction enzyme fragments of the PCR isolated Tfil gene cloned into Ml 3. These were sequenced by using the standard M13 forward sequencing primers with dye-primer chemistry and an Applied Biosystems 373 A Automated DNA Sequencer. The DNA sequence of Tfil polymerase and the derived amino acid sequence of the enzyme are shown in Fig 1. When compared with other available Thermus polymerase gene sequences, the Tfil polymerase sequence shows the following percentages of identical amino acids: Tfil vs. Taq = 84%; Tfil vs. Tfl = 82%. This represents a greater difference between Tfil polymerase and the others than is seen between Taq and Tfl (Taq vs. Tfl = 86% identity). An alignment of the deduced amino acid sequences of these enzymes is Fig 2.

The Tfil polymerase possesses several advantages over other polymerases, some of which advantages are outlined here. Characterisation of Tfil polymerase shows that its properties are clearly different from Taq polymerase, as Tfil polymerase has maximum activity in low salt buffers, possesses reverse transcriptase activity and has a conveniently greater affinity for dye- labelled dideoxynucleotide analogues. Using the 5'-3' exonuclease assay described in the methods section, no detectable exonuclease activity can be measured for Tfil polymerase or Taq polymerase.

Tfil polymerase has maximum activity between pH8.5 and 9.0 as demonstrated by comparing the yield of PCR products in different buffers (Fig 3). The enzyme has maximal activity in low salt buffers and has an absolute requirement for Mg2+ ions (Km 1.5mM) (Fig 4). The enzyme is completely, but reversibly inhibited by the addition of EDTA. The most suitable buffer for both DNA polymerase and Reverse Transcriptase activities is 50 mM Tris-HCl buffer pH 8.8 containing 1.5 mM MgCl2 and 0.01% Tween 20. In this buffer Tfil polymerase has a half-life at 94°C of approximately 28 minutes.

Reverse Transcriptase activity. The reverse transcriptase activity of thermophilic DNA polymerases has been described previously (Jones and Foulkes 1989. Nucl. Acids Res. 17, 8387-8388; Meyers and Gelfand 1991. Biochem. 30, 7661-7666) with the activity of Taq polymerase being poor compared with that of Tth polymerase. Tth polymerase shows similar reverse transcriptase activity to that of Tfil polymerase but is different in that Tth polymerase has greatest activity in buffers containing high salt (100 mM KC1). Furthermore, both Taq polymerase and Tth polymerase require Mn2+ supplied as MnCl2 for activity. Tfil polymerase shows the same high level of reverse transcriptase activity as Tth pol but differs in that no activity is obtained when MnCl2 was used instead of MgCl2 for reverse transcription. The use of Mg2+ as opposed to Mn2+ is a significant difference between Tth pol and Tfil polymerase. Reverse transcription in the presence of Mg2+ ions is preferable for two-step reactions where RNA is to be copied followed by DNA extension as Mn2+ ions are known to lower the fidelity of DNA synthesis (Beckman et al. 1985. Biochemistry 24, 5810-5817). Low fidelity DNA synthesis is likely to lead to mutated copies of the original template. In addition, Mn2+ ions have been implicated in an increased rate of RNA degradation, particularly at high temperatures and this can cause the synthesis of shortened products in a reverse transcription reaction.

Salt tolerance. Tfil polymerase shows greatest activity at low KCl concentrations but retains a greater percentage activity at high salt concentrations than does Taq polymerase which has maximal activity at 25 mM KCl in the solid phase DNA polymerase assay but loses activity rapidly as the concentration of KCl increase (Fig 5). Tfil polymerase was much more tolerant of high salt concentrations, maintaining 35% of its activity at 150 mM KCl whilst Taq polymerase showed 2% activity at this concentration. This result indicates that Tfil polymerase has far greater utility that Taq polymerase when high salt buffers are required.

DNA sequencing. There are two sequencing chemistries that can be used on the ABI 373A Automated DNA Sequencer (currently the most utilised DNA sequencer world- wide). Both methods incorporate fluorescent dyes to label terminated reactions and generate sequencing ladders which are detected by a laser/photomultiplier excitation/detection system. The chemistries use either a dye-labelled primer and standard dideoxynucleotides to terminate the sequence or an unlabelled primer and dye-labelled dideoxynucleotide terminators. The sequence obtained using dye-labelled primers is usually superior in both quality and read length than that acquired using dye-labelled terminators but dye-labelled terminators offer the advantage that any primer may be used to generate sequence data.

Tfil polymerase was able to generate long stretches of unambiguous sequence data when using dye-labelled primer and M13 single stranded templates. The quality of sequence obtained with Tfil polymerase with this chemistry similar to that produced by Taq polymerase. Good sequence data was obtained using 50 mM Tris-HCl buffer pH 8.8 containing 1.5 mM MgCl2 and 0.01% Tween 20 (v/v) with the ABI recommended reaction cycle. Good sequence data was obtained also with higher Mg2+, Tween 20 and Tris-HCl concentrations (up to 4 mM, 0.1% v/v and 135 mM respectively).

Greater differences were found between Taq polymerase and Tfil polymerase when sequencing data was generated using dye-labelled dideoxynucleotide terminators. Tfil polymerase incorporated the terminators more efficiently than Taq polymerase at the 60°C extension temperature recommended for Taq polymerase in dye-terminator sequencing, such that the sequence terminated prematurely and piled up at the beginning. Full length sequence was obtained by reducing the terminatoπdNTP ratio which would offer an advantage of economy as the cost of the dye-labelled terminators represent the major portion of the process cost.

PCR amplification of DNA. The polymerase was suitable for the efficient PCR amplification using plasmid or genomic DNA as template (Fig 6). As was found for the polymerase assay, increasing KCl concentrations in the PCR buffer caused a decrease in yield of PCR product. By varying the KCl, (NH4)2SO4, MgCl2 and Tween 20 concentrations in the PCR buffer (Fig 6) and measuring the yield of PCR product, it was found that the most suitable buffer for PCR amplification using Tfil polymerase was 50 mM Tris-HCl buffer pH 8.8 containing 1.5 mM MgCl2 and 0.01% Tween 20. For some templates, a higher MgCl2 and Tween 20 concentration (2 mM and 0.02% respectively) improved the PCR amplification by reducing the number and intensity of unwanted products.

PCR product was obtained over a wide range of pH values using the primers P4 and P5, with the greatest yield obtained at pH 8.5 to 9.0 (Fig 3). During PCR, the polymerase was estimated to have an extension rate of about 1000 bases/min. as determined by a primer extension assay (Carballeira et al. 1990. Biotechniques 9, 274-281) using the nucleotide and buffer conditions employed for PCR. Reverse transcription/ PCR amplifications. Tfil polymerase also shows very efficient reverse transcriptase activity on a variety of RNA templates. Using the protocol described in methods, Tfil polymerase was able to reverse transcribe and PCR amplify (RT/PCR) as little as 32 pg of template RNA (Fig 7). Greater sensitivity could be achieved by using more PCR cycles or by analysing a larger proportion of the reaction product on an agarose gel. The polymerase was also able to RT/PCR amplify target present in more complex samples. Fig 8 shows RT/PCR amplification from total cellular RNA of a 200 bp fragment from topoisomerase Ha message and the entire 800 bp message of a-lactalbumin. Possible contamination of the RNA template with DNA that potentially could be a target in the PCR reaction was tested by attempting to PCR amplify without initial reverse transcription. The absence of any PCR product using either Taq polymerase or Tfil polymerase with conditions that were optimal for PCR amplification showed that there was no contamination of the RNA template with DNA and that the product formed was from PCR amplification of cDNA generated in the first phase of the RT/PCR. Also, the buffer used for reverse transcription was found not to be suitable for PCR amplification (no product was obtained using the primers P4 and P5 with the plasmid template) and that the buffer used for PCR failed to give product in RT/PCR when it was used for the reverse transcription step.

DEPOSIT

Thermus filiformis is a named strain already deposited in the American Type Culture Collection under ATCC accession number 43280.

PHYLOGENY OF THE SPECIES Thermus filiformis.

The species Thermus filiformis is distinct from Thermus aquaticus, "Thermus fiavus" and "Thermus thermophilus" and this has been demonstrated by a number of methods. Moφhologically, Thermus filiformis is atypical of the genus in that it is filamentous and analysis of the 16S rRNA gene from this organism and others shows that it is part of a group of organisms unique to New Zealand (Fig 9). The complete 16S rRNA genes of twenty Thermus isolates have been sequenced (Saul et al. 1993. Int. J. System. Bacteriol. 43, In press) and the data subjected to a phylogenetic analysis using the method of maximum parsimony. The data suggest a close relationship between "T. fiavus" and "T. thermophilus" but that T. aquaticus and T. filiformis are more distally related to these organisms and to each other. The validity of the branches within the tree were assessed by "bootstrapping". Felsentein, 1985. Evolution 39, 783-791, suggests that confidence limits of the data should be set so that a branch should only be accepted if it occurs in more than 95% of the bootstrapped trees. T. filiformis, T. aquaticus and the "T. fiavus" /"T. thermophilus" pair are separated in 100% of the trees (Fig 9).

These results suggest that the cloned enzyme of this invention is a versatile thermostable DNA polymerase that is suitable for DNA synthetic activity from both DNA and RNA templates, as well as being highly suitable for automated DNA sequencing. Tfil polymerase performs both of these reaction in the same buffer, has a high salt tolerance and is suitable for use in cycled fluorescent DNA sequencing using both dye-primers and dye-terminators. Its greater affinity than existing enzymes for dye-terminators means that a reduction in consumption of these costly chemicals can be attained. All these properties in a single enzyme make Tfil polymerase a very useful tool for the molecular biologist.

CITED LITERATURE

Kaledin et al. 1980. Biokimiya 44, 644-651

Kaledin et al. 1981. Biokimiya 46, 1247- 1254

Kaledin et al. 1982. Biokimiya 47, 1515- 1521

Ruttimann, et al. 1985. Eur. J. Biochem 149, 41-46

Neuner et al. 1990. Arch. Microbiol. 153, 205-207

Lawyer et al. 1989. J. Biol. Chem. 264, 6427-6437

Engelke et α/. 1990. Anal. Biochem. 191

Lundberg et al. 1991. Gene 108, 1 -6

Perler et al. 1992. Proc. Natl. Acad. Sci. USA 89, 5577-5581 Brock etal. 1969. J. Bacteriol. 98, 289-297 Oshima and Imahori. 1974. J. Syst. Bacteriol. 24, 104-112 Saiki et al. 1972. Agric. Biol. Chem. 34, 2357-2366 Hudson et al. 1987. Int. J. Syst. Bacteriol. 37, 431-436 Saul et al. 1993. Int. J. Syst. Bacteriol. 43, 754-760 Jones, et al. 1989. Nucl. Acids Res. 17, 8387-8388 Meyers and Gelfand 1991. Biochem. 30, 7661-7666 Day et al. 1993 Anal. Biochem., 211; 174-176 Engelke et al. (1990) Anal. Biochem. 191: 396-400

Finally it will be appreciated that various alterations and modifications may be made to the foregoing without departing from the scope of this invention as claimed.

Claims

CLAIMS:
1. A purified heat-stable DNA polymerase enzyme (EC 2.7.7.7, a DNA nucleotidyltransferase DNA-directed enzyme), characterised in that it has reverse transcriptase activity in the presence of magnesium ions.
2. A DNA polymerase as claimed in claim 1, characterised in that it has reverse transcriptase activity in the substantial absence of manganese ions.
3. A DNA polymerase as claimed in claim 2, characterised in that it has been isolated from the eubacterium Thermus filiformis.
4. A DNA polymerase as claimed in claim 1, having an amino acid sequence Tfil substantially as shown in Fig 2.
5. A DNA polymerase as claimed in claim 1, having a pH-dependent activity as shown graphically in Fig 3.
6. A recombinant DNA sequence capable of encoding polymerase activity of the microorganism Thermus filiformis.
1. A recombinant DNA sequence as claimed in claim 6 wherein the DNA sequence is a recombinant DNA sequence capable of encoding amino acid residues 1 to 833 as shown in figure 1.
8. A recombinant DNA plasmid that comprises the DNA sequence substantially as shown in figure 1, inserted into plasmid vectors and which can be used to drive the expression of the thermostable DNA polymerase of Thermus filiformis in a host cell transformed with the plasmid.
9. A recombinant plasmid as claimed in claim 8, comprising the vector pUC18 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2300.
10. A recombinant plasmid as claimed in claim 8, comprising the vector pT7-7 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2303. 11. A host cell transformed with the recombinant DNA of the invention as claimed in claim 8.
12. The host cell as claimed in claim 11, being Escherischia coli I pNZ2300, the strain being designated PB5900.
13. The host cell as claimed in claim 11, being Escherischia coli I pNZ2300, the strain being designated PB5904.
14. A purified heat-stable DNA polymerase enzyme (EC 2.7.7.7, a DNA nucleotidyltransferase DNA-directed enzyme), isolated from the eubacterium Thermus filiformis.
15. A DNA polymerase as claimed in claim 14, having a molecular mass of about 93.4 KDa.
16. A DNA polymerase as claimed in claim 14, that is substantially in its native form.
17. A DNA polymerase as claimed in claim 14, that is substantially in its recombinant form.
18. A DNA polymerase as claimed in claim 14, having a dependency on magnesium ion concentration, as shown in Fig 4.
19. A DNA polymerase as claimed in claim 18, having reverse transcriptase activity in the substantial absence of manganese ions.
20. A DNA polymerase as claimed in claim 19, further comprising a DNA polymerase having an activity, referenced to Taq polymerase, as illustrated in Fig 5.
PCT/NZ1994/000135 1993-11-25 1994-11-23 Improved polymerase WO1995014770A1 (en)

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WO1997012991A1 (en) * 1995-09-22 1997-04-10 Terragen Diversity Inc. Method for isolating xylanase gene sequences from soil dna, compositions useful in such method and compositions obtained thereby
EP0821059A2 (en) * 1996-07-25 1998-01-28 THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH Method for reverse transcription
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
EP1152062A2 (en) * 2000-04-18 2001-11-07 F.Hoffmann-La Roche Ag High temperature reverse transcription using mutant DNA polymerases
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EP0592035A2 (en) * 1992-10-07 1994-04-13 Johnson & Johnson Clinical Diagnostics, Inc. Thermostable DNA polymerase composition comprising a temperature sensitive polymerase inhibitor, diagnostic test kits and methods of use

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WO1992006202A1 (en) * 1990-09-28 1992-04-16 F. Hoffmann-La Roche Ag PURIFIED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM $i(THERMOSIPHO AFRICANUS)
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WO1997012991A1 (en) * 1995-09-22 1997-04-10 Terragen Diversity Inc. Method for isolating xylanase gene sequences from soil dna, compositions useful in such method and compositions obtained thereby
US6441148B1 (en) 1995-09-22 2002-08-27 Terragen Diversity, Inc. Method for isolation of xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby
US5849491A (en) * 1995-09-22 1998-12-15 Terragen Diversity Inc. Method for isolating xylanase gene sequences from soil DNA, compositions useful in such method and compositions obtained thereby
EP0821059A3 (en) * 1996-07-25 1998-04-22 THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH Method for reverse transcription
US6013488A (en) * 1996-07-25 2000-01-11 The Institute Of Physical And Chemical Research Method for reverse transcription
US6221599B1 (en) 1996-07-25 2001-04-24 The Institute Of Physical And Chemical Research Method for improving heat stability of RNA
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EP0821059A2 (en) * 1996-07-25 1998-01-28 THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH Method for reverse transcription
US6372437B2 (en) 1996-07-25 2002-04-16 The Institute Of Physical & Chemical Research Method for improving heat stability of RNA
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6764839B2 (en) 1996-11-22 2004-07-20 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
EP1152062A2 (en) * 2000-04-18 2001-11-07 F.Hoffmann-La Roche Ag High temperature reverse transcription using mutant DNA polymerases
EP1152062A3 (en) * 2000-04-18 2002-09-11 F.Hoffmann-La Roche Ag High temperature reverse transcription using mutant DNA polymerases
US7179590B2 (en) 2000-04-18 2007-02-20 Roche Molecular Systems, Inc High temperature reverse transcription using mutant DNA polymerases
WO2007060707A2 (en) * 2005-11-23 2007-05-31 Diasorin Spa Reagents and method for simultaneous nucleic acid amplification and detection
WO2007060707A3 (en) * 2005-11-23 2007-10-18 Daniel Adlerstein Reagents and method for simultaneous nucleic acid amplification and detection

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