EP0730642A1 - Polymerase amelioree - Google Patents

Polymerase amelioree

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Publication number
EP0730642A1
EP0730642A1 EP95900945A EP95900945A EP0730642A1 EP 0730642 A1 EP0730642 A1 EP 0730642A1 EP 95900945 A EP95900945 A EP 95900945A EP 95900945 A EP95900945 A EP 95900945A EP 0730642 A1 EP0730642 A1 EP 0730642A1
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Prior art keywords
dna
polymerase
dna polymerase
recombinant
activity
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EP95900945A
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German (de)
English (en)
Inventor
Peter Leonard 3a Pukerangi Crescent BERGQUIST
Darren John Day
Moreland David 30 Laurie Avenue GIBBS
Rosalind Alison 30 Laurie Avenue REEVES
David James 5/4 Cowie Street SAUL
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PACIFIC ENZYMES Ltd
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PACIFIC ENZYMES Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • 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. • * *,
  • 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.
  • 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 K m at a high temperature.
  • 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).
  • the polymerase gene has been cloned and expressed (Lawy r et al.
  • 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.
  • thermostable enzymes in the PCR reaction, which is used to amplify existing nucleic acid sequences by a very large ratio.
  • 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.
  • T. flavus a species isolated from Waimangu Hot Springs, New Zealand (Hudson et al. 1987. Int. J. Syst. Bacteriol. 37, 431-436).
  • T. thermophilus nor “T.fiavus” are yet accepted as validly named species, whereas T. filiformis and T. aquaticus are accepted as distinct, valid species.
  • Thermus genus 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).
  • 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).
  • PCR polymerase chain reaction
  • RT PCR RNA rather than DNA templates
  • reverse transcription has been noted to varying degrees with Tth and Taq DNA polymerases (Jones, et al. 1989.
  • thermophilic DNA polymerases 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.
  • RT Moloney murine leukemia virus reverse transcriptase
  • avian myeloblastosis virus RT which are commonly used for cDNA synthesis
  • DNA polymerases 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.
  • a polymerase enzyme EC 2.7.7.7
  • 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.
  • 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.
  • a purified heat-stable DNA polymerase enzyme EC 2.7.7.7, a DNA nucleotidyltransferase DNA-directed enzyme
  • the invention comprises a recombinant DNA sequence that encodes DNA polymerase activity of the microorganism Thermus filiformis.
  • the DNA sequence is that shown in Fig 1.
  • the invention comprises a recombinant DNA sequence that encodes amino acid residues 1 to 833 as also shown in Fig 1.
  • 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.
  • the invention includes a recombinant plasmid comprising the vector pUC18 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2300.
  • the invention includes a recombinant plasmid comprising the vector pT7-7 carrying the Thermus filiformis DNA polymerase gene and designated pNZ2303.
  • the invention comprises a host cell transformed with the recombinant DNA of the invention.
  • the invention comprises such a host cell, being Escherischia c ⁇ /z ' /pNZ2300, the strain being designated PB5900.
  • the invention comprises such a host cell, being Escherischia coli/pNZ2303, the strain being designated PB5904.
  • the invention comprises a DNA polymerase having a molecular mass of about 93.4 KDa.
  • the invention comprises a DNA polymerase having a pH-dependent activity as shown in Fig 3.
  • the invention comprises a DNA polymerase having a dependency on magnesium ion concentration, as shown in Fig 4.
  • the invention comprises a DNA polymerase having Reverse Transcriptase activity in the substantial absence of manganese ions.
  • the invention comprises a DNA polymerase having an activity, referenced to Taq polymerase, as illustrated in Fig 5.
  • 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 ⁇ -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.
  • 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.
  • 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 my ⁇ D 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
  • 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.
  • 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.
  • 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 ⁇ -lactalbumin and using the dilutions as template for the RT/PCR reaction.
  • RT/PCR amplification of ⁇ -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 ⁇ -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.
  • 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 R ⁇ A transcript. Ten units of D ⁇ A polymerase were incubated with 200 ng of linearised plasmid or uncut plasmid, or 500 ng of R ⁇ A 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 R ⁇ A or plasmid D ⁇ A, 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 R ⁇ A gene from a thermophilic organism (p ⁇ Z2201). 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 ( ⁇ -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.
  • PCR and RT/PCR coupled amplifications 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 ⁇ - lactalbumin and topoisomerase Ha templates.
  • 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.
  • 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 ⁇ ngelke 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 ⁇ M 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.
  • 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 ⁇ M 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 Ec ⁇ RI and Sm ⁇ l, and the ligated products were transformed into E. coli strain DH.
  • Transformants were grown on L-agar containing 100 ⁇ g/ml ampicillin and IPTG/Xgal (5 mM and 33 ⁇ g ml respectively) to allow selection of recombinants.
  • 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 - Hindi ⁇ 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 ⁇ g/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 ⁇ g/ml each) and were grown at 32°C. Colonies were picked and grown in L-broth containing ampicillin and kanamycin (60 ⁇ g/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
  • Tfil DNA polymerase 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 ⁇ m filter to remove paniculate matter.
  • 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.
  • 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.
  • An alignment of the deduced amino acid sequences of these enzymes is Fig 2.
  • 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.
  • thermophilic DNA polymerases 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.
  • 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.
  • 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.
  • 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).
  • 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 terminato ⁇ dNTP ratio which would offer an advantage of economy as the cost of the dye-labelled terminators represent the major portion of the process cost.
  • 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).
  • 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).
  • RNA template was tested by attempting to PCR amplify without initial reverse transcription.
  • Thermus filiformis is a named strain already deposited in the American Type Culture Collection under ATCC accession number 43280.
  • Thermus filiformis is distinct from Thermus aquaticus, "Thermus fiavus” and "Thermus thermophilus” and this has been demonstrated by a number of methods. Mo ⁇ hologically, 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 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.

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Abstract

Enzyme thermophile, et plus particulièrement une polymérase d'ADN thermostable, isolée à partir de l'espèce Thermus néo-zélandaise appelée Thermus filiformis. On a également prévu des plasmides recombinés et des cellules hôtes transformées pouvant produire cette enzyme. L'enzyme se classe dans la classe EC 2.7.7.7: un type dirigé par ADN de nucléotidyltransférase d'ADN.
EP95900945A 1993-11-25 1994-11-23 Polymerase amelioree Ceased EP0730642A1 (fr)

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EP0851938A1 (fr) * 1995-09-22 1998-07-08 Terragen Diversity Inc. Procede d'isolement de sequences de gene de xylanase dans l'adn du sol, compositions utiles a ce procede et compositions ainsi obtenues
US6013488A (en) * 1996-07-25 2000-01-11 The Institute Of Physical And 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
US7179590B2 (en) * 2000-04-18 2007-02-20 Roche Molecular Systems, Inc High temperature reverse transcription using mutant DNA polymerases
AU2002247248B2 (en) * 2001-03-02 2007-07-05 University Of Pittsburgh Of The Commonwealth System Of Higher Education PCR method
EP1969140A2 (fr) * 2005-11-23 2008-09-17 DiaSorin S.p.A. Réactifs et méthode d'amplification et de détection simultanées d'acide nucléique

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US5338671A (en) * 1992-10-07 1994-08-16 Eastman Kodak Company DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody

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