WO2007127893A2 - Thermostable dna polymerase from thermotoga naphthophila and thermotoga petrophellia - Google Patents

Abstract

Disclosed are isolated, thermostable DNA polymerases from Thermotoga naphthophila and Thermotoga petrophellia respectively. These enzymes are useful for DNA sequencing, and polymerase chain reaction. Included within the scope of the present invention are various mutants (deletion and substitution) that retain thermostability and the ability to replicate DNA with substantially the same efficiency as the native Thermotoga naphthophila or Thermotoga petrophellia polymerase.

Description

THERMOSTABLE DNA POLYMERASE FROM THERMOTOGA NAPHTHOPHILA AND THERMOTOGA PETROPHELLIA

Cross-Reference to Related Applications This application claims priority to United States provisional patent application numbers 60/745,890 filed April 28, 2006 and 60/823,790 filed August 29, 2006; the entire disclosures of which are incorporated herein by reference.

Field of the Invention The present invention relates to novel thermostable DNA polymerases obtainable from the thermophilic organism Thermotoga naphthophila and Thermotoga petrophellia, respectively, to certain deletions and mutants of these enzymes, to genes and vectors encoding the wild type and mutant polymerases and their use in, polymerase chain reaction, and DNA sequencing.

Background of the Invention

DNA polymerases are a family of enzymes involved in DNA repair and replication. DNA polymerases have been isolated from E. coli (e.g. E. coli DNA polymerase I and the Klenow fragment thereof) and bacteriophageT4 DNA polymerase and more recently thermostable DNA polymerases have been isolated (e.g. from T. aquaticus, US Patent 4,889,818, and from T. litoralis). Thermostable DNA polymerases have been suggested (US Patent 4,683,195) for use in amplifying existing nucleic acid sequences in amounts that are large compared to that originally present. The polymerase chain reaction (PCR, US Patent 4,683,202) and strand displacement amplification (SDA) are two methods of amplifying nucleic acid sequences. PCR is based on the hybridization of oligonucleotide primers to specific sequences on opposite strands of the target DNA molecule, and subsequent extension of these primers with a DNA polymerase to generate two new strands of DNA which themselves can serve as a template for a further round of hybridization and extension. In PCR amplifications, the product of one cycle serves as the template for the next cycle such that at each repeat of the cycle the amount of the specific sequence present in the reaction can double leading to an exponential amplification process.

In reverse transcription/polymerase chain reaction (RT/PCR), a DNA primer is hybridized to a strand of the target RNA molecule, and subsequent extension of this primer with a reverse transcriptase generates a new strand of DNA, which can serve as a template for PCR. Preparation of the DNA template is preferably carried out at an elevated temperature to avoid early termination of the reverse transcriptase reaction caused by RNA secondary structure. Since most of the known, efficient reverse transcriptases come from animal viruses, there is a lack of efficient reverse transcriptases that act at elevated temperatures, e.g. above 50⁰C.

SDA differs from PCR in being an isothermal amplification process, i.e. all reactions occur at the same temperature without the need for elevated temperature to melt DNA strands. This is made possible by adoption of a reaction scheme which uses the ability of certain DNA polymerases when extending along a DNA template strand to displace any DNA molecules already hybridized to the template. In SDA this strand displacement is used to separate the double stranded DNA produced earlier in the reaction process and hence to maintain continuous amplification of the target DNA sequence (Walker, G.T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl. Acad. Sci. USA 89:392-396). SDA is therefore in principle more suited to use with large numbers of samples than PCR as the isothermal process, which is performed at temperatures of 37°C to 60 ⁰C, does not require stringent precautions to be taken to avoid evaporation and can be performed with simple temperature control equipment, for example in a standard laboratory incubator.

DNA polymerases, e.g. Sequenase, Klenow, Taq, etc, have also been extensively used in DNA sequencing, see for example "Molecular Cloning: A Laboratory Manual" (Sambrook, Fritsch, and Maniatis, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

Brief Description of the Invention Disclosed are thermostable DNA polymerases from Thermotoga naphthophila and Thermotoga petrophellia, respectively. These enzymes are useful for DNA sequencing, and polymerase chain reaction. Included within the scope of the present invention are various mutants (deletion and substitution) that retain thermostability and the ability to replicate DNA with substantially the same efficiency as the native polymerases.

Brief Description of the Drawings

Figure 1 is the DNA sequence from Thermotoga naphthophila encoding a full length thermostable DNA polymerase (SEQ ID NO:1).

Figure 2 is a contiguous open reading frame capable of encoding the full length polymerase from Thermotoga naphthophila (SEQ ID NO:2). Translation is of the open reading frame spanning SEQ ID NO:1 as shown in Figure 1, encoding native polymerase. Figure 3 is the amino acid sequence of the DNA polymerase from Thermotoga naphthophila, containing Y76C and F730Y mutations (SEQ ID NO:3).

Figure 4 is the amino acid sequence of the truncated version of DNA polymerase from Thermotoga naphthophila (SEQ ID NO:4). The second lysine was changed to Arginine for increased stability of the protein.

Figure 5 is a SDS-PAGE showing purification of the full-length Y76C, F730Y mutant Thermotoga naphthophila DNA polymerase enzyme. Lane 1, Low Molecular Weight protein standard; lane 2, Taq DNA polymerase as a positive control; lane 3, crude lysate; lane 4, Heparin column flow through; lanes 5-9, fractions 3-7 from the Heparin column purification, respectfully.

Figure 6 shows an endonuclease assay of the purified full-length Y76C, F730Y mutant Thermotoga naphthophila DNA polymerase enzyme. Lane 1, Molecular Weight

Standard (1 kb DNA ladder); lane 3, negative control (single-stranded DNA); lane 4, positive control (ss DNA); lane 5, 20 units Y76C, F730Y mutant enzyme (ss DNA); lane 6: 40 units Y76C, F730Y mutant enzyme (ss DNA); lane 8, negative control (double-stranded DNA); lane 9, positive control (ds DNA); lane 10: 20 units Y76C, F730Y mutant enzyme (ds DNA); lane 11 , 40 units Y76C, F730Y mutant enzyme (ds DNA). Lanes 2 and 7 were left blank.

Figure 7 shows the thermostability of full-length Y76C, F730Y mutant Thermotoga naphthophila DNA polymerase enzyme. Figure 8 shows sequencing results obtained using full length Y76C, F730Y mutant Thermotoga naphthophila DNA polymerase enzyme, compared to that of BigDye™ Terminator Cycle Sequencing kit (Applied Biosystems).

Figure 9 shows KCl tolerance test result of the full-length Y76C, F730Y mutant Thermotoga naphthophila DNA polymerase enzyme.

Figure 10 is an alignment of several DNA polymerase protein sequences. From the top, Thermotoga hypogea (SEQ ID NO: 13), Thermotoga maritime (SEQ ID NO: 14), Thermotoga naphthophila (SEQ ID NO :2), Thermus βliformis (SEQ ID NO : 15),

Thermusflavus (SEQ ID NO: 16), Thermus scotoductus (SEQ ID NO: 17) and Thermus thermophilus (SEQ ID NO: 18).

Figure 11 is the DNA sequence from Thermotoga petrophellia encoding a full length thermostable DNA polymerase (SEQ ID NO: 19).

Figure 12 is a contiguous open reading frame capable of encoding the full length polymerase from Thermotoga petrophellia (SEQ ID NO:20). Translation is of the open reading frame spanning SEQ ID NO: 19 as shown in Figure 11, encoding native polymerase.

Figure 13: Amino acid sequence comparison of the Thermotoga petrophellia DNA polymerase (SEQ ID NO:20) with that of Thermotoga naphthophila (SEQ ID NO:2). The comparison shows 13 differences between the two protein sequences. Figure 14: Sequence comparison of the nucleotide sequence that encodes the DNA polymerase enzyme of Thermotoga petrophellia (SEQ ID NO : 19) with that of Thermotoga naphthophila (SEQ ID NO:1).

Detailed Description of the Invention

In a first aspect, the present invention provides a purified DNA polymerase or fragment thereof having the DNA polymerase activity of Thermotoga naphthophila and having at least 90% amino acid homology, preferably at least 90% homology, most preferably at least 97 % amino acid homology, to at least a contiguous 40 amino acid sequence shown in Figure 2 (SEQ ID NO:2). Figure 2 represents the translation of the open reading frame of DNA sequence encoding a thermostable DNA polymerase from Thermotoga naphthophila (Figure 1) (SEQ ID NO:1) potentially encoding the native polymerase.

When used herein, the term amino acid homology means amino acid identity or conservative amino acid changes thereto. The DNA polymerase can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. The amino acid sequence will be substantially similar to the sequence shown in Figure 2, or fragments thereof. A sequence that is substantially similar will preferably have at least 80% homology (more preferably at least 90% and most preferably 98-100%) to the sequence of Figure 2.

By "identity" is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical or homologous residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.

The purified Thermotoga petrophellia DNA polymerase enzyme has a molecular weight of approximately 102,000 daltons when measured on SDS-PAGE. It possesses a 5 '-3' exonuclease activity. The temperature optimum of DNA synthesis is near 75°C under assay conditions. The optimum magnesium ion concentrations for DNA synthesis is 2mm

The term thermostable polymerase means an enzyme which is stable to heat (and heat resistant) and is suitable for use in SDA and/or sequencing at an elevated temperature, for example 70⁰C. The thermostable enzyme herein must satisfy a single criterion to be effective for the amplification reaction, i.e., the enzyme must not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect amplification. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. Preferably, the enzyme will not become irreversibly denatured at about 70⁰C but may become denatured at higher temperatures. The thermostable enzyme herein preferably has an optimum temperature at which it functions that is higher than about 40 ⁰C, which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) salt concentration and composition and (2) composition and length of primer, hybridization can occur at higher temperature (e.g., 45-70 ⁰C). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40⁰C, e.g., at 37 ⁰C, are also within the scope of this invention provided they are heat stable. Preferably, the optimum temperature ranges from about 50 to 80⁰C, more preferably 50-70⁰C. When used herein, the term a DNA polymerase or fragment thereof having the DNA polymerase activity of Thermotoga naphthophila means a DNA polymerase or fragment thereof (as hereinafter defined) which has the ability to replicate DNA with substantially the same efficiency as the enzyme encoded by the SEQ ID NO: 1. By substantially the same efficiency is meant at least 80% and preferably at least 90% of the efficiency of the enzyme encoded by SEQ ID NO:1 to incorporate deoxy- nucleotides.

The invention also encompasses a stable enzyme composition which comprises a purified thermostable DNA polymerase from Thermotoga naphthophila in a buffer. The Thermotoga petrophellia DNA polymerases of the present invention are preferably in a purified form. By purified is meant that the DNA polymerase is isolated from a majority of host cell proteins normally associated with it; preferably the polymerase is at least 10% (w/w), e.g. at least 50% (w/w) , of the protein of a preparation, even more preferably it is provided as a homogeneous preparation, e.g. homogeneous solution. Preferably the Thermotoga petrophellia DNA polymerase is a single polypeptide on an SDS polyacrylamide gel.

Buffers around neutral pH (5-9) such as 5-100 mM TrisHCl, HEPES or MES are suitable for use in the current invention.

In another aspect, the present invention provides a purified DNA polymerase or fragment thereof having the DNA polymerase activity of Thermotoga petrophellia and having at least 90% amino acid homology, preferably at least 95% homology, most preferably at least 97 % amino acid homology, to at least a contiguous 40 amino acid sequence shown in Figure 12 (SEQ ID NO:20). Figure 12 represents the translation of the open reading frame of DNA sequence encoding a thermostable DNA polymerase from Thermotoga petrophellia (Figure 11) (SEQ ID NO: 19) potentially encoding the native polymerase.

The amino acid sequence of the DNA polymerase of the Thermotoga petrophellia resembles that of Thermotoga naphthophila. In fact, there are just 13 amino acid sequence variations between the two sequences (Figure 13), therefore the two sequences share a sequence identity of about 98.5%. In contrast, the nucleotide sequences are 96.5% identical (Figure 14).

As discussed above, the DNA polymerase of Thermotoga petrophellia share similar amino acid sequence and structure as compared to the sequence of Thermotoga naphthophila. The properties of the DNA polymerase enzymes are therefore expected to be similar as well. Therefore, we predict that the Thermotoga petrophellia DNA polymerase has a molecular weight of approximately 102,000 daltons. It possesses a

5'-3' exonuclease activity. The temperature optimum of DNA synthesis is near 75°C under assay conditions. The optimum magnesium ion concentrations for DNA synthesis is 2mm. The invention also encompasses a stable enzyme composition which comprises a purified thermostable DNA polymerase from Thermotoga petrophellia in a buffer. The present invention also provides a gene encoding a Thermotoga naphthophila polymerase. Fig. 1 represents nucleotides of the cloned gene encoding the

Thermotoga naphthophila polymerase (SEQ ID NO:1). The present invention also provides a gene encoding a Thermotoga petrophellia polymerase. Figure 11 represents nucleotides of the cloned gene encoding the

Thermotoga petrophellia polymerase (SEQ ID NO: 19).

It has been found that the entire amino acid sequence of the polymerase is not required for enzymatic activity. Thus, for example, the exonuclease domain of the enzyme has been deleted to give an enzyme which retains enzyme activity . This exonuclease-free enzyme is analogous to the Klenow fragment of E. coli DNA polymerase I. Thus, the present invention also provides fragments of the polymerase which retain the DNA polymerase activity of Thermotoga naphthophila but have one or more amino acids deleted, preferably from the amino-terminus, while still having at least 80% amino acid homology to at least a 40 contiguous amino acid sequence shown in Figure 2 (SEQ ID NO:2).

The present invention also provides fragments of the polymerase which retain the DNA polymerase activity of Thermotoga petrophellia but have one or more amino acids deleted, preferably from the amino-terminus, while still having at least 80% amino acid homology to at least a 40 contiguous amino acid sequence shown in Figure 12 (SEQ ID NO:20).

In a further aspect, the present invention provides a thermostable DNA polymerase which corresponds to the DNA polymerase from Thermotoga naphthophila in which up to one third of the amino acid sequence at the amino- terminus has been deleted. In particular, fragments of Thermotoga naphthophila having N-terminal deletions (SEQ ID NO:4) have been found to retain enzyme activity.

It is preferred that the 5 '-3' exonuclease activity of the Thermotoga naphthophila DNA polymerase is removed or reduced. This may be achieved by deleting the amino acid region of the enzyme responsible for this activity, e.g. by deleting up to one third of the amino acid sequence at the amino terminus (See Figure 4, SEQ ID NO:4), or by appropriate amino acid changes (Y76C, See Figure 3, SEQ ID NO:3).

In yet another aspect, the present invention provides a thermostable DNA polymerase which corresponds to the DNA polymerase from Thermotoga petrophellia in which up to one third of the amino acid sequence at the amino- terminus has been deleted.

In addition to the N-terminal deletions and amino acid changes to remove the exonuclease activity, the enzymes may have conservative amino acid changes compared with the native enzymes which do not significantly influence thermostability or enzyme activity. Such changes include substitution of like charged amino acids for one another or amino acids with small side chains for other small side chains, e. g. ala for val. More drastic changes may be introduced at non-critical regions where little or no effect on polymerase activity is observed by such a change.

Joyce and Steitz, Annu. Rev. Biochem, 63:777-822, 1994, discuss various functions of DNA polymerases including the catalytic center, the binding site for the 3 ' terminus of the primer, and the dNTP binding site. In particular, it mentions mutations that affect the binding of dNTP in the ternary complex. European Patent Application 0655506 A discloses that the presence of a polar, hydroxyl containing amino acid residue at a position near the binding site for the dNTP substrate is important for the polymerase being able to efficiently incorporate a dideoxynucleotide. The modification of the dNTP binding site for the dNTP substrate in DNA polymerase obtainable from Thermotoga naphthophila by the inclusion of a polar, hydroxyl containing amino acid residue at a position near the binding site increases the efficiency of the polymerase to incorporate dideoxynucleotides. Preferably the polar, hydroxyl containing amino acid is tyrosine. It has also been found that replacing the phenylalanine at the critical position with tyrosine improves the incorporation of dideoxynucleotides when the enzyme is used for sequencing. In particular, a polymerase from Thermotoga naphthophila in which the exonuclease activity has been reduced e.g. by point mutation or deletion and which has the phenylalanine at the critical position replaced by an amino acid (e.g. tyrosine) which increases the efficiency of the enzyme to incorporate dideoxynucleotides at least 20 fold compared to the wild type enzyme, is a particularly preferred enzyme for use in sequencing.

Several modified Thermotoga naphthophila DNA polymerase sequences are provided, containing the FY mutation and other, preferable point mutations and truncations (See Figures 3 and 4, SEQ ID NO:3 and SEQ ID NO:4).

The DNA polymerases of the present invention can be constructed using standard techniques familiar to those who practice the art. By way of example, in order to prepare a polymerase with the phenylalanine to tyrosine mutation, mutagenic PCR primers can be designed to incorporate the desired Phe to Tyr amino acid change (FY mutation in one primer). Deletion of the exonuclease function is carried out by PCR to remove the amino terminus, or standard techniques of site directed mutagenesis to generate point mutations.

Improved expression of the DNA polymerases of the present invention can be achieved by introducing silent codon changes (i.e., the amino acid encoded is not changed). Such changes can be introduced by the use of mutagenic PCR primers. Silent codon changes such as the following increase protein production in E. coli: substitution of the codon GAG for GAA; substitution of the codon AGG, AGA, CGG or CGA for CGT or CGC; substitution of the codon CTT, CTC, CTA, TTG or TTA for CTG; substitution of the codon ATA for ATT or ATC; substitution of the codon GGG or GGA for GGT or GGC.

Genes encoding the DNA polymerases in which up to one third of the amino acid sequence at the amino terminus has been deleted and which have the exonuclease activity removed by point mutation and such polymerases which incorporate the phenylalanine to tyrosine modification are also provided by the present invention. In a yet further aspect, the present invention provides a host cell comprising a vector containing the gene encoding the DNA polymerases having an amino acid sequence corresponding to native Thermotoga naphthophila or differentiated from this in that it lacks up to one third of the N-terminal amino acids and optionally has mutations as shown in SEQ ID NO:3 and SEQ ID NO:4.

In a further aspect, the present invention provides a host cell comprising a vector containing the gene encoding the DNA polymerase having an amino acid sequence corresponding to native Thermotoga petrophellia or differentiated from this in that it lacks up to one third of the N-terminal amino acids and optionally has mutations as discussed above.

The present invention provides a method for determining the nucleotide base sequence of a DNA molecule. The method includes providing a DNA molecule, annealing with a primer molecule able to hybridize to the DNA molecule; and incubating the annealed molecules in a vessel containing at least one, and preferably four deoxynucleotide triphosphate, and a DNA polymerase of the present invention preferably one containing the phenylalanine to tyrosine mutation. Also provided is at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base. The method further includes separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined.

In preferred embodiments, the sequencing is performed at a temperature between 40 and 75°C.

In other preferred embodiments, the DNA polymerase has less than 1000, 250, 100, 50, 10 or even 2 units of exonuclease activity per mg of polymerase (measured by standard procedure, see below) and is able to utilize primers having only 4, 6 or 10 bases; and the concentration of all four deoxynucleoside triphosphates at the start of the incubating step is sufficient to allow DNA synthesis to continue until terminated by the agent, e.g. a ddNTP.

Preferably, more than 2, 5, 10 or even 100 fold excess of a dNTP is provided to the corresponding ddNTP.

In a related aspect, the invention features a kit or solution for DNA sequencing including a DNA polymerase of the present invention and a reagent necessary for the sequencing such as dITP, deaza dGTP, a chain terminating agent such as a ddNTP, and optionally a pyrophosphatase. The DNA polymerases of the present invention containing the phenylalanine to tyrosine mutation are suitably used in sequencing, preferably in combination with a pyrophosphatase. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention containing the phyenylalanine to tyrosine mutation in combination with a pyrophosphatase, preferably a thermostable pyrophosphatase from Thermoplasma acidophilum.

In another related aspect, the invention features a method for sequencing a strand of DNA essentially as described above with one or more (preferably 2, 3 or 4) deoxyribonucleoside triphosphates, a DNA polymerase of the present invention, and a first chain terminating agent. The DNA polymerase causes the primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each first DNA product having a chain terminating agent at its elongated end, and the number of molecules of each first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases. The method also features providing a second chain terminating agent in the hybridized mixture at a concentration different from the first chain terminating agent, wherein the DNA polymerase causes production of a second series of second DNA products differing in length of the elongated primer, with each second DNA product having the second chain terminating agent at its elongated end. The number of molecules of each second DNA product is approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and is distinctly different from the number of molecules of all the first DNA products having a length differing by no more than 20 bases from that of said second DNA products.

In preferred embodiments, three or four such chain terminating agents can be used to make different products and the sequence reaction is provided with a magnesium ion, or even a manganese or iron ion (e. g. at a concentration between 0.05 and 100 mM, preferably between 1 and 1OmM); and the DNA products are separated according to molecular weight in four or less lanes of a gel.

In another related aspect, the invention features a method for sequencing a nucleic acid by combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a DNA polymerase of the present invention, and at least two chain terminating agents in different amounts, under conditions favoring extension of the oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced. The method further includes separating the nucleic acid fragments by size and determining the nucleic acid sequence. The agents are differentiated from each other by intensity of a label in the primer extension products.

In another aspect, the invention features a method for polymerase chain reaction in the presence of a polymerase stabilizing agent, utilizing an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Thermotoga naphthophila or Thermotoga petrophellia and an exonuclease activity removed. The polymerase stabilizing agents include, but are not limited to, glycerol (10 to 50% final concentration), trimethylamine-N-oxide (TMANO; up to 4 M final concentration), and N-methylmorpholine-N-oxide (MMO; up to 3 M final concentration). Preferably, glycerol is used at a final concentration of 30%. By polymerase stabilizing agent is meant an agent which allows the use of the polymerase in PCR and RT/PCR. These agents reduce the denaturing temperature of the template and stabilize the polymerase. By stabilize is meant make temperature stable. By final concentration is meant the final concentration of the agent in the PCR or RT/PCR solution. The invention also features kits, with polymerase stabilizing agents, for polymerase chain reaction having an enzymatically active DNA polymerase with at least 80% identity in its amino acid sequence to the DNA polymerase of Thermotoga naphthophila or Thermotoga petrophellia and an exonuclease activity removed. The polymerase stabilizing agents include, but are not limited to, glycerol (10 to 50% final concentration), TMANO (up to 4 M final concentration), and MMO (up to 3 M final concentration). Preferably, glycerol is used at a final concentration of 30%. Also encompassed are solutions for use in polymerase chain reaction, having polymerase stabilizing agents including, but not limited to, glycerol (10 to 50% final concentration), TMANO (up to 4 M final concentration), and MMO (up to 3 M final concentration), and an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Thermotoga naphthophila or Thermotoga petrophellia and an exonuclease activity removed. Preferably, glycerol is used at a final concentration of 30%. In preferred embodiments the exonuclease activity is removed by an N-terminal deletion, by deleting up to one third of the amino acid sequence at the N-terminal, or by substitution of an amino acid in the amino terminal one third of the protein, and the glycerol concentration is 30%.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.

Examples

The following examples serve to illustrate the DNA polymerases of the present invention and are not intended to be limiting.

Example 1 : Cloning of native Thermotoga naphthophila polymerase and generation of mutant enzymes

The Thermotoga naphthophila bacterial strain was obtained from DSMZ. Five micro liters of the culture was amplified using the GenomiPhi kit (GE Healthcare). The amplified genomic DNA was used in the subsequent PCR reactions. The DNA polymerase gene was amplified under standard PCR conditions, using primers designed based on the available Thermotoga maritime consensus DNA sequence, with the introduction of restriction enzyme sites. The primer at N-terminus (with Kpn I site) is 5'- CCG GGT ACC ATG GCG AGG CTA TTT CTC - 3' (SEQ ID NO:5). The primer at the C-terminus (with Hind III site) is 5' - CCC AAG CTT TCA CGA CCA TGT TTT GCC - 3' (SEQ ID NO:6). The full length polymerase gene was cloned into pEXT20, a tac promoter expression vector (NCBI # U51557) at the Kpn I and Hind III Sites. The DNA polymerase protein was expressed in E. coli JM109 host cells.

The N-terminal truncated version of the Thermotoga naphthophila DNA polymerase was cloned from genomic DNA by pfu PCR using Kpn I and Hind III restriction sites and the following primers: Forward: 5'- CCG GGT ACC ATG AGA GAA CTG GAA TTC GCA TCC ATC -3' (SEQ ID NO:7); Reverse: 5'- CCC AAG CTT TCA CGA CCA TGT TTT GCC -3' (SEQ ID NO: 8). The second Lysine (K) was changed to Arginine (R) to increase the stability of the protein. Amplified product was cloned into pEXT20 vector as described above.

The point mutants were generated by using the Quickchange site-directed mutagenesis kit (Stratagene). The following primers were used: (1) for FY, forward: 5 '- GCT GGT AAG ATG GTG AAC TAC TCT ATC ATA TAC GGT GTA -3' (SEQ ID NO:9), and reverse: 5'- TAC ACC GTA TAT GAT AGA GTA GTT CAC CAT CTT ACC AGC -3' (SEQ ID NO: 10); (2) for YC, forward: 5'- CAC AAG CTC CTC GAG ACT TGC AAG GCT CAA AGA CCA AAG -3' (SEQ ID NO:11), and reverse: 5'- CTT TGG TCT TTG AGC CTT GCA AGT CTC GAG GAG CTT GTG -3 ' (SEQ ID NO: 12). Pfu High-Fidelity polymerase and standard PCR conditions were used.

Example 2: Purification of Cloned Enzymes from Thermotoga naphthophila

The full length and truncated enzyme clones were purified using similar protocols. The following describes, as an example, the fermentation and purification of the full length Y76C, F730Y mutations polymerase from Thermotoga naphthophila.

A single colony containing the pEXT20 vector with the DNA sequence encoding the full length Y76C, F730Y mutations polymerase was inoculated and grown in 22ml LB/Amp for 5hrs. The culture was then transferred to fresh 200ml 2X YT /Amp 100 and grown overnight at 37⁰C with shaking (250 rpm). The overnight culture was used to start 4 X IL fermentation using 2x YT medium, with a starting O. D.600=0.2. ImM IPTG was added to induce protein expression when the culture reaches O.D.₆₀o = 1.6. After a 2 hours induction, the cells were harvested by centrifugation. Sixteen grams of cell pellet was obtained from the 4 liter culture. Total 16g of cell paste was resuspended in 45 ml lysis buffer (50 mM Tris-Cl, pH 8.5; 100 mM NaCl; 1 mM EDTA; 0.1 % NP-40; 0.1 % Tween 20 and ImM PMSF), passed through Avestin cell disrupter at 17 kpsi. Lysis was carried out at 70 ⁰C for 20 min. Cool on ice for 30 minutes and centrifuge at 10,000 rpm for 20 min. The clear supernatant (200ml) is used for further purification experiment. A faint band of about 102 Kb is observed on an SDS gel, from the crude cell lysate (data not shown).

The Thermotoga naphthophila polymerase enzyme was purified from the clear lysate. Forty ml of the clear lysate (out of 200 ml total) was used to run directly on a pre-equilibrated Heparin column (pre-equilibrated with buffer A (5 OmM Tris 8.5, 1 mM EDTA, 5% glycerol) with 10OmM NaCl). The loading was slow due to the presence of high concentration of DNA in the lysate. After loading, the column was washed with buffer A, then eluted with gradient elution of 0-100% B (buffer A + IM NaCl) in 10 column volumes. Five ml fractions were collected. Figure 5 shows the result of the Heparin column purification of the full-length Y76C, F730Y mutant enzymes from Thermotoga naphthophila. Fractions 4, 5 6 and 7 show a clear band of approximately the right size (102 kD) with peak at fractions 5 and 6.

The rest of the clear supernatant (160ml) was treated with PEI to 0.3% per 120 OD₂₆o to remove contaminant DNA. The sample was kept on ice for 10 min before centrifuging at 10,000 rpm/20min to precipitate the DNA. The clear PEI treated supernatant was then treated with ammonium sulfate to 70%, and subsequently centrifuged to collect the protein containing pellet. The pellet was solubilized in 20ml of buffer A and dialyzed against buffer A for 3 hrs at 4 ⁰C. The dialyzed sample was then loaded onto a pre-equilibrated Heparin column, washed and eluted as described above. Fractions containing the expected band (enzyme) were then combined, dialyzed against buffer A. The dialyzed fractions were then loaded onto HT Q-Sepharose column pre-equilibrated with buffer A, washed with buffer A and eluted with gradient elution from 0-40% B in 15 column volume and 40-100% B in 4 column volumes. Five ml fractions were collected. SDS-gel of fractions from HT Q elution shows a band of the expected size, with the peak of the protein appearing in fractions 8 and 9 (data not shown).

Heparin fractions containing the purified protein (fractions 4, 5, 6 and 7) from the 40ml purification above were also combined, dialyzed and run through HT Q. The results are similar to the ones obtained for the 160 ml purification. Both fractions 8 off HT Q from the two purifications were combined and dialyzed against the storage buffer (5OmM Tris 8.5, ImM EDTA, 10OmM KCl, 0.5% NP-40, 0.5% Tween-20, 50% Glycerol). Both fractions 9 off HT Q from the two purifications were combined and dialyzed against the storage buffer. BCA assay of the semi-purified DNA polymerase protein gives a concentration of 168 ng/ul (fraction 8) and 167ng/ul (fraction 9),respectively. Polymerase assay using alpha ³³P[dATP] indicated that both fraction Q8 and Q9 have DNA polymerase activity.

Example 3: Characterization of Enzymes from Thermotoga naphthophila Endonuclease Contamination: No endonuclease contamination was detectable at 20 - 40 units of the purified full-length Y76C, F730Y mutant Thermotoga naphthophila enzyme, when incubated overnight with lμg DNA (both single- and double-stranded, Figure 6).

Thermostability Thermostability of the enzymes was assayed at 95⁰C, as 50% activity at time determined by Polymerase assay. Aliquots of 7μl of the enzyme in 8 tubes were identified as 0, 2min, 5min, 10, 20, 30, 45, and 60min. The tubes were incubated at 95⁰C according to the time marked on the tubes. After heating, the tubes were immediately kept on ice and the polymerase assay was performed as described below.

Polymerase assay was performed by taking 5μl of the 7 μl heated enzyme with 45 μl of reaction buffer containing 25mM TAPS, 25mM KcI, 2mM MgC12, ImM Beta-mercapto ethanol, 20OuM each dNTPs, and 20ug of sperm DNA and 0.4uCi alpha 33P dATP. The reaction mix was incubated at 74⁰C for 30min and the incorporations were measured. The counts show the polymerase activity of the enzyme (Figure 7).

DNA sequencing

The sequencing premix composition was formulated with DYEnamic ET terminators and dITP/dA, T, C (2500 μM dITP, 500 μM dCTP, 500 μM dATP, and 500 μM dTTP). The ratio of dNTP:ddNTP is 156. Each reaction composition also contains 20 units of enzyme mix from TS II or the Y76C, F730Y mutant enzyme from Thermotoga naphthophila.

5 pmoles of -40 Ml 3 primer, 200 ng Ml 3 DNA, 8 μl of terminator premix

composition and water were added to a total volume of 20 μl. Reaction mixtures were cycled through 95°C, 20 seconds; 50⁰C, 30 seconds; and 60⁰C, 60 seconds, repeated 30 times. Reactions were then held at 4°C until purification and analysis.

The samples were purified and analyzed according to manufacturer's instructions, and run on ABI 3100 capillary sequencing instrument (Applied Biosystems). The resulting electropherograms using enzyme mix from TS II and Thermotoga naphthophila polymerase are shown in Figure 8. It shows that in a sequencing reaction, the Y76C, F730Y mutant enzyme from Thermotoga naphthophila performs similarly to that of TS II.

KCl Tolerance KCl tolerance assay was performed as 50% activity at mM KCl as determined by Pol assay. 5μl of enzyme was mixed with 45ul of polymerase reaction buffer as described earlier with various KcI concentrations (0, 25, 50, 75, 100, 150, 200 and 300 mM KCl.) KCl tolerance plot for the Y76C, F730Y mutant enzyme from Thermotoga naphthophila is shown in Figure 9. The enzyme is more active in low salt (<50 mM KCl).

Example 4: Analysis of the polymerase gene sequence from Thermotoga petrophellia The Thermotoga petrophellia bacterial strain was obtained from ATCC. Five micro liters of the culture was used to amplify the genomic DNA using the GenomiPhi kit. The amplified genomic DNA was used in the subsequent cloning and PCR reactions. Briefly, several known polymerase gene sequences from Thermotoga and Taq were aligned. Based on the alignment result, degenerate primers were designed from conserved sequences. PCR amplification was performed followed by sequencing of the PCR product, if PCR product was of desired length. If a polymerase like sequence is obtained, further primers were designed based on the sequence, and primer walking was used to obtain the full length sequence of the Thermotoga petrophellia DNA polymerase gene. The DNA polymerase gene was finally amplified under standard PCR conditions. Upper primer 5" -ATG GCG AGA CTA TTT CTC TTT - 3' (SEQ ID NO:21), and lower Primer 5" - ACT CAC GAC CAT GTT TTG CCG -3' (SEQ ID NO:22) were used to amplify the full length gene. PCR product was cleaned using EXO SAPit (GE Healthcare). The full length gene sequence was determined by cycle sequencing.

Example 5: Cloning and Purification of the Enzymes from Thermotoga petrophellia To express the Thermotoga petrophellia polymerase gene for protein analysis, the gene is first cloned from PCR amplified fragments into pEXT20, a tac promoter expression vector (NCBI # U51557). The Thermotoga petrophellia DNA polymerase protein is expressed in E. coli JM 109 host cells. The following describes, as an example, the fermentation and purification of the full length polymerase. A single colony containing the pEXT20 vector with the DNA sequence encoding the full length Thermotoga petrophellia polymerase is inoculated and grown in 22ml LB/ Amp for 5hrs. The culture is then transferred to fresh 200ml 2X YT /Amp 100 and grown overnight at 37⁰C with shaking (250 rpm). The overnight culture is used to start 4 X IL fermentation using 2x YT medium, with a starting O.D.600=0.2. ImM IPTG is added to induce protein expression when the culture reaches O.D.₆₀o = 1.6. After a 2 hours induction, the cells are harvested by centrifugation.

Sixteen grams of cell pellet is obtained from the 4 liter culture. Total 16g of cell paste is re-suspended in 45 ml lysis buffer (50 mM Tris-Cl, pH 8.5; 100 mM NaCl; 1 mM EDTA; 0.1 % NP-40; 0.1 % Tween 20 and ImM PMSF), passed through Avestin cell disrupter at 17 kpsi. Lysis is carried out at 70 ⁰C for 20 min. Cool on ice for 30 minutes and centrifuge at 10,000 rpm for 20 min. The clear supernatant (200ml) is used for further purification experiment.

The Thermotoga petrophellia polymerase enzyme is purified from the clear lysate. Forty ml of the clear lysate (out of 200 ml total) is used to run directly on a pre-equilibrated Heparin column (pre-equilibrated with buffer A (5 OmM Tris 8.5, 1 mM EDTA, 5% glycerol) with 10OmM NaCl). The loading is expected to be slow due to the presence of high concentration of DNA in the lysate. After loading, the column is washed with buffer A, then eluted with gradient elution of 0-100% B (buffer A + IM NaCl) in 10 column volumes. Five ml fractions are collected for subsequent analysis by SDS-PAGE.

The rest of the clear supernatant (160ml) is treated with PEI to 0.3% per 120 OD260 to remove contaminant DNA. The sample is kept on ice for 10 min before centrifuging at 10,000 rpm/20min to precipitate the DNA. The clear PEI treated supernatant is then treated with ammonium sulfate to 70%, and subsequently centrifuged to collect the protein containing pellet. The pellet is solubilized in 20ml of buffer A and dialyzed against buffer A for 3 hrs at 4 ⁰C. The dialyzed sample is then loaded onto a pre-equilibrated Heparin column, washed and eluted as described above. Fractions containing the expected band (enzyme) are then combined, dialyzed against buffer A. The dialyzed fractions are then loaded onto HT Q-Sepharose column pre-equilibrated with buffer A, washed with buffer A and eluted with gradient elution from 0-40% B in 15 column volume and 40-100% B in 4 column volumes. Five ml fractions are collected. SDS-gel analysis of fractions from HT Q elution is performed.

Heparin fractions containing the purified protein from the 40ml purification above are combined, dialyzed and run through HT Q. The results are similar to the ones obtained for the 160 ml purification. Fractions showing the highest concentration of the expected band from the two purifications are combined and dialyzed against the storage buffer (5OmM Tris 8.5, ImM EDTA, 10OmM KCl, 0.5% NP-40, 0.5% Tween-20, 50% Glycerol). BCA assay of the semi-purified DNA polymerase protein is performed to determine the protein concentration of the sample. Polymerase assay using alpha ³³P[dATP] is performed to test for DNA polymerase activity. Example 6: Characterization of Enzymes from Thermotoga petrophellia

Thermostability

Thermostability of the enzymes is assayed at 95⁰C, as 50% activity at time determined by polymerase assay. Aliquots of 7μl of the Thermotoga petrophellia enzyme in 8 tubes are identified as 0, 2min, 5min, 10, 20, 30, 45, and 60min. The tubes were incubated at 95⁰C according to the time marked on the tubes. After heating, the tubes are immediately kept on ice and the polymerase assay is performed as described below.

Polymerase assay is performed by taking 5μl of the 7 μl heated Thermotoga petrophellia enzyme with 45 μl of reaction buffer containing 25mM TAPS, 25mM KcI,

2mM MgC12, ImM Beta-mercapto ethanol, 20OuM each dNTPs, and 20ug of sperm

DNA and 0.4uCi alpha 33P dATP. The reaction mix is incubated at 74⁰C for 30min and the incorporations are measured. The counts show the polymerase activity of the enzyme.

DNA Sequencing

The sequencing premix composition is formulated with DYEnamic ET terminators and dITP/dA, T, C (2500 μM dITP, 500 μM dCTP, 500 μM dATP, and 500 μM dTTP). The ratio of dNTP:ddNTP is 156. Each reaction composition also contains 20 units of enzyme mix from TS II or the DNA polymerase enzyme from Thermotoga petrophellia.

5 pmoles of -40 Ml 3 primer, 200 ng Ml 3 DNA, 8 μl of terminator premix

composition and water are added to a total volume of 20 μl. Reaction mixtures are cycled through 95°C, 20 seconds; 50⁰C, 30 seconds; and 60⁰C, 60 seconds, repeated 30 times. Reactions are then held at 4°C until purification and analysis. The samples are purified and analyzed according to manufacturer's instructions, and run on ABI 3100 capillary sequencing instrument (Applied Biosystems). The resulting electropherograms using enzyme mix from TS II and Thermotoga petrophellia polymerase are compared to show the effectiveness of the Thermotoga petrophellia polymerase as a sequencing enzyme.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, kits, solutions, and molecules, described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Other embodiments are within the following claims.

Claims

What is claimed is:

1. An isolated, novel thermostable DNA polymerase protein comprising: a) a DNA polymerase from Thermotoga naphthophila having amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or b) a polypeptide having a sequence with 90% similarity to that of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

2. An isolated, nucleic acid sequence encoding a novel thermostable DNA polymerase, comprising: a) a nucleotide having the sequence of SEQ ID NO: 1 ; or b) a polynucleotide having a sequence with 90% similarity to that of SEQ ID NO:1.

3. A vector comprising a nucleic acid sequence encoding the DNA polymerase of claim 1.

4. A transformed cell comprising a vector of claim 3.

5. A vector comprising a nucleic acid sequence of claim 2.

6. A transformed cell comprising a vector of claim 5.

7. In a method for sequencing a DNA strand, the improvement comprises using the novel DNA polymerase of claim 1 as the sequencing enzyme.

8. A novel thermostable DNA polymerase protein comprising: a) a DNA polymerase from Thermotoga petrophellia having amino acid sequence of SEQ ID NO:20; or b) a polypeptide having a sequence with 98% similarity to that of SEQ ID NO:20.

9. A nucleic acid sequence encoding a novel thermostable DNA polymerase, comprising: a) a nucleotide having the sequence of SEQ ID NO : 19; or b) a polynucleotide having a sequence with 90% similarity to that of SEQ

ID NO:19.

10. A vector comprising a nucleic acid sequence encoding the DNA polymerase of claim 8.

11. A transformed cell comprising a vector of claim 10.

12. A vector comprising a nucleic acid sequence of claim 9.

13. A transformed cell comprising a vector of claim 12.

14. In a method for sequencing a DNA strand, the improvement comprises using the novel DNA polymerase of claim 8 as the sequencing enzyme.