WO2007076461A1

WO2007076461A1 - Thermostable dna polymerase from thermus scotoductus

Info

Publication number: WO2007076461A1
Application number: PCT/US2006/062528
Authority: WO
Inventors: Renee Rech; Scott Hamilton; Carl W. Fuller
Original assignee: Ge Healthcare Bio-Sciences Corp.
Priority date: 2005-12-22
Filing date: 2006-12-22
Publication date: 2007-07-05

Abstract

Disclosed is a thermostable DNA polymerase from Thermus scotoductus. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA, for DNA sequencing, reverse transcription 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 Thermus scotoductus polymerase.

Description

THERMOSTABLE DNA POLYMERASE FROM THERMUS SCOTODUCTUS

Cross-Reference to Related Applications

This application claims priority to United States provisional patent application number 60/753,076 filed December 22, 2005; the entire disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to novel thermostable DNA polymerases obtainable from the thermophilic organism Therm us scotoductus, to certain deletions and mutants of this enzyme, to genes and vectors encoding the wild type and mutant polymerases and their use in strand displacement activity, polymerase chain reaction, DNA sequencing and as reverse transcriptases.

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 trans cription/polymcrasc chain reaction (RTVPCR), 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 canied 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 50°C. 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. ScL USA 89:392-396). SDΛ 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 60⁰C, 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 is a thermostable DNA polymerase from Thermits scotoductus. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA, for DNA sequencing, reverse transcription 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 Thermus scotoductus polymerase.

Brief Description of the Drawings

Figure 1 is the DNA sequence from Thermus scotoductus 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 Thermus scotoductus (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 a DNA sequence encoding the DNA polymerase from Thermus scotoductus, containing FY and ER mutations (SEQ TD NO:3).

Figure 4 is the amino acid sequence of the DNA polymerase from Thermus scotoductus, containing FY and ER mutations (SEQ ID NO:4).

Figure 5 is the polynucleotide sequence encoding the DNA polymerase from Thermus scotoductus, containing YC, FY and ER mutations (SEQ ID NO:5).

Figure 6 is the amino acid sequence of the DNA polymerase from Thermus scotoductus, containing YC, FY and ER mutations (SEQ ID NO:6). The YC mutation decreased exonuclease activity.

Figui-e 7 is DNA sequence encoding a truncated version of a DNA polymerase from Thermus scotoductus (SEQ ID NO:7).

Figure 8 is the amino acid sequence of the truncated version of DNA polymerase from Thermus scotoductus (SEQ ID NO: 8).

Figure 9 is DNA sequence encoding a truncated version of a DNA polymerase from Thermus scotoductus, containing FY and ER mutations (SEQ ID NO:9).

Figure 10 is the amino acid sequence of the truncated version of DNA polymerase from Thermus scotoductus, containing FY and ER mutations (SEQ ID NO: 10). Figure 1 1 is a SDS-PAGE showing purified enzymes.

Figure 12 shows the thermostability of full length and truncated mutant enzymes.

Figure 13 shows sequencing results obtained using full length and truncated mutant enzymes, compared to that of BigDye™ Terminator Cycle Sequencing kit (Applied Biosystcms) (SEQ ID NO:21).

Figure 14 shows KCl tolerance test results of several of these enzymes.

Figure 15 shows the pH range for active enzymes.

Figure 16 is an alignment of several DNA polymerase protein sequences (SEQ ID NO:

22 - SEQ ID NO:28).

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 Thermus scotoductus and having at least 80% amino acid homology, preferably at least 90% 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 Thermus scotoductus (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 Hie 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 enzyme of the present invention has a molecular weight of approximately 93,000 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 and manganese ion concentrations for DNA synthesis are 1 mM and 0.5 mM respectively.

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 7O⁰C. 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 40⁰C, 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., 45-70⁰C). 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 40⁰C₅ e.g., at 37°C, 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 Thermus scotoductus 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 Thermus scotoductus in a buffer. The 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 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.

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

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 and also has reverse transcriptase activity making it useful for making cDNA. This exonuclease-firee enzyme is analogous to the Klcnow fragment of is', coli DNA polymerase I. Thus, the present invention also provides fragments of the polymerase which retain the DNA ~ polymerase activity of Thermus scotoductus but have one or more amino acids deleted, preferably from the amino-terminus, wbile 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) .

In a further aspect, the present invention provides a thermostable DNA polymerase which corresponds to the DNA polymerase from Thermus scotoductus in which up to one third of the amino acid sequence at the amino- terminus has been deleted. In particular, fragments of Thermus scotoductus having N-terminal deletions (SEQ ID NO:8 and SEQ ID NO: 10) have been ftrand to retain enzyme activity.

It is preferred that the 5'-3' exonuclease activity of the DNA polymerase is removed or reduced. This maybe 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, or by appropriate amino acid changes (YC, See Figures 5 and 6) (SEQ ID NO:5 and SEQ ID NO:6). Tn addition to theN-terminal deletions and amino acid changes to remove the cxonuclease activity, the enzyme may have conservative amino acid changes compared with the native enzyme 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 number 0655506A 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. Applicant has discovered that the modification of the dNTP binding site for the dNTP substrate in DNA polymerase obtainable from Thermus scotoductus 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 Thermus scotoductus 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 DNA polymerase sequences are provided, containing the FY mutation and other, preferable point mutations and truncations (See Figures 3 through 6 and 9 through 10) (SEQ ID NO:3 through SEQ ID NO:6 and SEQ ID NO:9 through SEQ ID NOrIO).

The DMA 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 ΛGG, 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 polymerase from Thermus scotoductus 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 polymerase activity of the present invention, e.g., encoding an amino acid sequence corresponding to native Thermus scotoductus 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 through SEQ TD NO:10.

The DNA polymerases of the present invention are suitably used in SDA, preferably in combination with a thermostable restriction enzyme. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention in combination with a thermostable restriction enzyme, for example BsoBI from Bacillus stearothermophilus. The invention also features a kit or solution for SDA comprising a DNA polymerase of the present invention in combination and a thermostable restriction enzyme. The polymerases of the present invention are also useful in methods for generating and amplifying a nucleic acid fragment via a strand displacement amplification (SDA) mechanism. The method generally compiises: a) specifically hybridizing a first primer 5¹ to a target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region; b) extending the 3' ends of the hybridized material using a DNA polymerase of the present invention, preferably one in which the exonuclease activity has been removed, in the presence of three dNTPs and one dNTPα S; c) nicking at the hemiphosphorothioale recognition site with a restriction enzyme, preferably; d) extending the 3' end at the nick using a DNA polymerase of the present invention, displacing the downstream complement of the target strand; and e) repeating steps (c) and (d).

This SDA method proceeds at a linear amplification rate if one primer is used as above. However, if two primers are used which hybridize to each strand of a double-stranded DNA fragment, then the method proceeds exponentially (Walker, G.T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl. Acad. ScL USA 89:392-396).

The present invention also 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 dcoxynucleoside 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 Thermoplάsma 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. TWA prodiicts 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 teπninating 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 niM, 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 a further aspect, the present invention provides a method for preparing complementary DNA by combining an oligonucleotide primer, a sample of RNA, a DNA polymerase of the present invention, and between one and four deoxyribonucleoside phosphates, under conditions favoring preparation of the complementary DNA. , . . .

The DNA polymerases of the present invention which act as reverse transcriptases lack appreciable, and preferably have no RNaseH activity and, as such, are useful in RTYPCR, the generation of hybridization probes and RNA sequencing. Tn a yet further aspect, the present invention provides a purified, thermostable reverse transcriptase having a reverse transcriptase activity of greater than 1000 units per milligram. Preferably, the reverse transcriptase lacks RNaseH activity; the reverse transcriptase is from Thermus scotoductus; the reverse transcriptase has an N-terminal deletion or amino acid changes that remove the exonuclease function. In a further aspect, the invention features a method for reverse transcription/polymerase chain reaction (RT/PCR) which utilizes a DNA polymerase of the present invention and a DNA polymerase suitable for PCR in the same reaction vessel. Preferably, the DNA polymerase of the present invention is from Thermus scotoduclus, the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease activity, the DJMA polymerase suitable for PCR is Taq DNA polymerase. In another aspect, the present invention features a kit or solution for RT/PCR comprising a DNA polymerase of the present invention and a DNA polymerase sui table for PCR. Preferably, the DNA polymerase of the present invention is from Thermus scotoductus, the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease function, and the DNA polymerase suitable for PCR is Taq DNA polymerase.

In another aspect, the invention features a method for polymerase chain reaction in the presence of a polymerase stabilizing agent, utilizing an enzymaticaUy aetive DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Thermus scυloductus 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 Hie 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 Thermits scotoductus 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 ot^'Thermm scotoductus 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 substitutition of an amino acid in the amino terminal one third of the protein, and the glycerol concentration is 30%. Applicants have discovered that the DNA polymerases of the present invention can be used to carry out polymerase chain reaction (PCR) when the reaction is carried out in the presence of a polymerase stabilizing agent, such as glycerol, TMANO, or MMO, and the like. Preferably, the glycerol concentration is in the range of 10 to 50%, and most preferably it is at a final concentration of 30%. TMANO and MMO can be used at concentrations up to 4 M and 3 M final concentration, respectively. These agents have been shown to stabilize the polymerase from Thermus scotoductus. Thus, the polymerases of the present invention are also suitable for reverse transcription/polymerase chain reaction (RT/PCR) under these conditions. Thus, in another aspect the invention features a method for reverse transcription/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 Thermus scotoductus and an exonuclease activity removed. In preferred embodiments, 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%. The invention also features kits for reverse transcription/polymerase chain reaction, with a polymerase stabilizing agent, having an enzymatically active DNA polymerase with at least 80% identity in its amino acid sequence to the DNA polymerase of Thermus scotoductus and an exonuclease activity removed. Also encompassed are solutions for use in reverse transcription/polymerase chain reaction comprising a polymerase stabilizing agent and an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Thermus scotoductus and an exonuclease activity removed. In preferred embodiments, 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%.

In preferred embodiments the exonuclease activity is removed by having an N-terminal deletion, by deleting up to one third of the amino acid sequence at the N-tcrminal, by substitutition 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 polymerase and generation of mutant enzymes The Thermus scotoductus bacterial strain obtained from DSMZ was dispensed in Medium 965 (DSM, Germany) and lysed with KOH, Tris, EDTA (blood GenomiPhi protocol, GE Healthcare) and the sample was heat treated at 95°C for 3 min and the GenomiPhi reaction started. The amplified genomic DNA was cleaned by passing through a G-50 column. The polymerase gene was then amplified by PCR and cloned into PKK vector at the EcoRI and HindIII Site. The primers were designed using the known genome sequence, with the introduction of restriction enzyme sites. The primer at N-terminus (with EcoRI site) is 5 '- CGG AAT TCA TGC TGC CCC TCT TTG AGC CC - 3 ' (SEQ ID NO: 11). The primer at the C-terminus (with HindIII site) is 5 ' - CCC AAG CTT CTA GGC CTT GGC GGA AAG - 3' (SEQ TD NO:12). The PCR product was generated using Pfu High-Fidelity DNA polymerase and cloned into PKK vector. The truncated version was cloned from genomic DNA by PCR using EcoRI and Kpnl restriction sites and the following primers: Forward: 5'- GGA ATT CAT GGA GAG GCT GGA GTT CGG -3' (SEQ ID NO:13); Reverse: 5'- AAA GGT ACC AAC GAC TAT ACC CCA CGA CC -3' (SEQ ID NO:14). Amplified product was cloned into PKK vector.

The point mutants were generated by using the Quickchange site-directed mutagenesis kit (Stratagene). The following primers were used: (1) for FY, forward: 5'- GCC AAG ACC ATC AAC TAC GGC GTC CTC TAC GGC -3' (SEQ ID NO:15), and reverse: 5'- GCC GTA GAG GAC GCC GTA GTT GAT GGT CTT GGC -3' (SEQ ID NO: 16); (2) ER, forward: 5 '- CAC CGG CTT TCG GGA GAG CTG GCC ATC CCC TAC -3' (SEQ ID NO:17), and reverse: 5'- GTA GGG GAT GGC CAG CTC TCC CGA AAG CCG GTG -3' (SEQ ID NO:18); (3) YC, forward: 5'- AGA CCT ACG AGG CCT GCA AGG CGG GGC GGG C -3' (SEQ ID NO: 19), and reverse: 5'- GCC CGC CCC GCC TTG CAG GCC TCG TAG GTCT -3' (SEQ ID NO:20). Pfu High-Fidelity polymerase and standard PCR conditions were used.

Example 2: Purification of Cloned Enzymes

The full length and truncated enzyme clones were fermented using 2X LB medium. Overnight cultures of the plasmid-containing strains were inoculated into 2X LB medium. The polymerase expression was induced at 1.0 OD by adding TPTG. The cells were harvested after growth overnight at 37°C.

Pellet cells by centrifugation. The cell pastes containing full length or truncated enzymes were resuspended in 4 vomme lysis buffer (50 mM Tris-Cl, pH 8.5; 100 mM NaCl; 1 mM EDTA; 0.2% NP-40; 0.2% Tween 20), passed through Avestin cell disrupter 2x 5 to lOlcpsi. Lysis was carried out at 70⁰C for 10 min. Cool on ice for 30 minutes and centrifuge 10,000 rpm. Adjust conductivity to 9mS/cm.

The supernatant was loaded onto 5ml HiTrap Heparin Sepharose HP. The column was washed with 10 column volumes with Buffer A (5 OmM Tri s pH 8.3 ; 1 mM EDTA; 5OmM NaCl). The column was further washed with gradient: 0 - 60% Buffer B (5OmM Tris pH 8.3; ImM EDTA; IM NaCl) in 10 column volumes. Elution was continued by stepping up to 100% buffer B for 5 column volumes. 5ml fractions were collected. Fractions showing polymerase activity were dialyzed overnight against Buffer A so that the conductivity is less than 10mS/cm. For delta Thermus scotoductus FYER: Load sample onto 5ml HiTrap SP

Sepharose column, wash with 10 column volumes of Buffer A. Elution was continued by stepping up to 100% buffer B and collect 5ml fraction. Take peak fractions from SP and dialyze overnight against Buffer A until conductivity is less than lOmS/cm. Load sample onto 5ml HiTrap Q Sepharose HP. Wash with 10 column volumes Buffer A. Gradient: 0 — 60% Buffer B in 10 column volumes. Elution was continued by stepping up to 100%B for 5 column volumes. 5ml fractions were collected.

For Thermus scotoductus FYER and Thermus scotoductus YCFYER: Load sample onto 5ml HiTrap Q Sepharose HP. Wash with 10 column volumes Buffer A, then with gradient: 0 — 60% Buffer B in 10 column volumes. Elution was continued by stepping up to 100%B for 5 column volumes. 5ml fractions were collected.

For all, pool the fractions containing the polymerase activity and dialyze against the final storage buffer (20 mM Tris-Cl, pH 8.5, 25 mM KCL, 0.1 mM EDTA, 50% glycerol, 0.5% NP-40, 0.5% Tween 20 and ImM DTT). Figure 11 shows SDS-PAGE gel of the purified enzymes. The proteins are analyzed using Agilent Protein 200 Plus assay on a BioAnalyzer, and their purity determined (Table 1). Table 1 : Purification of T. scotoductus DNA polymerases

Example 3: Characterization of Enzymes Polymerase Enzyme Activity

Activity of purified enzymes is determined and result shown in Table 2. Specific activity is higher for enzyme from delta T. scotoductus FYER, as compared to T. scotoductus YCFYER or T. scotoductus FYER.

Table 2: Polymerase activity as shown by DAPI assay units and BCA Protein Determination Assay from Pierce.

Thermostability

Thermostability of the enzymes was assayed at 95° C, as 50% activity at time determined by DAPI assay. The enzyme was diluted to 20ng/μl in enzyme dilution buffer (25mM Tris pH 8.0, 5OmM KCl, 0.5% (v/v) Tween 20, and 0.5% (v/v) NP-40). A 20μl reaction was set up with, the following final composition (5OmM Tris pH 8.0, 5mM MgCl₂, 50ng Ml 3 DNA, 50μM dNTPs and 20ng enzyme). The reaction was • incubated at 95°C for 0, 1, 2, 4, 6, 8, 10 and 12 minutes. DAPI assay was performed by taking 5μl of the 20μl reaction, adding it to

195μl DAPI reaction buffer (8OmM Tris pH 9.0, 2.4mM MgCl₂, 5μM FAS42 template, 7.5μM DAPI and 250μM dNTPs). DAPI plate assay was run at 37°C, and the maximum slopes were plotted. As shown in Figure 12, the truncated FYRR enzyme (delta) is more stable than the full length FYER enzyme, while the full length YCFYER is least stable, with thermostability as less then 0.5 min.

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 BigDye™ Terminator Cycle Sequencing kit or T ' hermus scoloduclus polymerase.

5 pmoles of -40 M13 primer, 200 ng M13 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 BigDye™ Terminator Cycle Sequencing kit and Thermus scotoductus polymerase are shown in Figure 13. It shows that in a sequencing reaction, the enzyme from Thermits scotoductus performs similarly to that of enzyme from the BigDye™ Terminator Cycle Sequencing kit.

Endomiclease Contamination:

No endonuclease contamination was detectable at 20 units of any of the tested purified enzymes, when incubated overnight with 1 μg DNA (both single- and double-stranded). The purified enzymes tested include the Delta Thermus scotoductus FYER, the Thermus scotoductus FYER, and the Thermus scotoductus YCFYER.

Exonuclease Activity:

Exonuclease activity was also tested. When 20 units of Thermus scotoductus YCFYER was incubated overnight with either single- or double-stranded nucleic acid substrate, no exonuclease activity was detected. In comparison, slight activity was detected for Delta Thermus scotoductus FYER. A third enzyme, Thermus scotoductus FYER, shows exonuclease activity under these testing conditions.

KCl Tolerance

KCl tolerance assay was performed as 50% activity at niM KCl as determined by DAPI assay. Enzyme was diluted to 2ng/μl. 5μl of diluted enzyme was mixed with

170μl of DAPI reaction buffer. Make total reaction volume to 200μl with water and

2.5M KCl so that the final reaction has 0, 25, 50, 75, 100, 150, 200 and 30OmM KCl.

DAPl reaction buffer formulation is the same as described in the thermostability section.

KCl tolerance plot is shown in Figure 14. pH Range for Activity

DAPI plate assay. Dilute enzyme to lng/μl, mix 5μl of enzyme with 195μl DAPI reaction buffer of varying pH values (4.5 to 10). Figure 15 shows the pH range for several of the polymerases mutants.

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 maybe 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 maybe 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. A novel thermostable DNA polymerase protein comprising: a) a DNA polymerase from Thermus scotoductus having amino acid. sequence of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID

NO: 8 or SEQ ID NO: 10; or b) a polypeptide having a sequence with 80% similarity to that of SEQ TD NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO: 10.

2. A nucleic acid sequence encoding a novel thermostable DNA polymerase, comprising: a) a nucleotide having the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9; or b) a polynucleotide having a sequence with 90% similarity to that of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID

NO:10.

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.