A NOVEL DNA POLYMERASE FROM

CALDICELL ULOSIR UPTOR KRISTJANSSONII

Cross-Reference to Related Applications This application claims priority to United States provisional patent application number 60/744,045 filed March 31 , 2006; the entire disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention The present invention relates to novel DNA polymerases obtainable from the thermophilic organism Caldicellulosiruptor kristjanssonii, 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, 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 for use in amplifying existing nucleic acid sequences in amounts that are large compared to that originally present (US Patent 4,683,195). 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, product of one cycle serves as template for the next cycle such that at each repeat of the cycle the amount of 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 50 ⁰ 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. Sci. USA 89:392-396). SDA is therefore in principle more suited to use with large number of samples than PCR as the isothermal process, which is performed at temperatures of 37°C to 60 ⁰ C, 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 novel DNA polymerase from Caldicellulosiruptor kristjanssonii. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA, for DNA sequencing, and polymerase chain reaction. Included within the scope of the present invention are various mutants (e.g. deletion and substitution) that retain the ability to replicate DNA as the native Caldicellulosiruptor kristjanssonii polymerase.

Brief Description of the Drawings

Figure 1 is the DNA sequence from Caldicellulosiruptor kristjanssonii encoding a full length DNA polymerase (SEQ ID NO:1).

Figure 2 is a contiguous open reading frame capable of encoding the full length polymerase from Caldicellulosiruptor kristjanssonii (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 Caldicellulosiruptor

kristjanssonii, containing Y71C, F684Y and D698R mutations (SEQ ID NO:3).

Figure 4 is the amino acid sequence of the DNA polymerase from Caldicellulosiruptor kristjanssonii, containing Y71C, F684Y and D698R mutations (SEQ ID NO:4).

Figure 5 is a truncated amino acid sequence of the DNA polymerase from Caldicellulosiruptor kristjanssonii, containing F684Y and D698R mutations and deletion of the N-terminal 270 amino acids (SEQ ID NO:5).

Figure 6 is a truncated amino acid sequence of the DNA polymerase from

Caldicellulosiruptor kristjanssonii, containing F684Y and D698R mutations and deletion of the N-terminal 271 amino acids (SEQ ID NO: 6).

Figure 7 is a truncated amino acid sequence of the DNA polymerase from Caldicellulosiruptor kristjanssonii, containing F684Y and D698R mutations and deletion of the N-terminal 272 amino acids (SEQ ID NO: 7).

Figure 8 is a multi-sequence alignment of several DNA polymerase protein sequences. Included in the alignment are DNA polymerase sequence from Ckristjanssonii (SEQ ID NO: 19), Anaerocellum thertnophilum (SEQ ID NO: 20), Caldicellulosiruptor saccharolyticus (SEQ ID NO: 21), Thermoanaerobacter ethanolicus ATCC 33223 (SEQ ID NO: 22), Thermoanaerobacter tengcongensis MB4 (SEQ ID NO: 23), Clostridium perfringens str. 13 (SEQ ID NO: 24), and Dictyoglomus thermophilum (SEQ ID NO: 25)

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 Caldicellulosiruptor kristjanssonii and having at least 90% amino acid homology, preferably at least 95% homology, more preferably at least 98% 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 DNA polymerase from Caldicellulosiruptor kristjanssonii (Figure 1; SEQ ID NO:1) 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 enzyme of the present invention has a molecular weight of approximately 97,000 daltons when measured on SDS-PAGE.

When used herein, the term a DNA polymerase or fragment thereof having the DNA polymerase activity of Caldicellulosiruptor kristjanssonii 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 deoxynucleotides.

The invention also encompasses a stable enzyme composition which comprises a purified DNA polymerase from Caldicellulosiruptor kristjanssonii 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. Fig. 1 represents nucleotides of the cloned gene encoding the polymerase of the present invention (SEQ ID NO: 1).

It is understood 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 Caldicellulosiruptor kristjanssonii 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) . In a further aspect, the present invention provides a DNA polymerase which corresponds to the DNA polymerase from Caldicellulosiruptor kristjanssonii in which up to one third of the amino acid sequence at the amino- terminus has been deleted. In particular, fragments of Caldicellulosiruptor kristjanssonii having N-terminal 270 - 272 amino acid deletions have been generated (Figures 5, 6 and 7; SEQ ID NO:5, 6, and 7).

It is preferred that the 5 '-3' exonuclease activity of the 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, or by appropriate amino acid changes (Y71C, See Figures 3 and 4; SEQ ID NO:3 and SEQ ID NO:4).

In addition to the N-terminal deletions and amino acid changes to remove the exonuclease 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 VaI.

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. 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 Caldicellulosiruptor kristjanssonii 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 F684Y mutation and other, preferable point mutations and truncations (See Figures 3 through 7; SEQ ID NO:3 - 7).

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 polymerase from Caldicellulosiruptor kristjanssonii 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

Caldicellulosiruptor kristjanssonii 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 ID NO:7.

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 comprises: a) specifically hybridizing a first primer 5' 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 hemiphosphorothioate 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 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 Caldicellulosiruptor kristjanssonii 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 Caldicellulosiruptor kristjanssonii 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 Caldicellulosiruptor kristjanssonii 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%.

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 Caldicellulosiruptor kristjanssonii bacterial strain was obtained from

ATCC (ATCC 700841) as a freeze-dried culture which was dissolved in 1 ml of sterile 4% solution of NaCl. Genomic DNA amplification was performed as described for purified DNA sample (GenomiPhi™ protocol, GE Healthcare). The polymerase gene was then isolated by PCR using degenerate primers designed based on known DNA polymerase sequences from other species. The final cloned DNA polymerase gene was

amplified by PCR and cloned into PKK vector at the SacII and HindIII Site (Restriction Site SacII was received in PKK vector by site directed mutagenesis). The primers used for the final cloning of the complete DNA polymerase gene were designed using the known gene sequence, with the introduction of restriction enzyme sites. The primer at N-terminus (with SacII site) is 5 '- TCC CCG CGG ATG AAA CTG GTT ATA TTC GAT GGA - 3' (SEQ ID NO: 8). The primer at the C-terminus (with HindIII site) is 5' - CCC AAG CTT CTA TTT TGT CTC ATA CCA GTT CAG TCC - 3' (SEQ ID NO:9). The PCR product was generated using Pfu High-Fidelity DNA polymerase and 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'- GTC AAG CTA AAG CAG TGA ATT ATG GTA TAG TTT ATG GG -3' (SEQ ID NO:10), and reverse: 5'- CCC ATA AAC TAT ACC ATA ATT CAC TGC TTT AGC TTG AC -3' (SEQ ID NO: 11); (2) DR, forward: 5'- ATG GTC TTG CAA GAC GTG TAA AAA TTT CGC -3' (SEQ ID NO: 12), and reverse: 5'- GCG AAA TTT TTA

CAC GTC TTG CAA GAC CAT-3' (SEQ ID NO:13); (3) YC, forward: 5'- AGC GAA TAC CAA GAA TGC AAA GCT AAC AGA -3' (SEQ ID NO: 14), and reverse: 5'- TCT GTT AGC TTT GCA TTC TTG GTA TTC GCT -3' (SEQ ID NO:15). Pfu High-Fidelity polymerase and standard PCR conditions were used. The N-terminal deletion versions of the FY/DR mutant were generated. The truncated clones start at amino acid sequence 271, 272, and 273 of SEQ ID NO:2, repectively . These clones were generated from the double FY/DR mutant by PCR using SacII and HindIII restriction sites. The reverse primer used is the primer of SEQ ID NO:9, while the forward primer for truncated clone start at amino acid sequence position 271, is 5'- TCC CCG CGG ATG ATT TTG GTC CAG TTA GAG -3' (SEQ

ID NO: 16); for truncated clone start at amino acid sequence position 272, is 5'- TCC CCG CGG ATG TTG GTC CAG TTA GAG -3' (SEQ ID NO: 17); for truncated clone start at amino acid sequence position 273, is 5 '- TCC CCG CGG ATG GTC CAG TTA GAG TTC AAA AGT ATA -3 ' (SEQ ID NO: 18). Amplified products were cloned into PKK vector.

Example 2: Purification of Cloned Enzymes

The full length enzyme clones were fermented using 2X LB medium. Overnight cultures of the plasmid-containing strains were inoculated into 2X YT medium. The polymerase expression was induced at 1.0 OD by adding ImM IPTG. The cells were harvested after growth 2 hours at 37 ⁰ C.

Pellet cells by centrifugation. The cell paste containing full length enzymes was resuspended in 4 volume of lysis buffer (50 mM Tris-Cl, pH 7.5; 50 mM NaCl; 0.5 mM EDTA; 0.2% NP-40; 0.2% Tween 20, ImM PMSF). Lysis was carried by heat at 70 ⁰ C for 20 min, then freeze at -80 ⁰ C for 10 min. After defrost the cell lysate was centrifuged at 6,000 rpm for 20 min. The clear supernatant had conductivity of 1.8mS/cm ² . The pH was adjusted to 8.1.

The supernatant was loaded onto a 5ml HiTrap Q. The column was washed with 10 column volumes with Buffer A (5OmM Tris pH 8.0; ImM EDTA; ImM DTT). Elution was done by gradient from 0 to 100% Buffer B (5OmM Tris pH 8.0; ImM EDTA; ImM DTT, IM NaCl) in 20 fraction volumes. Elution was continued by stepping up to 100% buffer B for 5 column volumes. 5ml fractions were collected.

Fractions were assayed for polymerase activity. Those fractions showing polymerase activity were concentrated and analyzed on a SDS-PAGE gel. A protein of the expected size (97,000 kD) can be detected.

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.