WO2001018213A1 - Chimeric thermostable dna polymerases - Google Patents

Abstract

This invention relates to a chimeric DNA polymerase comprising a 3'→5' exonuclease domain from one DNA polymerase and a DNA polymerase domain from a heterologous DNA polymerase enzyme, together with nucleic acid molecules encoding such chimeric DNA polymerases. The invention further relates to expression vectors and host cells which can express such chimeric DNA polymerases and the use of such chimeric DNA polymerases in various molecular biology techniques.

Description

CHIMERIC THERMOSTABLE DNA POLYMERASES

This invention relates to novel chimeric DNA polymerase molecules and their use in molecular biology techniques such as those involving primer extension or chain extension, for example second strand DNA synthesis, the polymerase chain reaction (PCR) and DNA sequencing.

DNA polymerases are essential cellular enzymes that are involved in replication of cellular DNA, repair of DNA damage, genetic recombination etc. An organism will in general contain several types of DNA polymerases . Thus the eubacterium Escherichia coli possesses three different DNA polymerases : DNA polymerase I that participates in DNA replication and repair, DNA polymerase II where the main cellular function is still unknown, and DNA polymerase III, which is the main DNA polymerase involved in the replication of the E. coli genome .

Based on homology to the Escherichia coli DNA polymerases, DNA polymerases from other species can be divided into four families : families A, B and C which show homology with the E. coli DNA polymerases I, II and III, respectively and Family X which show no homology with any of the E. coli DNA polymerases.

Most prokaryotic and eukaryotic DNA polymerases share the same fundamental type of synthetic activity. That is, that under suitable conditions with respect to pH, temperature, nucleoside triphosphate concentrations etc . they will synthesize a DNA strand which is complementary to a template strand, starting from a double stranded region in the DNA template.

This synthetic activity makes various DNA polymerases useful in a variety of molecular biology and gene technology procedures . This is especially the case for family A polymerases . Examples of polymerases from this family which are used as gene technology tools are the DNA polymerase I enzyme from E. coli (and the Klenow fragment thereof, see below) , the DNA polymerase I enzyme from Thermus aqua icus (Taq) , Thermus thermophilus (Tth) , Bacillus caldotenax (Bca) , Bacillus stearothermophilus (Bst) and Thermotoga mari tima (Tma) .

The detailed structure of E. coli DNA polymerase I is known. Three domains are formed by folding of the polypeptide chain: an N-terminal domain with 5 '-3' exonuclease activity (5'->3' exo domain), a central domain with S'→Ξ' exonuclease activity (3 '-5¹ exo domain) , and a C-terminal domain with polymerase activity (polymerase domain) .

Except for some polymerases which lack the N- terminal domain, such as the bacteriophage T7 DNA polymerase, the same three domain structure is found for other family A DNA polymerases. However, for some DNA polymerases (for example Taq, Tth, Bca and Bst polymerases) the central 3' -5' exo domain lacks the 3' -5' exonuclease (proofreading) activity associated with this domain in the E. coli polymerase I enzyme.

The ability of DNA polymerases to synthesise a DNA strand which is complementary to a template strand, starting from a double stranded region in the DNA template, is a property also sometimes referred to as chain extension or primer extension. Chain extension or primer extension is the mainstay of many current molecular biology protocols. Thus, the DNA polymerase enzymes which catalyse chain extension are important tools in many molecular biology techniques. Due to the ability to catalyse chain extension DNA polymerase enzymes can be used as tools in techniques which involve synthesis of DNA fragments, and also for example techniques such as the polymerase chain reaction (PCR) amplification of DNA fragments and DNA sequencing.

Many of the molecular biology techniques in which DNA polymerases are used as technological tools involve the subjection of biological samples to heat. Many known DNA polymerases are not stable when heated and are thus not useful for these techniques. The sensitivity of DNA polymerases to heat, or in other words, the thermostability of the DNA polymerases, varies depending on the particular DNA polymerase in question. However, in general a DNA polymerase is considered to be thermostable/thermophilic if it can withstand heating to temperatures in the range of 50-70°C. Examples of thermostable family A polymerases include Taq, Tth, Bst, Bca, Cau and Tma.

From the above discussion, it is apparent that there is a need in the art for novel DNA polymerases which are thermostable.

Surprisingly, it has now been found that novel DNA polymerases which exhibit proof-reading (3 ' -5 ' exonuclease) properties, can be produced by generating chimeric DNA polymerases comprising a 3 '-5' exonuclease domain from one DNA polymerase enzyme and a DNA polymerase domain from a different DNA polymerase enzyme . Such novel chimeric DNA polymerases are produced by inserting one or more domains which exhibit catalytic activity, for example DNA polymerase activity, 5' -3' exonuclease activity or 3 '-5' exonuclease activity, from one DNA polymerase into the molecular framework of another, different, DNA polymerase.

In particular, it has been found that a novel chimeric DNA polymerase may be produced which combines the property of thermostability, with the property of a 3' -5' exonuclease (proofreading) activity.

Some modified DNA polymerases with altered properties are known in the art. For example, Klenow et al. 1970 (PNAS 65, 168-175), describe a DNA polymerase with increased polymerase activity and reduced exonuclease activity, produced by removing the N- terminal 5 '-3' exonuclease domain from E. coli DNA polymerase I . In addition, Tabor et al . (PNAS USA 84, 4767-4771, 1987; JBC 262, 15330-15333, 1987; JBC 264, 6447-6458, 1989) describe a DNA polymerase with reduced or eliminated 3' -5' exonuclease activity, produced either by chemical treatment or by point mutation or deletion of a specific histidine residue (His¹²³) in T7 DNA polymerase .

Tabor et al . 1995 (PNAS USA 92, 6339-6343) showed that the mutation of a single phenylalanine residue to tyrosine in either E. coli DNA polymerase I or Taq DNA polymerase lead to a reduced discrimination of the DNA polymerase against dideoxyribonucleotides .

Park et al . 1997 (Mol. Cells 7, 419-424) showed that 3' -5' exonuclease activity can be introduced into Taq polymerase by point mutation of 4 residues in the inactive 3 '-5' exonuclease domain, in order to facilitate the binding of metal ions, which is known to be important for exonuclease activity.

Hoffman La Roche AG (EP-A-892058) described the production of mutant DNA polymerases displaying improved properties in nucleic acid sequencing reactions, such as improved incorporation of dideoxynucleoside triphosphates (ddNTPs) . The described DNA polymerases consist of an N-terminal region derived from the 5 '--3' exonuclease domain of a Thermus species DNA polymerase and a C-terminal region derived from the 3' -5' exonuclease and polymerase domains of Tma DNA polymerase. It is preferred in the chimeric polymerases of EP-A-892058 to inactivate the exonuclease activities by mutation.

However, although the production of modified DNA polymerases is documented in the art, chimeric DNA polymerases such as those disclosed herein which comprise a 3 ' —5 ' exonuclease domain from one DNA polymerase enzyme and a DNA polymerase domain from a different DNA polymerase enzyme, have not been produced.

Indeed, given what is known in the prior art with regard to the structure and interaction of the 3 main domains which make up DNA polymerases, it is surprising and unexpected that the chimeric DNA polymerases of the present invention which comprise a 3 '-5' exonuclease domain from one DNA polymerase enzyme and a DNA polymerase domain from a different DNA polymerase enzyme can exhibit the ability to bind DNA together with DNA polymerase activity and 3' -5' exonuclease activity.

In this regard, it is well known in the art that the 5' -3' exonuclease domain can be separated from the other domains by treatment with proteolytic enzymes, indicating that the junction between the 5 '-3' exonuclease domain and the 3 '-5' exonuclease domain is open and easily accessible (see for instance Klenow H. & Henningsen, I. Proc Natl Acad Sci USA 65: 168-175, 1970) . Studies on the crystal structure of Taq DNA polymerase (Kim et al . , 1995, Nature 376, 612-616) show that the 5' -3' exonuclease domain forms a structure that is separate from the other two domains with only 850 A² surface area in contact with the 3' -5' exonuclease domain. Moreover, the document by Kim et al . (and the document by Joyce and Steitz, 1994, Annu. Rev. Biochem. 63, 777-822) disclose that the 5' -3' exo domain is able to function catalytically after having been proteolytically removed from the other two domains. Similarly, after proteolysis the two remaining domains (i.e. the 3'-5' exonuclease domain and the DNA polymerase domain) function in a way that is not substantially different from the way they function in the intact enzyme (this is demonstrated in the Klenow fragment of E. coli DNA polymerase I and in corresponding truncated forms of other DNA polymerases) . It is thought that this apparent independent function of the 5' -3' exonuclease domain from the other two domains of the DNA polymerase enzyme might well be due to the lack of interaction between the S'-^¹ exonuclease domain and the other domains . With regard to the 3 '-5' exonuclease domain however, it is known (Joyce and Steitz, supra) that this domain cooperates with the polymerase domain for the binding of DNA and that removal of the 3' -5' exo domain weakens duplex DNA binding to the polymerase . On the other hand, removal of the polymerase domain leaves an exonuclease domain with no detectable enzymatic or single-stranded DNA-binding activity. Furthermore, Korolev et al . , 1995. PNAS. USA 92, 9264-9268 shows that 2960 A² or 21% of the surface of the small domain (3 '-5' exo) is buried at the interface between this domain and the polymerase domain of Taq polymerase, and that the corresponding figure for the E. coli enzyme is 2730 A² or 14.7%. The figures illustrating the structures of Taq polymerase and the E. coli DNA polymerase in the documents discussed supra show clearly that the 3" -5' exo domain is much more integrated with the polymerase domain than is the 5' -3' exo domain.

The present invention therefore provides a chimeric DNA polymerase comprising a 3^!-5' exonuclease domain from one DNA polymerase enzyme and a DNA polymerase domain from a heterologous DNA polymerase enzyme .

The DNA polymerase enzymes for use as sources for the different domains in the present invention may be any DNA polymerase enzymes, whether native or modified e.g. by mutagenesis. As mentioned above many DNA polymerase enzymes are known in the art and described in the literature and any of these may be used to provide the domain (s) which make up the chimeric DNA polymerases of the invention.

"domain" as used herein refers to a part of a DNA polymerase polypeptide that folds into a separate distinct unit. The polypeptide chain can traverse forwards and backwards within the domain, whereas neighbouring domains are usually connected by one or at most two polypeptide chain segments. "domain" as used herein can be regarded as a separate folding unit, i.e. that the presence or absence of a given domain will not significantly affect the folding and structure of the other domains in the protein.

Examples of "domains" in accordance with the present invention include the 3' -5' exo domain, the 5'->3' exo domain and the polymerase domains of family A DNA polymerases .

Also included within the term "domain" are separate folding units within a domain, also termed sub-domains. Examples of such structures in accordance with the present invention are the finger, thumb and palm subdomains in the polymerase domain of family A DNA polymerases.

Furthermore, the domain (s) making up the chimeric DNA polymerases of the present invention may be native (ie. as they occur in nature) or they may be modified.

The term "modified" as used herein in relation to DNA polymerase domains includes all forms of amino acid sequence modification, and thus includes single or multiple amino acid substitution (s) , addition or deletion, be this of single amino acids or longer amino acid sequences e.g. truncations, insertions, etc. Also included are sequences where the amino acids have been chemically modified, including by glycosylation, or other chemical substitution of amino acid residues.

Furthermore the domain (s) making up the chimeric DNA polymerases of the invention may be wholly or partly synthetic, i.e. may wholly or partly comprise amino acids which have been generated chemically.

The chimeric DNA polymerases of the invention may be produced by the insertion of one or more domains from a DNA polymerase enzyme into a heterologous DNA polymerase enzyme. "insertion" as used herein refers to the positioning of a domain (s) from one DNA polymerase enzyme into a different DNA polymerase enzyme. In the context of the present invention the term "insert" can take the form of a replacement or a substitution of a domain from one DNA polymerase with a functionally equivalent or homologous domain (e.g. a corresponding domain) from a different DNA polymerase enzyme. For example, a preferred chimeric DNA polymerase of the invention is produced by the replacement of the 3'-*5' exonuclease domain (and optionally the 5' -3' exonuclease domain) of one DNA polymerase enzyme with functionally equivalent domain (s) (i.e. a 3'-5' exonuclease domain and optionally a 5'-*3' exonuclease domain) from a heterologous DNA polymerase enzyme. Or, viewed another way, a preferred chimeric DNA polymerase of the invention is produced by the replacement of the DNA polymerase domain (and optionally the 5'-3' exonuclease domain) of one DNA polymerase enzyme with functionally equivalent domain (s) from a heterologous DNA polymerase enzyme . In a most preferred chimeric DNA polymerase of the invention the 3' -5' exonuclease domain and the 5 ' —3 ' exonuclease domain of the DNA polymerase enzyme are replaced with a 3' -5' exonuclease domain from a heterologous DNA polymerase enzyme. Thus, it can be seen that the term "insert" as used herein also includes the replacement or substitution of more than one domain from a DNA polymerase with a domain from a different DNA polymerase enzyme. However, also included is the possibility of adding an extra new domain into an existing DNA polymerase enzyme.

"Corresponding" or "functionally equivalent" etc. domains of different polymerase enzymes may readily be identified by sequence alignments, according to techniques well known in the art.

A chimeric polymerase according to the invention may also be prepared by "joining" together selected domains from two or more polymerase enzymes. Thus, each of the three primary domains (5' -3' exo, 3 '-5' exo and polymerase) may be derived from a different polymerase enzyme .

The insertion of a domain (s) or joining of domains according to the present invention, is carried out so that the inserted domain (s) /joined domains and the domain (s) comprising the heterologous DNA polymerase enzyme are operatively joined.

"operatively joined" as used herein in the context of protein domains, means that the domains are joined in such a way that they function together in the same way as in a naturally occurring DNA polymerase enzyme.

"heterologous DNA polymerase enzyme" as used herein refers to a DNA polymerase enzyme encoded by a different nucleic acid sequence. Thus a "heterologous DNA polymerase enzyme" according to the present invention includes DNA polymerase enzymes of the same, or different species of organism, providing the DNA polymerase enzymes are encoded by different nucleic acid sequences. In effect, the invention requires that a native DNA polymerase enzyme be modified to introduce a new catalytic domain (s) which is not naturally present in that enzyme - this catalytic domain (s) may be native (ie. naturally occurring) or modified or synthetic, and it may be derived from any other DNA polymerase enzyme .

In one aspect of the present invention the chimeric DNA polymerase further comprises a 5'->3' exonuclease domain. Such a 5' -3' exonuclease domain can be derived from the same DNA polymerase enzyme as the 3' -5' exonuclease domain is derived from, or can be derived from the same DNA polymerase enzyme as the DNA polymerase domain is derived from. Alternatively, such a 5 '-3' exonuclease domain can be derived from a completely different DNA polymerase enzyme.

In yet another aspect of the present invention, at least one of the inserted domain or domains from the heterologous DNA polymerase, is derived from a thermostable DNA polymerase. Preferably the thermostable DNA polymerase is selected from the group comprising Taq, Tth, Bca, Bst, Cau, Tma, Pfu, Vent and Deep Vent DNA polymerases. Preferably the chimeric DNA polymerase of the present invention is a thermostable DNA polymerase .

"thermostable DNA polymerase" as used herein refers to a DNA polymerase which can be heated to temperatures of at least 50°C, preferably temperatures in the range of approximately 50°C-70°C (even more preferably up to 95°C) for an extended period of time, such as for example 30 minutes, without extensive loss of catalytic activity. In this context, "without extensive loss of catalytic activity" means that at least 40%, more preferably 50%-70%, even more preferably at least 75, 80, 85, 90 or 95% of the original activity is retained, measured under suitable conditions regarding temperature, pH, ionic strength etc., as compared with the original activity of the polymerase enzyme before exposure to heat. Suitable conditions may be any condition known in the art to be appropriate for, or conducive to the polymerase reaction. Exemplary conditions include the presence of a polymerase buffer which has a pH of 9.0 at 25°C and comprises 50 mM KC1 and 5 mM MgCl₂ and measurement of polymerase activity at a temperature of 70°C. Such catalytic activity may be any catalytic activity associated with a DNA polymerase. Examples of catalytic activity which may be associated with a DNA polymerase include, for example, the DNA polymerase activity, 3'-5' exonuclease activity and 5 ' —3 ' exonuclease activity. A polymerase enzyme according to the invention may thus be "thermostable" with regard to a selected catalytic activity and this means that the enzyme is stable to heat (e.g. up to a temperature of 50°C, 60°C or 70°C or more) and has an elevated temperature reaction optimum.

Preferably the thermostable DNA polymerase can withstand heating to higher temperatures, for example 92°C to 95°C. Such polymerases would thus be useful in techniques such as PCR. PCR (and variants thereof) is a technique which is well known and described in the art and allows in vi tro amplification of a DNA sequence limited by two synthetic oligonucleotides, or primers (described in more detail below) .

During the PCR process, samples need to be heated to high temperatures (approximately 92-95°C) . For practical efficiency, it is thus necessary that the DNA polymerase enzyme used in the reaction must encounter such temperatures without being irreversibly inactivated.

Examples of family A DNA polymerases which can withstand heating to approximately 95°C and consequently are usable in PCR, are Taq DNA polymerase and Tth DNA polymerase. In addition, some family B DNA polymerases from the thermophilic Archae bacteria are used, for instance Pfu DNA polymerase from Pyrococcus furiosus , Vent polymerase from Thermococcus li toralis and Deep Vent polymerase from Pyrococcus species GB-D.

However, although the DNA polymerases from Taq and Tth are thermostable and can withstand heating to approximately 95°C, these enzymes (as mentioned above) , do not exhibit a 3' -5' exonuclease (proofreading) activity. This lack of proofreading activity means that difficulties arise when such polymerases are used to amplify long DNA fragments.

Firstly, due to the lack of a proofreading activity, Taq and Tth DNA polymerases cannot remove misincorporated nucleotides and subsequent elongation of a growing chain with a 3 ' nucleotide that is not complementary to the corresponding nucleotide in the template strand is a slow and inefficient process.

Moreover, difficulties arise in the amplification of long fragments, since for these there is a high possibility that errors are introduced into the growing chains that will stop chain elongation before the other primer site has been reached. For this reason 4-5 kilobases (kb) is commonly regarded as an upper size limit for fragments that can be amplified with Taq and Tth DNA polymerase .

Unlike the two family A polymerases, Taq and Tth, the family B polymerases mentioned above have a 3 '-5' exonuclease (proofreading) activity. However, notwithstanding the presence of this proofreading activity, problems still arise with the amplification of long DNA fragments. One of the reasons for this is that the 3'-5' exonuclease activities of these enzymes are so strong that the unannealed DNA primers used in the PCR method are degraded during the PCR process . Another reason is the low processivity of these family B polymerase enzymes.

The current solution to overcome the problems associated with the DNA polymerases known in the art, is to use combinations of DNA polymerases when trying to amplify long DNA fragments (Barnes, W.M. (1994) PNAS USA 91: 2216-2220). For example, Taq polymerase in combination with small amounts of a proofreading polymerase such as Pfu is sometimes used.

It can be seen from the above discussion, that the provision of a DNA polymerase having both a 3'-5' proofreading activity and a thermostability at temperatures of 92-95°C would be extremely advantageous.

It is envisaged that the thermostability of chimeric DNA polymerases of the invention, which are thermostable in the range of 50°C-70°C, may be improved (i.e. the temperature to which the polymerase may be heated without a loss in catalytic activity is increased) by further modification of the polymerases. Such modification may take any form, including genetic manipulation of the chimeric enzyme involving techniques well known and documented in the art such as for example single or multiple nucleotide or amino acid substitution, addition, mutation or deletion. Other well known methods in the art could be used to improve the thermostability of the chimeric DNA polymerases of the invention, such as, for example, the addition of stabilising compounds such as trehalose and trimethylamine-N-oxide (Carninci, P., et al . , PNAS USA, 1998, Vol. 95: 520-524 and Baskakov, I. and Bolen, D. , J. Biol Chem, 1998, Vol 273: 4831-4834) to the reaction mix, or the introduction of random C-terminal tails in the enzymes (Matsuura, T., et al . , Nature Biotech. 1999, Vol 17: 58-61) .

In another aspect of the present invention, at least one of the inserted domain or domains from the heterologous DNA polymerase, is derived from a family A DNA polymerase^'.

Preferably, the family A DNA polymerase is selected from the group comprising E. coli polymerase I, Taq, Tth, Bca, Bst, Cau, Tma and T7 bacteriophage DNA polymerases.

Preferably, the part of the chimeric DNA polymerase which exhibits the DNA polymerase activity is derived from Taq DNA polymerase.

Preferably, the part of the chimeric DNA polymerase which exhibits the 3'-5' exonuclease activity is derived from Cau DNA polymerase.

More preferably, the chimeric DNA polymerase comprises a domain exhibiting DNA polymerase activity derived from Taq DNA polymerase and a domain exhibiting 3' -5' exonuclease activity derived from Cau DNA polymerase. More preferably, the chimeric DNA polymerases of the invention have the amino acid sequence shown in either SEQ ID NO: 2 or 4.

In an alternative preferred embodiment the part of the chimeric DNA polymerase which exhibits the 3' -5' exonuclease activity is derived from Tma DNA polymerase, and hence an additional preferred chimeric DNA polymerase comprises a domain exhibiting DNA polymerase activity derived from Taq DNA polymerase and a domain exhibiting 3' -5' exonuclease activity derived from Tma DNA polymerase .

The chimeric DNA polymerases of the invention may be prepared using techniques which are standard or conventional in the art. Generally these will be based on genetic engineering techniques, but protein manipulation techniques or proteolytic digestion to release a selected domain and chemical coupling is also possible, using known techniques.

As mentioned above, one of the methods which may be used to prepare the DNA polymerases of the invention is through genetic engineering techniques. Thus, a genetic construct is prepared, using standard recombinant DNA techniques, encoding a desired chimeric DNA polymerase and comprising appropriate nucleotide sequences (nucleotide fragments) encoding the various domains, etc. so as to express the desired chimeric polypeptide as a complete "finished" molecule. Thus, the appropriate nucleotide sequences may be ligated together, or inserted into one another etc.

Such a genetic construct or gene designed to encode the desired polypeptide (i.e. a chimeric DNA polymerase enzyme) may then be inserted into an expression vector construct. Such expression vectors include appropriate control sequences such as for example translational (e.g. start and stop codons, ribosomal binding sites) and transcriptional control elements (e.g. promoter- operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecule encoding the desired polypeptide of the invention. Optional further components of such vectors include for example replication origins, selectable markers, secretion signalling and processing sequences.

Such expression vectors may include plasmids, cosmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art, including bacterial (e.g. E. coli) , Baculovirus, yeast or mammalian expression systems. One preferred vector for use in an E. coli expression system in accordance with the present invention is the plasmid pTrc99A (Pharmacia Biotech, Uppsala, Sweden) which provides an inducible tac promoter and suitable transcription termination signals, as well as ori sequences for autonomous replication and an ampicillin resistance gene for selection.

A variety of techniques are known and may be used to introduce such vectors into suitable host cells for expression. Such host cells may for example include prokaryotic cells such as E.coli, eukaryotic cells such as yeasts or the baculovirus insect cell system, transformed mammalian cells, germ line or somatic cells, or genetically engineered cell lines. Suitable techniques by which such vectors may be introduced into such cells are well known and documented in the literature .

Once the vector construct has been introduced into an appropriate host cell, the chimeric DNA polymerase according to the invention can be produced by culturing the host cells under conditions which allow the expression of the chimeric polypeptide by the host cell. Depending on the control sequences present in the vector construct and the host cell used, transcription and expression of the coding sequence may be initiated by adding a suitable inducer to the culture medium. An example of such an inducer which may be used in the present invention is IPTG. Once a sufficient amount of the chimeric DNA polymerase has been produced by the host cells, depending on the control sequences contained in the vector construct, the chimeric DNA polymerase of the invention can be isolated either from the host cells or the culture medium.

Some vector constructs will contain control sequences that direct the produced protein to the periplasmic space or out into the growth medium, in which case isolation will entail removal of the host cells and recovery of the polymerase from the medium. With other vector constructs the produced protein will remain in the host cell, in which case it must be recovered, for example by lysis of the host cells and purification of the proteins by methods well known and documented in the art . Such recombinantly produced proteins may also be produced by the host cell in an insoluble form and end up in so-called inclusion bodies. In this case the protein can be recovered from the inclusion bodies using methods well known and documented in the art, for example by isolating the inclusion bodies from lysed cells, solubilization of the protein in the inclusion body by using reagents such as urea or guanidinium thiocyanate, and removal of the reagent under conditions that allow refolding of the protein.

For example, to prepare the genetic construct encoding the chimeric DNA polymerase of the invention, separate DNA fragments encoding the desired domains of the appropriate chimeric polypeptide may be produced and joined together, for example by ligation. While carrying out this procedure, one or more of the DNA fragments may already be joined to the intended vector construct. Alternatively, while carrying out this procedure, none of the DNA fragments may be joined to the intended vector construct, but joined to the intended vector construct in a subsequent step.

The domain encoding DNA fragments can be obtained by techniques which are well known and documented in the art. For example by restriction enzyme cutting, if necessary after the introduction of suitable restriction sites or other sequences that are not present in the starting sequences. Such restriction sites or other sequences can be introduced by for example site directed mutagenesis or PCR amplification (optionally using tailed primers) . Alternatively the domain encoding DNA fragments may be prepared by PCR amplification from a template DNA molecule using appropriate primers. Such PCR amplification can also be used to introduce any required restriction sites or other sequences into the DNA fragments .

After the DNA fragments have been obtained, they may be purified using techniques which are well known and documented in the art, for example by subjecting the DNA fragment to electrophoresis in a gel medium (for example an agarose gel) , isolating the part of the gel containing said DNA fragment and purification of the fragment from the gel medium by methods known in the art (for example using the Geneclean kit produced by BIO 101, Inc., 1070 Joshua Way, Vista, CA 92083, USA or the kits produced by Qiagen, Qiagen GmbH, Max-Volmer-Strasse 4, 40724 Hilden, Germany) . After the DNA fragments have been obtained and, if necessary, purified, the fragments are joined together for example by incubation with a suitable DNA ligase, such as for instance T4 DNA ligase, under conditions and for a period of time that allows joining together of the fragments. Ligation to the linearised vector can be performed in the same or a separate ligation reaction.

In order for the genetic construct encoding the desired chimeric polypeptide to express the chimeric polypeptide as a complete "finished" molecule, it is important that the DNA fragments encoding the domains of the desired chimeric polypeptide are joined in such a way that the domains of the chimeric polypeptide expressed by said construct are operatively joined, that is joined in such a way that they function together in the same way as in a native polypeptide molecule. In native proteins, domains are often linked by flexible loops or unstructured regions in the polypeptide chain. Therefore, one possibility is that the ends of the DNA fragments encoding the domains of the desired chimeric DNA polymerase encode amino acids that are found in such linking regions in the native polymerases. This should facilitate the correct orientation of the polypeptide domains relative to each other and increase the likelihood that the joined domains will be able to function in a concerted way.

For some DNA polymerases, such as the Klenow fragment, Taq DNA polymerase and Bca DNA polymerase, the 3 -dimensional structure is known from X-ray crystallography. Careful examination of the 3- dimensional model enables suitable choices of start and end amino acid residues for the portion of the polypeptide chain that will be used in the chimeric enzyme to be made. For other family A polymerases the 3 -dimensional structure has not yet been experimentally determined. For these, carefully adjusted alignment of sequences might be helpful to identify suitable ends to be used for the DNA fragments. Sequence alignments can be made using techniques well known and documented in the art, for example with the help of computer programs like the Clustal series (Thompson et al . , 1994 Nucleic Acids Research 22: 4673-4680). If so desired, such computer programs can also take into account the secondary structure of sequences, where such structures are known. These alignments can then for example be used to make a 3 -dimensional model of polymerases for which no experimental structure is available, by using protein modelling techniques known in the art, or be used to identify residues that correspond to suitably placed residues in the polypeptides for which an experimental structure is known.

A further aspect of the present invention provides nucleic acid molecules comprising a nucleotide sequence which encodes a chimeric DNA polymerase of the invention. Preferably such nucleic acid molecules comprise the sequence as defined in SEQ ID NO. 1 or 3 , or a fragment thereof encoding a functionally active product, or a sequence which is degenerate, substantially homologous with or which hybridises with the sequence as defined in SEQ ID NO. 1 or 3 or with the sequence complementary thereto, or a fragment thereof encoding a functionally active product.

"Functionally active product" as used herein refers to any chimeric product encoded by said sequence which exhibits DNA polymerase activity.

"Substantially homologous" as used herein includes those sequences having a sequence homology of approximately 60% or more, e.g. 70%, 75%, 80% or 85% or more and also functionally equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By "functionally equivalent" in this sense is meant nucleotide sequences which encode catalytically active polypeptides, ie. having DNA polymerase activity.

For determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, J. D., D.G. Higgins, et al . (1994). "CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice". Nucleic Acids Res 22: 4673-4680). One especially useful feature of this program is that, in cases where structure information is available for one or more members of the alignment, it can be set to use such information to improve the quality of the alignment. Programs that compare and align pairs of sequences, like ALIGN (E. Myers and W. Miller, "Optical Alignments in Linear Space", CABIOS (1988) 4: 11-17), FASTA (W.R. Pearson and D. J. Lipman (1988) , "Improved tools for biological sequence analysis", PNAS 85:2444- 2448, and W.R. Pearson (1990) "Rapid and sensitive sequence comparison with FASTP and FASTA" Methods in Enzymology 183:63-98) and gapped BLAST (Altschul, S.F., T.L. Madden, et al . (1997) . "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs". Nucleic Acids Res. 25: 3389-3402) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, J. of Mol. Biology, 1993, Vol. 233: 123-38; Holm, Trends in Biochemical Sciences, 1995, Vol 20: 478-480; Holm, Nucleic Acid Research, 1998, Vol. 26: 316-9).

By way of providing a reference point, sequences according to the present invention having 60%, 70%, 75%, 80%, 85% homology etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, Montpellier, France) .

Sequences which "hybridise" are those sequences binding (hybridising) under non-stringent conditions (e.g. 6 x SSC, 50% formamide at room temperature) and washed under conditions of low stringency (e.g. 2 x SSC, room temperature, more preferably 2 x SSC, 42°C) or conditions of higher stringency (e.g. 2 x SSC, 65°C) (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2).

Generally speaking, sequences which hybridise under conditions of high stringency are included within the scope of the invention, as are sequences which, but for the degeneracy of the code, would hybridise under high stringency conditions.

A further aspect of the present invention provides an expression vector capable of expressing a chimeric DNA polymerase of the invention. Preferably, the expression vector comprises a nucleic acid molecule of the invention. Possible types and structures of such expression vectors according to the invention are described above.

Thus, a yet further aspect of the present invention provides a host cell expressing a chimeric DNA polymerase of the invention. Examples of possible host cells which may be used to express the chimeric DNA polymerase of the invention are described above.

Thus, a yet further aspect of the present invention provides a method of producing a chimeric DNA polymerase of the invention, comprising the steps of (i) growing a host cell containing a nucleic acid molecule encoding a chimeric DNA polymerase of the invention under conditions suitable for the expression of the chimeric DNA polymerase; and (ii) isolating the chimeric DNA polymerase from the host cell or from the growth medium. A further aspect of the present invention provides the use of the chimeric DNA polymerases of the invention in molecular biology and gene technology techniques. The chimeric DNA polymerases of the present invention are particularly useful in those molecular biology techniques involving chain or primer extension and requiring a thermostable enzyme. Such techniques include for example second strand DNA synthesis, PCR amplification, DNA sequencing, nucleic acid based assays, etc. The proof-reading (3 '-5' exonuclease) activity of the chimeric DNA polymerases of the present invention makes the polymerases particularly useful in techniques where the fit between the primer and the template DNA is not exact. For example, the use of a proof-reading chimeric enzyme of the invention with some degree of thermostability, would be an advantage in second strand cDNA synthesis using a consensus primer, as in cases where sequence information for the cDNA is not available and the primer is made taking sequence information from homologous enzymes from other species into consideration. For the same reason the chimeric DNA polymerases of the invention will be useful in DNA sequencing with consensus primers . This is because polymerases have problems extending primers that do not anneal properly at the 3' end. A polymerase without proof-reading capability would not be able to deal with this problem, whereas a proof-reading enzme (such as that of the present invention) could remove any mismatches at the 3 ' end and replace them with properly matched nucleotides. A further aspect of the present invention provides kits for use in molecular biology and gene technology techniques comprising a chimeric DNA polymerase of the invention. Preferably said kit comprises at least a chimeric DNA polymerase of the invention together with one or more primers which hybridise to strands of target DNA in order to provide a substrate for the chain extension reaction which may be catalysed by the chimeric DNA polymerase.

The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

Figure 1 shows the activity of CauTaq DNA polymerase version 2 at various temperatures. The enzyme reaction was performed at the given temperatures for 5 minutes. Polymerase activity is given as per cent of the activity at the optimal temperature, 55°C. For comparison, the results from corresponding experiments with Cau DNA polymerase and Taq DNA polymerase are shown.

Figure 2 shows time courses for inactivation of CauTaq DNA polymerase version 2 at various temperatures. The enzyme was preincubated at the stated temperatures for various periods of time before polymerase activity was measured. Polymerase activity, relative to the activity before preincubation, is given as a function of preincubation time.

Figure 3 demonstrates thermostable exonuclease activity in versions 1 and 2 of CauTaq DNA polymerase. The data shown is output from an ALF Express DNA sequencer and show the size (in bases) of Cy5 -labelled fragments . Samples labeled Match contained the Match primer and samples labeled Mismatch the Mismatch primer. Samples labeled Alul were treated with Alul before electrophoretic analysis. The results demonstrate that the CauTaq polymerases are able to edit mismatched nucleotides and extend the edited primer. For comparison, the data for Taq polymerase demonstrate that this enzyme is unable to edit the mismatch as well as to extend the Mismatch primer.

Figure 4. Temperature optimum for the editing/elongation reactions. The polymerases were diluted and analyzed in the presence of Mismatch primer. The enzyme dilution was sufficient to repair and extend about 50% of the primer present under standard conditions. The ratio between the amount of 18 nucleotides long product from the Alul digestion and the amount of a 30^' nucleotides long Cy5-labelled oligonucleotide, added in equal amounts to all samples together with the formamide prior to electrophoresis, was used as a measure of the ability of the enzymes to edit and extend the primer at the temperatures being tested. The results demonstrate that the temperature optimum of this activity is lower for CauTaq polymerase version 2 than for Cau DNA polymerase (≤ 60°C and 65- 70°C, respectively) .

Example 1 : Construction of an expression vector coding for CauTag polymerase version 1

Cloning of Tag DNA polymerase

The following amplification primers were designed:

5' - Taq: CACGAATTCGGGATGCTGCCCCTCTTTGAGCCCAAG (SEQ ID NO. 5)

3' - Taq: GTGAGATCTATCACTCCTTGGCGGAGAGCCAGTC(SEQ ID NO . 6)

The primers were based on the published sequence of Taq DNA polymerase (Lawyer et al . (1989) J. Biol. Chem. 264, 6427-6437) and were designed to introduce a EcoRI site in the 5' end of the gene and a Bglll site in the 3' end. Thermus aquaticus strain YT-1, obtained from American Type Culture Collection (ATCC) , was grown in Castenholz medium, after which genomic DNA was isolated using the method of Chen and Ku (1993) Nucl . Acids Res. 21, 2260.

A PCR reaction mixture was set up containing approximately 0.05 μg genomic Tag-DNA, 20 pmoles each of the primers above. 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl2, 2 U Taq DNA polymerase (Boehringer- Mannheim) , and 2.5 μl 10 x PCR buffer (Boehringer- Mannheim) . The polymerase gene was amplified using 35 PCR cycles, each consisting of 94°C (30 seconds) , 45°C (30 seconds), and 72°C (1 minute). The resulting 2.6 kb DNA fragment was digested with the restriction endonucleases EcoRI and Bglll , gel purified and ligated into the expression plasmid pTrc99A (Pharmacia, Sweden) that previously had been digested with EcόRI and BamHl, using a 20 μl ligation mixture with T4 DNA ligase (Promega) according to the recommendations of the producer. The ligation mix was introduced into E. coli strain INVαF¹ made competent according to the procedure of Inoue et al . (1990) Gene 96, 23-28. Bacteria containing the correct construct were identified by isolation of the plasmid, restriction enzyme mapping and DNA sequencing. The plasmid, in the following designated pTaq, contains an open reading frame coding for native Taq, except that the N-terminal sequence is MEFGML rather than MRGML in the native sequence, due to the choice of cloning procedure.

Introduction of a NruJ site in pTaq

To facilitate the introduction of foreign sequences into pTaq a restriction site was introduced in the part of the sequence that codes for the part of the polypeptide chain that links the 3 '-5' exo and the polymerase domain together, based on molecular modelling with the known 3D structure of the Klenow fragment as a template. Site- directed mutagenesis was performed using a U.S.E. Mutagenesis Kit (Pharmacia Biotech, Sweden) according to the instructions of the manufacturer, except that the concentration of the mutagenic primer was 25 times higher than recommended, and that the pTaq template was denatured with alkali rather than heat. The mutagenesis primer was GCCTCTCCACCTCG,CGATAAAGCCAAAGGA SEQ ID NO. 7, which is complementary to nucleotide 1277-1306 in the T. aquaticus polK gene sequence in the EMBL DNA sequence database (accession number D32013), except for the two underlined nucleotides in the sequence above. After final transformation of product DNA into E. coli strain DH5 made competent as described above, 12 plasmid containing colonies were grown up in 5 ml LB medium containing 50 μg/ml ampicillin, and DNA was isolated by a boiling method (Sambrook, J. , Fritsch, E. F. , and Maniatis, T. (1989) Molecular cloning. A laboratory manual, 2. ed. , Cold Spring Harbor Laboratory Press) and analysed by treatment with Seal and ivrul . Of 12 colonies, 11 contained plasmid with an insert of the expected size. Of these, all lacked the Seal site, indicating that the selection primer in the U.S.E. kit had worked, while 9 had a ivrul site, indicating that the wanted mutation had been introduced in these. One culture was chosen and used for the preparation of larger amount of mutated pTaq, which then was verified by DNA sequencing. This mutated version of pTaq will in the following be called pTaqMut .

PCR amplification of the 3 '-5' exo domain from Cau DIVA polymerase

The following amplification primers were designed:

5'-exo3: CACGAATTCACAGCCGAACCGCCACCGGT (SEQ ID NO. 8) 3' -exo3: TAATACAGATCGTGCAGCGC (SEQ ID NO. 9)

5 ' -exo3 corresponds to part of the sequence of the cloned Cau DNA polymerase, described in Tvermyr et al . Genetic analysis: Biomolecular Engineering 14: 75-83 (1998). In the polymerase clone, pCauPol , it is placed in the 5 ' end of the part of the sequence that codes for the 3' -5' exo domain, according to sequence alignments. 5'-exo3 in addition contains an EcoRI site. 3 ' -exo3 is complementary to an area in the Cau DNA polymerase clone assumed to code for C-terminal part of the Cau 3'-5' exo domain .

These primers were used to amplify DNA coding for the 3 ' -5 ' exo domain by using cloned Cau DNA polymerase as a template in a PCR reaction performed as described above, except that the annealing temperature was 55°C and that 30 cycles were used. The PCR product was analysed in a 1% agarose gel. The part of the gel containing the 0.6 kb product was cut out and DNA purified using a Quiaquick kit (Qiagen) .

Introduction of DNA coding for Cau 3 ' -5 ' exonuclease domain into pTaqM t

The PCR product obtained above was treated with the restriction endonuclease EcoRI and purified using a Qiaquick kit as above. The purified, BcoRI-cut fragment was ligated into the large DNA fragment from pTaqMut that had been cut with EcoRI and Nrul to release the fragment coding for the two exonuclease domains and gel purified as described above. After transformation and plating of E. coli DH5α, plasmid DNA was prepared from colonies by boiling as described above. Restriction fragment analysis showed that 10 of these plasmids gave the anticipated pattern. One of these was chosen and its sequence verified by DNA sequencing. In the following, this plasmid coding for the 3 '-5' exo domain from Cau DNA polymerase linked to the polymerase domain from Tag DNA polymerase is called pCauTaql (SEQ ID NO. 1) . The encoded amino acid sequence is shown in SEQ ID NO. 2.

Example 2: Construction of an expression vector coding for CauTag polymerase version 2

When the experimentally determined 3D structure of Taq DNA polymerase' became available, it indicated that the border between the 3' -5' exo domain and the polymerase domain is placed approximately 8 amino acid residues closer to the N-terminal than assumed during the construction of pCauTaql . Codons for these residues were introduced by designing a new 3 ' primer for the amplification of the Cau 3 '-5' exo domain:

3*-exo3b: CGATAAAGCCAAAGGAGCCTCTCTTCAGCCTCTAACTGAC (SEQ ID NO. 10)

The first 23 5' nucleotides in this sequence are complementary to sequence in the Tag gene, while the rest is complementary to sequence from the Cau clone. Amplification of DNA using cloned Cau DNA polymerase gene as a template and primer 5'-exo3 (see above) and 3 ' -exo3b as primers should give a fragment coding for the 3 '-5' exo domain from Cau DNA polymerase and in addition the 8 missing amino acid residues from the Taq polymerase domain.

Amplification was performed as described above, and the amplification product was cut with EcoRI , purified and cloned into EcoRI/Nrul cut pTaqMut in the same manner as for pCauTaql. The new construct, pCauTaq2 (SEQ ID NO. 3) , was isolated and the sequence verified by DNA sequencing. The encoded amino acid sequence is shown in SEQ ID NO . 4 .

Example 3 : Expression of CauTag DNA polymerases

pCauTaql and pCauTaq2 were separately transformed into the E. coli host strain INV F. Bacteria containing pCauTaql or 2 , or the vector pTrc99A without an insert, were grown in 0.11 1 LB broth containing 100 μg/ml ampicillin by adding 0.5 ml of an overnight culture to 1 1 of the medium. The culture was grown to OD₆₀₀ approximately 0.6, and expression of the plasmid-encoded polypeptides was induced by the addition of IPTG to a final concentration of 125 μg/ml. After 16 hours induction the cells were harvested by centrifugation and washed with 10 ml of buffer A (50 mM Tris-HCl pH 7.9, 50 mM dextrose, 1 mM EDTA) . The cells from each culture were again recovered by centrifugation and suspended in 3 ml pre-lysis buffer (buffer A + 4 mg/ml lysozyme) . After 15 min at room temperature an equal volume of lysis buffer (10 mM Tris-HCl pH 7.9, 50 mM KC1, 1 mM EDTA, 1 mM PMSF, 0.5% Tween 20, 0.5% Nonidet P40) was added, and the lysis mixture was incubated at 37°C for 60 min. After centrifugation for 10 min at 4°C and 15000 rpm the cleared lysate was transferred to a clean tube, and 0.7 ml 10 x storage buffer (0.5 M Tris-HCl pH 8.0. 1 M NaCl, 10 mM EDTA, 5 mM DTT) was added, followed by 100% glycerol to give a final concentration of 50% (v/v) glycerol. These extracts were stored at -20°C until further use.

SDS-PAGE analysis of the extracts showed clearly that a protein band of apparent molecular weight 95 kDa was present in lysates from bacterial cells transfected with a pCauTaq construct, but not in lysates from cells transfected with pTrc99A without an insert (results not shown) . Example 4. Demonstration of DNA polymerase activity in the chimeric constructs, and characterization of the activity

Since analysis of cleared lysates prepared from mock transfected E. coli cultures showed that endogenous activity from E. coli DNA polymerases could not be detected, the cleared lysates containing the chimeric constructs were considered suitable for detection and initial characterization of polymerase activity in the constructs .

Detection of DNA polymerase activity.

In a 0.5 ml microcentrifuge tube, 10 μl of 10 X polymerase buffer (500mM KC1, lOOmM Tris-HCl (pH 9.0 at 25°C) , 50 mM MgCl₂, 1% Triton^® X-100) was mixed with 2.5 μl of a dNTP mix with dATP, dGTP and dCTP, each in a concentration of 2 mM, 0.25 μl ³H-dTTP (1 μCi/μl) , 10 μg of activated DNA (nicked by partial digestion with nuclease (Aposhian and Kornberg (1962) J. Biol. Chem 237, 519), and MilliQ water to a final volume of 23 μl . After temperature equilibration for 0.5 minutes at 70°C, 2 μl of the cleared lysate, suitably diluted with storage buffer, was added and incubation at 70 °C continued for another 30 minutes. DNA was precipitated by addition of 1 ml of ice cold 10% TCA and 10 μl bovine serum albumin (100 mg/ml) as a carrier. Precipitated material was pelleted at 13000 x g for 30 min at 4°C. The pellet was recovered and washed by addition of 1 ml 10% TCA, careful vortexing and centrifugation again as above .

This washing step was repeated twice. The pellet was resuspended in 0.5 ml 10% TCA, vortexed and incubated at 90°C for 25 minutes for hydrolysis of DNA. After centrifugation, 450 μl of the supernatant is mixed with 3 ml Ultima Gold scintillation fluid and radioactivity measured in a liquid scintillation counter, washed and the amount of incorporated ³H-dTMP measured by scintillation counting. The amount of incorporated dTTP was calculated from the ratio of incorporated radioactivity relative to total added radioactivity. Table 1 below shows that whereas significant DNA polymerase activity was not detected in an extract from E. coli cells transfected with pTrc99A (the radioactivity recovered in this sample was 42 cpm) , extracts derived from cells transfected with either of the pCauTaq constructs possessed such activity. This shows that the chimeric CauTaq DNA polymerases encoded by the two pCauTaq constructs possess thermostable DNA polymerase activity.

Table 1. Polymerase activity at 70°C in various bacterial extracts.

The temperature optimum of the chimeric polymerases.

The optimal temperatures for the polymerase activities was determined using a DNA polymerase assay as described above, but incubating at various temperatures between 25 and 85°C. Also, the amount of ³H-dTMP was increased to 0.5 μl (0.5 μCi) and the incubation time reduced to 5 minutes . The data from this experiment are given in Figure 1. For comparison, the Figure includes the temperature profiles of the polymerase activity of the ancestral enzymes, Tag polymerase and Cau polymerase. The results shown in Figure 1 indicate that CauTaq DNA polymerase has optimal polymerase activity at 55°C, while Cau polymerase and Taq polymerase have optimal activity at 65 and 75°C, respectively.

Thermal inactivation of the polymerase activity.

CauTaq DNA polymerase version 2 was diluted in reaction mixtures corresponding to the ones used for the DNA polymerase assay above, but without added activated DNA as a template. After incubation at various temperatures for various time intervals, the reaction tubes were transferred to a heating block at 55°C, which is the optimal temperature of the polymerase activity of the enzyme. 10 μg of activated DNA was added, and remaining polymerase activity was determined as described above. For comparison, Taq polymerase and Cau polymerase were treated in the same way at various temperatures and time intervals, before remaining polymerase activity was quantified at 70°C as above.

The results shown in Figure 2 indicate that the polymerase activity of CauTaq DNA polymerase version 2 had a half-life at 55°C of 15 minutes. At 65°C the half- life was reduced to about 7.5 minutes. At 70°C and above, the polymerase activity was quickly lost within the first 5 minutes of incubation.

For Taq polymerase a half-life for the polymerase activity at 95°C of about 30 minutes was observed, in accordance with published results (Lawyer, F.C., Stoffel, S., Saiki, R.K., Chang, S.Y., Landre, P.A. , Abramson, R.D. and Gelfand, D.H. (1993) Genome Research. 2(4) :275-87) . The polymerase activity of Cau polymerase had a half-life of about 4 minutes at 75°C and about 2 minutes at 80°C (results not shown) .

Example 5. Demonstration of 3' -5' exonuclease activity in the chimeric constructs, and characterization of the activity

3' -5' exonuclease activity in the cleared lysates was detected using an assay system described in Tvermyr et al , supra . Elongation of primers labelled with the fluorescent label Cy5 using M13mpl8 single-stranded DNA as a template was measured using an ALF Express automated DNA sequencer and the Fragment Manager software (both from Pharmacia Biotech, Uppsala, Sweden) . Two primers were used:

Match primer: Cy5-ACGACGGCCAGTGCCAAGCT [SEQ ID NO. 11]

Mismatch primer: Cy5-ACGACGGCCAGTGCCAAGCA [SEQ ID NO. 12]

The Match primer is complementary to part of the M13mpl8 template. The Mismatch primer is identical to the Match primer except for the nucleotide in the 3', end, where the T in the Match primer is exchanged with an A in the Mismatch primer. The position in M13mpl8 to which the primers anneal was chosen in such a way that the 3 ' ends of the primers overlap with a recognition site for the restriction endonuclease Alul in the template sequence. Further Alul sites are found 63 and 88 nt downstream of the 5' end of the primers. Primer elongation followed by cutting of the double-stranded product with Alul would then give a 18 nt (nucleotides) long Cy5-labelled product, which could be detected and quantified using the DNA sequencer. Any extension of the Mismatch primer would give a double-stranded product that would not be cut by Alul in this position, due to the mismatch introduced by the primer. Cutting in the other Alul sites would instead give a 63 nt long Cy5-labelled product . With the perfectly matched primer annealed to the template DNA, any DNA polymerase would be able to extend it and in this way remove the 20 nt Cy5-labelled oligonucleotide from the reaction mix. Complete cutting with Alul should then give a 18 nt labelled fragment in an amount that corresponds to the amount of elongated primer in the reaction mix. Any unextended primer will remain at 20 nt even after Alul treatment, since the 18 nt A u site is too close to the end of the primer for the enzyme to cut. Using the primer with the 3' mismatch, polymerases would be expected to give different species of labelled fragments depending on its catalytic capabilities:

a) If the polymerase is unable to repair the mismatch and unable to extend the mismatched primer, the amount of Cy5-labelled 20mer should remain constant. Treatment with Alul will not influence this, since the Alul site is destroyed by the mismatch.

b) If the polymerase is unable to repair the mismatch but able to extend the mismatched primer the amount of Cy5-labelled 20mer should decrease. Treatment with Alul should give a 63 nt labelled fragment, since the Alu site in position 18 is destroyed by the mismatch.

c) If the polymerase has proofreading activity one would expect results similar to those obtained with the perfectly matched primer, i.e. removal of 20 nt fragment upon polymerase-catalysed elongation and appearance of a 18 nt fragment upon treatment with Alul .

It should be noted that this assay depends on both the exonuclease activity and the polymerase activity of the DNA polymerases . Because of this it is not suited for quantitative assessments of any of these activities separately. Detection of 3' -5' exonuclease activity

For each investigated polymerase 4 reactions were made, two with each primer. One reaction from each set was treated with Alul prior to analysis of labelled reaction products by electrophoresis in a fluorescence-based DNA sequencer. The reactions were set up as follows: The relevant primer was annealed to the M13 template by heating 0.2 μg M13mpl8 DNA and 0.2 pmol Match or Mismatch primer in 33 mM Tris-HCl pH 8.0 , 40 mM NaCl, 8 mM MgCl₂, total volume 6 μl, to 70°C for 3 min and cooling to 20°C over a period of 20 minutes. Then 1 μl of a solution containing 1.25 mM of the four dNTPs was added, and the mixtures were equilibrated at 70°C for 1 minute . DNA polymerase in an amount corresponding to approximately 0.5 U of polymerase activity were added in a volume of 1 μl, and the reaction mixtures were incubated at 70°C for 10 minutes. Samples to be analysed by restriction cutting then were provided with 0.5 μl 1 M NaCl and 0.5 μl Alul (New England Biolabs, 10 U/μl) and incubated at 37°C for 2-3 hours. After addition of 4 μl formamide and 0.25 μl Cy5 Size Marker 50-500 (Pharmacia Biotech) or other suitable Cy5- labelled oligonucleotides as size markers and quantitative standards, all samples were heated at 95°C prior to fragment size fractionation in a 20% polyacrylamide gel containing 7 M urea and 1 x TBE at 55°C in an ALF Express DNA Sequencer (Pharmacia Biotech) . Relative amounts and sizes of Cy5-labelled products were estimated using the Fragment Manager software from the same provider, by comparison with the mobilities and peak sizes of the added standards.

The results obtained are shown in Figure 3. In addition to the two chimeric construction of the invention, cleared lysate from E. coli transfected with pTaq and expressing Taq polymerase was analysed in the same way. The results show clearly that both of the constructs according to the invention have a 3' -5' exonuclease activity at 70°C that efficiently removes the mismatch and allows efficient elongation of the proof-read Mismatch primer. Tag polymerase shows, as expected, no signs of proofreading activity and is in addition not able to elongate the Mismatch primer.

The temperature optimum of the editing/extending activity

For quantitative estimation of the ability of the enzymes to edit mismatched bases and extend the edited primers, the DNA polymerases were diluted until only about 20-50% of the Mismatch primer was edited and extended under standard conditions. The diluted enzymes were then analysed in a standard exonuclease assay as above, except that the incubation temperature was varied. As a measure of the editing/extending capability we used the amount of 18 nucleotides long product formed by Alul digestion of extension products, relative to a fixed amount of Cy5-labelled oligonucleotide added to the reactions before loading on the polyacrylamide gel. In this way it is possible to compensate for any differences between the efficiency of the detectors associated with separate lanes in the gel .

The results in Figure 4 show that the temperature profile of CauTaq DNA polymerase version 2 with regard to editing/extending activity is similar to that of the polymerase activity of this enzyme. This indicates that the 3 '-5' exonuclease activity has a temperature optimum corresponding to that of the polymerase activity, or higher. Compared to the stability of the 3' -5' exonuclease activity of Cau DNA polymerase, the donor of this activity in CauTag polymerase, the temperature optimum of CauTaq polymerase is lower (≤ 60°C and 65- 70°C, respectively) .

Claims

Claims

1. A chimeric DNA polymerase comprising a 3'-»5' exonuclease domain from one DNA polymerase enzyme and a DNA polymerase domain from a heterologous DNA polymerase enzyme .

2. The chimeric DNA polymerase of claim 1 wherein said DNA polymerase further comprises a 5' -3' exonuclease domain .

3. The chimeric DNA polymerase of claim 1 or claim 2 wherein at least one of the domains is derived from a thermostable DNA polymerase .

4. The chimeric DNA polymerase of claim 3 wherein the thermostable DNA polymerase is selected from the group comprising Taq, Tth, Bca, Bst, Cau, Tma, Pfu, Vent and Deep Vent DNA polymerases .

5. The chimeric DNA polymerase of any one of claims 1 to 4 wherein said chimeric DNA polymerase is a thermostable DNA polymerase .

6. The chimeric DNA polymerase of any one of claims 1 to 5 wherein at least one of the domains is derived from a family A DNA polymerase.

7. The chimeric DNA polymerase of claim 6 wherein the family A DNA polymerase is selected from the group comprising E. coli polymerase I, Taq, Tth, Bca, Bst, Cau, Tma and T7 bacteriophage DNA polymerases.

8. The chimeric DNA polymerase of any one of claims 1 to 7 wherein the part of the chimeric DNA polymerase which exhibits the DNA polymerase activity is derived from Taq DNA polymerase .

9. The chimeric DNA polymerase of any one of claims 1 to 8 wherein the part of the chimeric DNA polymerase which exhibits the 3'-5' exonuclease activity is derived from Cau DNA polymerase or Tma DNA polymerase .

10. The chimeric DNA polymerase of any one of claims 1 to 9 wherein the 3' -5' exonuclease domain is derived from Cau DNA polymerase or Tma DNA polymerase and the DNA polymerase domain derived from Taq DNA polymerase .

11. The chimeric DNA polymerase of any one of claims 1 to 10 having the amino acid sequence shown in either SEQ ID NO: 2 or 4.

12. A nucleic acid molecule comprising a nucleotide sequence which encodes a chimeric DNA polymerase as defined in any one of claims 1 to 11.

13. The nucleic acid molecule of claim 12 wherein said molecule comprises the sequence as defined in SEQ ID NO. 1 or 3 , or a fragment thereof encoding a functionally active product, or a sequence which is substantially homologous with or which hybridises with the sequence as defined in SEQ ID NO. 1 or 3 or with the sequence complementary thereto, or a fragment thereof encoding a functionally active product.

14. An expression vector capable of expressing a chimeric DNA polymerase as defined in any one of claims 1 to 11.

15. The expression vector of claim 14 comprising a nucleic acid molecule as defined in claim 12 or claim 13.

16. A host cell expressing a chimeric DNA polymerase as defined in any one of claims 1 to 11.

17. A method of producing a chimeric DNA polymerase comprising the steps of (i) growing a host cell containing a nucleic acid molecule encoding a chimeric DNA polymerase as defined in any one of claims 1 to 11 under conditions suitable for the expression of the chimeric DNA polymerase; and (ii) isolating the chimeric DNA polymerase from the host cell or from the growth medium.

18. Use of the chimeric DNA polymerase as defined in any one of claims 1 to 11 in molecular biology and gene technology techniques.

19. The use as claimed in claim 18 wherein the technique is second strand DNA synthesis, PCR amplification, or DNA sequencing,

20. A kit for use in molecular biology and gene technology techniques comprising a chimeric DNA polymerase as defined in any one of claims 1 to 11.