# Patent

## Title
Modified DNA polymerases

## Abstract

The present invention provides, among other things, modified DNA polymerases containing amino acid alterations based on mutations identified in directed evolution experiments designed to select enzymes that are better suited for applications in recombinant DNA technologies.

## Background
The present application is a national phase entry of international application serial number PCT/US2009/063169, filed Nov. 3, 2009 which claims priority to U.S. Provisional patent application Ser. No. 61/110,883, filed Nov. 3, 2008, the entire disclosure of which is incorporated herein by reference.
The present specification makes reference to a Sequence Listing (submitted electronically as a .txt file named “Sequence Listing.txt” on May 3, 2011). The .txt file was generated on Nov. 9, 2009 and is 122 kb in size. The entire contents of the Sequence Listing are herein incorporated by reference.


BACKGROUND OF THE INVENTION
DNA polymerases are a family of enzymes that use single-stranded DNA as a template to synthesize the complementary DNA strand. In particular, DNA polymerases can add free nucleotides to the 3′ end of a newly-forming strand resulting in elongation of the new strand in a 5′-3′ direction. Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For example, many DNA polymerases have 3′→5′ exonuclease activity. These polymerases can recognize an incorrectly incorporated nucleotide and the 3′→5′ exonuclease activity of the enzyme allows the incorrect nucleotide to be excised (this activity is known as proofreading). Following nucleotide excision, the polymerase can re-insert the correct nucleotide and replication can continue. Many DNA polymerases also have 5′→3′ exonuclease activity.
DNA polymerases, like other natural enzymes, have evolved over millions of years to be efficient in their natural cellular environment. Many of them are almost perfectly adapted to work in that environment. In such an environment the way that the protein can evolve is constrained by a number of requirements; the protein has to interact with other cellular components, it has to function in the cytoplasm (i.e., particular pH, ionic strength, in the presence of particular compounds, etc.) and it cannot cause lethal or disadvantageous side effects that detract from the fitness of the parent organism as a whole.
When DNA polymerases are removed from their natural environment and used in industrial or research applications, the environment and conditions under which the enzyme is operating is inevitably vastly different than those in which it evolved. Many of the constraints that limited the evolutionary direction the protein could take fall away. Therefore, there is vast potential for improvement of DNA polymerases for use in industrial or research applications.
SUMMARY OF THE INVENTION
The present invention provides improved DNA polymerases that may be better suited for applications in recombinant DNA technologies. Among other things, the present invention provides modified DNA polymerases based on directed evolution experiments designed to select mutations that confer advantageous phenotypes under environment and conditions used in industrial or research applications.
In one aspect, the present invention provides modified DNA polymerases containing one or more amino acid alterations (e.g., substitution, deletion or insertion) corresponding to one or more positions selected from the positions identified in Tables 1, 2, 3, 4, 5, or 6 relative to the corresponding wild-type or parental enzyme. In some embodiments, such amino acid alterations alter (e.g., increase or decrease) enzyme activity, fidelity, processivity, elongation rate, stability, primer-dimer formation, salt resistance, solubility, expression efficiency, folding robustness, thermostability, polymerization activity, concentration robustness, resistance to impurities, strand-displacement activity, knock-out of uracil read-ahead function, nucleotide selectivity, and/or other properties and characteristics involved in the process of DNA polymerization.
In some embodiments, modified DNA polymerases of the invention contain amino acid alterations at one or more positions corresponding to F752, F591, E668, G638, E734, E377, T609, P454, E582 and/or G715 of SEQ ID NO:16 (the Kofu amino acid sequence shown in the Sequences section).
In some embodiments, modified DNA polymerases of the invention contain one or more amino acid substitutions selected from Tables 2, 3, 4, 5, or 6. In some embodiments, modified DNA polymerases of the invention contain one or more amino acid substitutions selected from F752Y, F591L, F591I, E668V, G638R, G638V, E734K, E377K, T6091, T609A, P454S, E582K and/or G715R.
In some embodiments, the present invention provides modified DNA polymerases containing one or more amino acid alterations (e.g., substitutions, deletions or insertions) at one or more positions selected from the positions corresponding to E377, V356, E386, F591, G638, E668, E734, E738, F752, and/or W772 of SEQ ID NO:16, wherein the one or more amino acid alterations increase the enzyme activity of the DNA polymerases. In some embodiments, modified DNA polymerases in accordance with the invention contain one or more amino acid substitutions selected from the substitutions corresponding to F752Y, F591L, F591I, G638V, G638R, E668V, E734K, V356M, E738G, E386K, W772R, and/or E377K of SEQ ID NO:16.
In some embodiments, the present invention provides modified DNA polymerases containing one or more amino acid alterations (e.g., substitutions, deletions or insertions) at one or more positions selected from the positions corresponding to D346, V356, E377, A494, A550, F591, G638, E668, E734, and/or E738, of SEQ ID NO:16, wherein the one or more amino acid alterations increase the DNA binding affinity of the DNA polymerases. In some embodiments, modified DNA polymerases in accordance with the invention contain one or more amino acid substitutions selected from the substitutions corresponding to F591I, F591L, A550V, E377K, A494V, E734K, G638V, G638R, E668V, D346G, V356M, E738G, E734G, and/or E734N of SEQ ID NO:16.
In some embodiments, the present invention provides modified DNA polymerases containing one or more amino acid alterations (e.g., substitutions, deletions or insertions) at one or more positions selected from the positions corresponding to S376, R410, E582, E652, A679, or T680 of SEQ ID NO.14, wherein the one or more amino acid alterations decrease the DNA binding affinity of the DNA polymerase. In some embodiments, modified DNA polymerases in accordance with the invention contain one or more amino acid alterations selected from the substitutions corresponding to R410H, E582K, E652K, A679V, A679T, S376G, and/or T680I of SEQ ID NO.16.
In some embodiments, the present invention provides modified DNA polymerases containing one or more amino acid alterations (e.g., substitutions, deletions or insertions) at positions corresponding to S376, V441, F591, G638, E668, T680, and/or F752, of SEQ ID NO.14, wherein the one or more amino acid alterations decrease the fidelity of the DNA polymerase.
In some embodiments, modified DNA polymerases of the invention contain one or more amino acid substitutions selected from the substitutions corresponding to F591L, F752Y, F591I, E668V, V441I, G638R, S376G and/or T680I, of SEQ ID NO.16.
In some embodiments, modified DNA polymerases of the present invention are modified from a naturally-occurring polymerase (e.g., a naturally-occurring euryarchaeal family B polymerase) including, but not limited to, the naturally-occurring polymerases isolated from P. kodakaraensis, P. furiosus, T. gorgonarius, T. zilligii, T. litoralis “VentTM”, P. GB-D “Deep Vent”, T. 9N-7, T. aggregans, T. barossii, T. fumicolans, T. celer, Pyrococcus sp. strain ST700, T. pacificus, P. abysii, T. profundus, T. siculi, T. hydrothermalis, Thermococcus sp. strain GE8, T. thioreducens, P. horikoshii or T. onnurineus NA1, or truncated versions thereof.
In some embodiments, modified DNA polymerases of the invention are modified from a recombinant or engineered DNA polymerase including, but not limited to, chimeric DNA polymerases, fusion polymerases, and other modified polymerases (e.g., polymerases that contain deletions, substitutions or insertions but retain polymerase activity). In some embodiments, modified DNA polymerases of the invention are modified from a chimeric DNA polymerase containing SEQ ID NO.16.
In another aspect, the present invention provides methods of engineering modified DNA polymerases based on various mutations described herein. In some embodiments, methods of the invention include steps of: (a) modifying a DNA polymerase by introducing one or more amino acid alterations at one or more positions corresponding to the positions identified in Table 1; (b) determining the enzyme activity, fidelity, processivity, elongation rate, stability, primer-dimer formation, salt resistance, and/or solubility of the modified DNA polymerase from step (a). In some embodiments, the present invention provides various modified DNA polymerases engineered according to the methods described herein.
The present invention also provides kits and compositions containing various modified polymerases described herein and uses thereof. In addition, the present invention provides nucleotide sequences encoding various modified polymerases described herein and vectors and/or cells containing the nucleotide sequences according to the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are for illustration purposes only not for limitation.
FIG. 1 depicts an alignment of exemplary naturally-occurring type B DNA polymerases and exemplary chimeric DNA polymerases, Kofu and Pod.



DEFINITIONS
Amino acid: As used herein, term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical without adversely affecting their activity. Amino acids may participate in a disulfide bond. The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. It should be noted that all amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus.
Base Pair (bp): As used herein, base pair refers to a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
Chimeric polymerase: As used herein, the term “chimeric polymerase” (also referred to as “chimera”) refers to any recombinant polymerase containing at least a first amino acid sequence derived from a first DNA polymerase and a second amino acid sequence derived from a second DNA polymerase. Typically, the first and second DNA polymerases are characterized with at least one distinct functional characteristics (e.g., processivity, elongation rate, fidelity). As used herein, a sequence derived from a DNA polymerase of interest refers to any sequence found in the DNA polymerase of interest, or any sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in the DNA polymerase of interest. A “chimeric polymerase” according to the invention may contain two or more amino acid sequences from related or similar polymerases (e.g., proteins sharing similar sequences and/or structures), joined to form a new functional protein. A “chimeric polymerase” according to the invention may contain two or more amino acid sequences from unrelated polymerases, joined to form a new functional protein. For example, a chimeric polymerase of the invention may be an “interspecies” or “intergenic” fusion of protein structures expressed by different kinds of organisms.
Complementary: As used herein, the term “complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base-pairing. It is known that an adenine nucleotide is capable of forming specific hydrogen bonds (“base pairing”) with a nucleotide which is thymine or uracil. Similarly, it is known that a cytosine nucleotide is capable of base pairing with a guanine nucleotide.
DNA binding affinity: As used herein, the term “DNA-binding affinity” typically refers to the activity of a DNA polymerase in binding DNA nucleic acid. In some embodiments, DNA binding activity can be measured in a two band-shift assay. For example, in some embodiments (based on the assay of Guagliardi et al. (1997) J. Mol. Biol. 267:841-848), double-stranded nucleic acid (the 452-bp HindIII-EcoRV fragment from the S. solfataricus lacS gene) is labeled with 32P to a specific activity of at least about 2.5×107 cpm/μg (or at least about 4000 cpm/fmol) using standard methods. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, NY) at 9.63-9.75 (describing end-labeling of nucleic acids). A reaction mixture is prepared containing at least about 0.5 μg of the polypeptide in about 10 μl of binding buffer (50 mM sodium phosphate buffer (pH 8.0), 10% glycerol, 25 mM KCl, 25 mM MgCl2). The reaction mixture is heated to 37° C. for 10 min. About 1×104 to 5×104 cpm (or about 0.5-2 ng) of the labeled double-stranded nucleic acid is added to the reaction mixture and incubated for an additional 10 min. The reaction mixture is loaded onto a native polyacrylamide gel in 0.5× Tris-borate buffer. The reaction mixture is subjected to electrophoresis at room temperature. The gel is dried and subjected to autoradiography using standard methods. Any detectable decrease in the mobility of the labeled double-stranded nucleic acid indicates formation of a binding complex between the polypeptide and the double-stranded nucleic acid. Such nucleic acid binding activity may be quantified using standard densitometric methods to measure the amount of radioactivity in the binding complex relative to the total amount of radioactivity in the initial reaction mixture. Other methods of measuring DNA binding affinity are known in the art (see, e.g. Kong et al. (1993) J. Biol. Chem. 268(3):1965-1975).
Elongation rate: As used herein, the term “elongation rate” refers to the average speed at which a DNA polymerase extends a polymer chain. As used herein, a high elongation rate refers to an elongation rate higher than 25 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s). As used in this application, the terms “elongation rate” and “speed” are used inter-changeably.
Enzyme activity: As used herein, the term “enzyme activity” refers to the specificity and efficiency of a DNA polymerase. Enzyme activity of a DNA polymerase is also referred to as “polymerase activity,” which typically refers to the activity of a DNA polymerase in catalyzing the template-directed synthesis of a polynucleotide. Enzyme activity of a polymerase can be measured using various techniques and methods known in the art. For example, serial dilutions of polymerase can be prepared in dilution buffer (e.g., 20 mM Tris.Cl, pH 8.0, 50 mM KCl, 0.5% NP 40, and 0.5% Tween-20). For each dilution, 5 μl can be removed and added to 45 μl of a reaction mixture containing 25 mM TAPS (pH 9.25), 50 mM KCl, 2 mM MgCl2, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.1 mM dCTP, 12.5 μg activated DNA, 100 μM [α-32P]dCTP (0.05 μCi/nmol) and sterile deionized water. The reaction mixtures can be incubated at 37° C. (or 74° C. for thermostable DNA polymerases) for 10 minutes and then stopped by immediately cooling the reaction to 4° C. and adding 10 μl of ice-cold 60 mM EDTA. A 25 μl aliquot can be removed from each reaction mixture. Unincorporated radioactively labeled dCTP can be removed from each aliquot by gel filtration (Centri-Sep, Princeton Separations, Adelphia, N.J.). The column eluate can be mixed with scintillation fluid (1 ml). Radioactivity in the column eluate is quantified with a scintillation counter to determine the amount of product synthesized by the polymerase. One unit of polymerase activity can be defined as the amount of polymerase necessary to synthesize 10 nmole of product in 30 minutes (Lawyer et al. (1989) J. Biol. Chem. 264:6427-647). Other methods of measuring polymerase activity are known in the art (see, e.g. Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3.sup.rd ed., Cold Spring Harbor Laboratory Press, NY)).
Fidelity: As used herein, the term “fidelity” refers to the accuracy of DNA polymerization by template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an inaccurate nucleotide, i.e., a nucleotide that is not incorporated at a template-dependent manner). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3′-5′ exonuclease activity of a DNA polymerase. The term “high fidelity” refers to an error rate less than 4.45×10−6 (e.g., less than 4.0×10−6, 3.5×10−6, 3.0×10−6, 2.5×10−6, 2.0×10−6, 1.5×10−6, 1.0×10−6, 0.5×10−6) mutations/nt/doubling. The fidelity or error rate of a DNA polymerase may be measured using assays known to the art. For example, the error rates of DNA polymerases can be tested using the lacI PCR fidelity assay described in Cline, J. et al. (96) NAR 24: 3546-3551. Briefly, a 1.9 kb fragment encoding the lacIOlacZa target gene is amplified from pPRIAZ plasmid DNA using 2.5 U DNA polymerase (i.e. amount of enzyme necessary to incorporate 25 nmoles of total dNTPs in 30 min at 72° C.) in the appropriate PCR buffer. The lacI-containing PCR products are then cloned into lambda GT10 arms, and the percentage of lacI mutants (MF, mutation frequency) is determined in a color screening assay, as described (Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) Gene 180: 1-8). Error rates are expressed as mutation frequency per by per duplication (MF/bp/d), where by is the number of detectable sites in the lad gene sequence (349) and d is the number of effective target doublings. Similar to the above, any plasmid containing the lacIOlacZa target gene can be used as template for the PCR. The PCR product may be cloned into a vector different from lambda GT (e.g., plasmid) that allows for blue/white color screening.
Fusion DNA polymerase: As used herein, the term “fusion DNA polymerase” refers to any DNA polymerase that is combined (e.g., covalently or non-covalently) with one or more protein domains having a desired activity (e.g., DNA-binding, stabilizing template-primer complexes, hydrolyzing dUTP). In some embodiments, the one or more protein domains are derived from a non-polymerase protein. Typically, fusion DNA polymerases are generated to improve certain functional characteristics (e.g., processivity, elongation rate, fidelity, salt-resistance, etc.) of a DNA polymerase.
Modified DNA polymerase: As used herein, the term “modified DNA polymerase” refers to a DNA polymerase originated from another (i.e., parental) DNA polymerase and contains one or more amino acid alterations (e.g., amino acid substitution, deletion, or insertion) compared to the parental DNA polymerase. In some embodiments, a modified DNA polymerases of the invention is originated or modified from a naturally-occurring or wild-type DNA polymerase. In some embodiments, a modified DNA polymerase of the invention is originated or modified from a recombinant or engineered DNA polymerase including, but not limited to, chimeric DNA polymerase, fusion DNA polymerase or another modified DNA polymerase. Typically, a modified DNA polymerase has at least one changed phenotypes compared to the parental polymerase.
Mutation: As used herein, the term “mutation” refers to a change introduced into a parental sequence, including, but not limited to, substitutions, insertions, deletions (including truncations). The consequences of a mutation include, but are not limited to, the creation of a new character, property, function, phenotype or trait not found in the protein encoded by the parental sequence.
Mutant: As used herein, the term “mutant” refers to a modified protein which displays altered characteristics when compared to the parental protein.
Joined: As used herein, “joined” refers to any method known in the art for functionally connecting polypeptide domains, including without limitation recombinant fusion with or without intervening domains, inter-mediated fusion, non-covalent association, and covalent bonding, including disulfide bonding, hydrogen bonding, electrostatic bonding, and conformational bonding.
Nucleotide: As used herein, a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3′ or 5′ position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a “base sequence” or “nucleotide sequence,” and is represented herein by a formula whose left to right orientation is in the conventional direction of 5′-terminus to 3′-terminus.
Oligonucleotide or Polynucleotide: As used herein, the term “oligonucleotide” is defined as a molecule including two or more deoxyribonucleotides and/or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be derived synthetically or by cloning. As used herein, the term “polynucleotide” refers to a polymer molecule composed of nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.
Polymerase: As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3′-end of the primer annealed to a polynucleotide template sequence, and will proceed toward the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides.
Primer: As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, e.g., in the presence of four different nucleotide triphosphates and thermostable enzyme in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the thermostable enzyme. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 nucleotides, although it may contain more or few nucleotides. Short primer molecules generally require colder temperatures to form sufficiently stable hybrid complexes with template.
Processivity: As used herein, “processivity” refers to the ability of a polymerase to remain attached to the template and perform multiple modification reactions. “Modification reactions” include but are not limited to polymerization, and exonucleolytic cleavage. In some embodiments, “processivity” refers to the ability of a DNA polymerase to perform a sequence of polymerization steps without intervening dissociation of the enzyme from the growing DNA chains. Typically, “processivity” of a DNA polymerase is measured by the length of nucleotides (for example 20 nts, 300 nts, 0.5-1 kb, or more) that are polymerized or modified without intervening dissociation of the DNA polymerase from the growing DNA chain. “Processivity” can depend on the nature of the polymerase, the sequence of a DNA template, and reaction conditions, for example, salt concentration, temperature or the presence of specific proteins. As used herein, the term “high processivity” refers to a processivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240 nts, 260 nts, 280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400 nts, or higher) per association/disassociation with the template. Processivity can be measured according the methods defined herein and in WO 01/92501 A1.
Synthesis: As used herein, the term “synthesis” refers to any in vitro method for making new strand of polynucleotide or elongating existing polynucleotide (i.e., DNA or RNA) in a template dependent manner Synthesis, according to the invention, includes amplification, which increases the number of copies of a polynucleotide template sequence with the use of a polymerase. Polynucleotide synthesis (e.g., amplification) results in the incorporation of nucleotides into a polynucleotide (i.e., a primer), thereby forming a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules. “DNA synthesis,” as used herein, includes, but is not limited to, PCR, the labeling of polynucleotide (i.e., for probes and oligonucleotide primers), polynucleotide sequencing.
Template DNA molecule: As used herein, the term “template DNA molecule” refers to a strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction.
Template-dependent manner: As used herein, the term “template-dependent manner” refers to a process that involves the template dependent extension of a primer molecule (e.g., DNA synthesis by DNA polymerase). The term “template-dependent manner” typically refers to polynucleotide synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of polynucleotide is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)).
Thermostable enzyme: As used herein, the term “thermostable enzyme” refers to an enzyme which is stable to heat (also referred to as heat-resistant) and catalyzes (facilitates) polymerization of nucleotides to form primer extension products that are complementary to a polynucleotide template sequence. Typically, thermostable stable polymerases are preferred in a thermocycling process wherein double stranded nucleic acids are denatured by exposure to a high temperature (e.g., about 95 C) during the PCR cycle. A thermostable enzyme described herein effective for a PCR amplification reaction satisfies at least one criteria, i.e., the enzyme do not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for denaturation will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° C. to about 96° C. for a time depending mainly on the temperature and the nucleic acid length, typically about 0.5 to four minutes. Higher temperatures may be tolerated as the buffer salt concentration and/or GC composition of the nucleic acid is increased. In some embodiments, thermostable enzymes will not become irreversibly denatured at about 90° C.-100° C. Typically, a thermostable enzyme suitable for the invention has an optimum temperature at which it functions that is higher than about 40° C., which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) magnesium and salt, concentrations and (2) composition and length of primer, hybridization can occur at higher temperature (e.g., 45° C.-70° C.). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40° C. (e.g., at 37° C.) are also with the scope of this invention provided they are heat-stable. In some embodiments, the optimum temperature ranges from about 50° C. to 90° C. (e.g., 60° C.-80° C.).
Wild-type: As used herein, the term “wild-type” refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, among other things, modified DNA polymerases containing amino acid alterations based on mutations identified in directed evolution experiments designed to select enzymes that are better suited for applications in recombinant DNA technologies.
As described in the Examples section, the present inventors have successfully developed directed DNA polymerase evolution experiments by mimicking the typical or less-than typical environments and conditions under which an enzyme is usually used or expected to be used in real-life industrial or research applications. Typically, no specific selection pressure is deliberately applied in the directed evolution experiments to increase the diversity of mutations that are selected for.
As discussed in the Examples, various mutations have been observed during the selection process (see Table 1). Many mutations are selected for a wide range of advantages relating to enzyme characteristics including, but not limited to, expression efficiency, solubility and folding robustness, thermostability, polymerization activity, processivity, speed (elongation rate), concentration robustness, resistance to impurities, fidelity, avoidance of primer-dimers, strand-displacement activity, knock-out of uracil read-ahead function, nucleotide selectivity, and other properties and characteristics involved in the process of DNA polymerization (see Table 2).
It is contemplated that the mutations identified herein confer a variety of phenotypes that would make DNA polymerases better suited for applications in recombinant DNA technologies. For example, mutations identified in accordance with the present invention may confer enzymatic phenotypes related to the selective advantages described herein. Indeed, the present inventors have identified or expect to identify mutant polymerases that express well, are more soluble, that display higher activity, fidelity, processivity and/or speed, that are active over a wide range of concentrations, that are resistant to impurities, that work over a range of concentrations and have a higher fidelity, and other phenotypes that may not be immediately measurable. Since many of these phenotypes may depend on the manner in which the DNA and polymerase interact, it is contemplated that many of the mutations identified in accordance with the present invention may affect DNA-polymerase binding characteristics.
In addition, it is contemplated that mutations identified according to the present invention may confer enzymatic phenotypes not directly related to the selective advantages described herein. For example, some phenotypes may confer no advantage, but merely be a side effect of the advantageous mutation. In addition, some mutants may display phenotypes that could be considered disadvantageous. For example, some mutations confer an advantage (for example, high activity), but this advantage comes at a cost (for example, high error-rate). If the advantage outweighs the disadvantage, the mutation will still be selected for. Such mutations may have commercial uses. For example, a low fidelity enzyme could be used in error prone PCR (e.g., for mutagenesis).
Exemplary mutations having specific phenotypes are shown in Tables 3, 4, 5 and 6.
It is further contemplated that, since many DNA polymerases have similar sequences, structures and functional domains, mutations and/or the positions where mutations occur identified herein can serve as bases for modification of DNA polymerases in general. For example, same or similar mutations, as well as other alterations, may be introduced at the corresponding positions in various DNA polymerases to generate modified enzymes that are better adapted for recombinant use.
DNA Polymerases
DNA polymerases in accordance with the present invention may be modified from any types of DNA polymerases including, but not limited to, naturally-occurring wild-type DNA polymerases, recombinant DNA polymerase or engineered DNA polymerases such as chimeric DNA polymerases, fusion DNA polymerases, or other modified DNA polymerases (e.g., DNA polymerases that contain deletions (N- or C-terminal or internal deletions), substitutions or insertains but retain polymerase activity).
Naturally-Occurring DNA Polymerases
In some embodiments, DNA polymerases suitable for the invention are naturally-occurring DNA polymerases (e.g., thermostable DNA polymerases). Typically, DNA polymerases are grouped into six families: A, B, C, D, X and Y. Families A, B, C are grouped based on their amino acid sequence homologies to E. coli polymerases I, II, and III, respectively. Family X has no homologous E. coli polymerases. In some embodiments, DNA polymerases suitable for the present invention are family B DNA polymerases. Family B polymerases include, but are not limited to, E. coli pol II, archaeal polymerases, PRD1, phi29, M2, T4 bacteriophage DNA polymerases, eukaryotic polymerases α, Δ, ε, and many viral polymerases. In some embodiments, DNA polymerases suitable for the invention are archaeal polymerases (e.g., euryarchaeal polymerases).
Suitable exemplary archaeal polymerases include, but are not limited to, DNA polymerases from archaea (e.g., Thermococcus litoralis (VentTM, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep VentTM, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738, BAA06142)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: O29753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus spp. GE8 (GenBank: CAC12850), Thermococcus spp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus spp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus spp. GE23 (GenBank: CAA90887), Pyrococcus spp. ST700 (GenBank: CAC12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: □58295), Desulforococcus species TOK, Desulfurococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)). Additional representative temperature-stable family A and B polymerases include, e.g., polymerases extracted from the thermophilic bacteria Thermus species (e.g., favus, Huber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus. 
Typically, appropriate PCR enzymes from the archaeal family B DNA polymerase group are commercially available, including Pfu (Stratagene), KOD (Toyobo), Pfx (Life Technologies, Inc.), Vent (New England BioLabs), Deep Vent (New England BioLabs), Tgo (Roche), and Pwo (Roche). Suitable DNA polymerases can also be derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures. In some embodiments, suitable archaea exhibit maximal growth temperatures of >80-85° C. or optimal growth temperatures of >70-80° C. Additional archaea related to those listed above are described in the following references: Archaea: A Laboratory Manual (Robb, F. T. and Place, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995.
DNA polymerases suitable for the present invention include DNA polymerases that have not yet been isolated.
Chimeric DNA Polymerases
In some embodiments, chimeric DNA polymerases suitable for the invention include any DNA polymerases containing sequences derived from two or more different DNA polymerases. In some embodiments, chimeric DNA polymerases suitable for the invention include chimeric DNA polymerases as described in co-pending application entitled “Chimeric DNA polymerases” filed on even date, the disclosures of which are hereby incorporated by reference. In some embodiments, chimeric DNA polymerases suitable for the invention contain sequences derived from Pfu and KOD DNA polymerases. In particular embodiments, a chimeric DNA polymerase suitable for the invention contains an amino acid sequence as shown in SEQ ID NO:16 (the Kofu amino acid sequence shown in the Sequences section). In some embodiments, a chimeric DNA polymerase suitable for the invention contains an amino acid sequence as shown in SEQ ID NO:15 (the Pod amino acid sequence shown in the Sequences section).
Chimeric DNA polymerases suitable for the invention also include the chimeric DNA polymerases described in U.S. Publication Nos. 20020119461, 20040058362 and U.S. Pat. No. 7,560,260, herein incorporated by reference in their entireties.
Fusion DNA Polymerases
Suitable fusion DNA polymerases include any DNA polymerases that are combined (e.g., covalently or non-covalently) with one or more protein domains having a desired activity (e.g., DNA-binding, dUTP hydrolysis or stabilizing template-primer complexes). In some embodiments, the one or more protein domains having the desired activity are derived from a non-polymerase protein. Typically, fusion DNA polymerases are generated to improve certain functional characteristics (e.g., processivity, elongation rate, fidelity, salt-resistance, dUTP tolerance etc.) of a DNA polymerase. For example, DNA polymerase has been fused in frame to the helix-hairpin-helix DNA binding motifs from DNA topoisomerase V and shown to increase processivity, salt resistance and thermostability of the fusion DNA polymerase as described in Pavlov et al., 2002, Proc. Natl. Acad. Sci. USA, 99:13510-13515. Fusion of the thioredoxin binding domain to T7 DNA polymerase enhances the processivity of the DNA polymerase fusion in the presence of thioredoxin as described in WO 97/29209, U.S. Pat. No. 5,972,603 and Bedford et al. Proc. Natl. Acad. Sci. USA 94: 479-484 (1997). Fusion of the archaeal PCNA binding domain to Taq DNA polymerase results in a DNA polymerase fusion that has enhanced processivity and produces higher yields of PCR amplified DNA in the presence, of PCNA (Motz, M., et al., J. Biol. Chem. May 3, 2002; 277 (18); 16179-88). Also, fusion of the sequence non-specific DNA binding protein Sso7d or Sac7d from Sulfolobus sulfataricus to a DNA polymerase, such as Pfu or Taq DNA polymerase, was shown to greatly increase the processivity of these DNA polymerases as disclosed in WO 01/92501 A1, which is hereby incorporated by reference in its entirety. Additional fusion polymerases are described in US Publication No. 20070190538A1, which is incorporated herein by reference.
Commercially available exemplary fusion polymerases include, but are not limited to, PhusionTM (Finnzymes and NEB, sold by BioRad as iProof) which is a chimeric Deep VentTM/Pfu DNA polymerase fused to a small basic chromatin-like Sso7d protein (see, U.S. Pat. No. 6,627,424, U.S. Application Publication NOs. 20040191825, 20040081963, 20040002076, 20030162173, 20030148330, and Wang et al. 2004, Nucleic Acids Research, 32(3), 1197-1207, all of which are hereby incorporated by reference); PfuUltraTM II Fusion (Stratagene) which is a Pfu-based DNA polymerase fused to a double stranded DNA binding protein (U.S. Application No. 20070148671, which is incorporated by reference); Herculase II Fusion (Stratagene) which is a Herculase II enzyme fused to a DNA-binding domain; and Pfx50 (Invitrogen) which is a DNA polymerase from T. zilligii fused to an accessory protein that stabilizes primer-template complexes.
Generation of Modified DNA Polymerases of the Invention
Modified DNA polymerases can be generated by introducing one or more amino acid alterations into a DNA polymerase at the positions corresponding to the positions described herein (e.g., positions identified in Tables 1, 2, 3, 4, 5 and 6).
Corresponding positions in various DNA polymerases can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above. An example of an alignment is shown in FIG. 1.
Alterations may be a substitution, deletion or insertion of one or more amino acid residues. Appropriate alteration for each position can be determined by examining the nature and the range of mutations at the corresponding position described herein. In some embodiments, appropriate amino acid alterations can be determined by evaluating a three-dimensional structure of a DNA polymerase of interest (e.g., parental DNA polymerase). For example, amino acid substitutions identical or similar to those described in Tables 1, 2, 3, 4, 5, or 6 can be introduced to a DNA polymerase. Alternative amino acid substitutions can be made using any of the techniques and guidelines for conservative and non-conservative amino acids as set forth, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution. As used herein, “non-conservative substitution” refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln. Insertions or deletions may optionally be in the range of 1 to 5 amino acids.
Appropriate amino acid alterations allowed in relevant positions may be confirmed by testing the resulting modified DNA polymerases for activity in the in vitro assays known in the art or as described in the Examples below.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)), inverse PCR with mutations included in the primer sequence, or other known techniques can be performed on the cloned DNA to produce desired modified DNA polymerases.
In some embodiments, alterations suitable for the invention also include chemical modification including acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
Modified DNA polymerases according to the invention may contain one or more amino acid alterations at one or more positions corresponding to those described in Tables 1, 2, 3, 4, 5, or 6. Modified DNA polymerases according to the invention may also contain additional substitutions, insertions and/or deletions independent of the mutations observed or selected in the directed evolution experiments. Thus, in some embodiments, a modified DNA polymerase according to the invention has an amino acid sequence at least 70%, including at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to the corresponding wild-type (or naturally-occurring) DNA polymerase.
“Percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a modified sequence that are identical with the amino acid residues in the corresponding parental sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity are similar to the alignment for purposes of determining corresponding positions as described above.
Methods well known in the art may be applied to express and isolate modified DNA polymerases. Many bacterial expression vectors contain sequence elements or combinations of sequence elements allowing high level inducible expression of the protein encoded by a foreign sequence. For example, expression vectors are commercially available from, for example, Novagen (www.emdbiosciences.com/html/NVG/AllTables.html#).
As an example, bacteria expressing an integrated inducible form of the T7 RNA polymerase gene may be transformed with an expression vector bearing a modified DNA polymerase gene linked to the T7 promoter. Induction of the T7 RNA polymerase by addition of an appropriate inducer, for example, isopropyl-p-D-thiogalactopyranoside (IPTG) for a lac-inducible promoter, induces the high level expression of the chimeric gene from the T7 promoter.
Appropriate host strains of bacteria may be selected from those available in the art by one of skill in the art. As a non-limiting example, E. coli strain BL-21 is commonly used for expression of exogenous proteins since it is protease deficient relative to other strains of E. coli. For situations in which codon usage for the particular polymerase gene differs from that normally seen in E. coli genes, there are strains of BL-21 that are modified to carry tRNA genes encoding tRNAs with rarer anticodons (for example, argU, ileY, leuW, and proL tRNA genes), allowing high efficiency expression of cloned chimeric genes (several BL21-CODON PLUSTM cell strains carrying rare-codon tRNAs are available from Stratagene, for example). Additionally or alternatively, genes encoding DNA polymerases may be codon optimized to facilitate expression in E. coli. Codon optimized sequences can be chemically synthesized.
There are many methods known to those of skill in the art that are suitable for the purification of a modified DNA polymerase of the invention. For example, the method of Lawyer et al. (1993, PCR Meth. & App. 2: 275) is well suited for the isolation of DNA polymerases expressed in E. coli, as it was designed originally for the isolation of Taq polymerase. Alternatively, the method of Kong et al. (1993, J. Biol. Chem. 268: 1965, incorporated herein by reference) may be used, which employs a heat denaturation step to destroy host proteins, and two column purification steps (over DEAE-Sepharose and heparin-Sepharose columns) to isolate highly active and approximately 80% pure DNA polymerase.
Further, modified DNA polymerase may be isolated by an ammonium sulfate fractionation, followed by Q Sepharose and DNA cellulose columns, or by adsorption of contaminants on a HiTrap Q column, followed by gradient elution from a HiTrap heparin column
Applications of Modified DNA Polymerases of the Invention
Modified DNA polymerases of the present invention may be used for any methods involving polynucleotide synthesis. Polynucleotide synthesis methods are well known to a person of ordinary skill in the art and can be found, for example, in Molecular Cloning second edition, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). For example, modified DNA polymerases of the present invention have a variety of uses in recombinant DNA technology including, but not limited to, labeling of DNA by nick translation, second-strand cDNA synthesis in cDNA cloning, DNA sequencing, whole-genome amplification and amplifying, detecting, and/or cloning nucleic acid sequences using polymerase chain reaction (PCR).
In some embodiments, the invention provides enzymes that are better suited for PCR used in industrial or research applications. PCR refers to an in vitro method for amplifying a specific polynucleotide template sequence. The technique of PCR is described in numerous publications, including, PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications, by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification, H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584, each of which is herein incorporated by reference.
Modified DNA polymerases with higher processivity, elongation rate and/or fidelity are expected to improve efficiency and success rate of long-range amplification (higher yield, longer targets amplified) and reduce the amount of required DNA template.
Various specific PCR amplification applications are available in the art (for reviews, see for example, Erlich, 1999, Rev Immunogenet., 1: 127-34; Prediger 2001, Methods Mol. Biol. 160: 49-63; Jurecic et al., 2000, Curr. Opin. Microbiol. 3: 316-21; Triglia, 2000, Methods Mol. Biol. 130: 79-83; MaClelland et al., 1994, PCR Methods Appl. 4: S66-81; Abramson and Myers, 1993, Current Opinion in Biotechnology 4: 41-47; each of which is incorporated herein by references).
As non-limiting examples, the present invention can be used in PCR applications including, but are not limited to, i) hot-start PCR which reduces non-specific amplification; ii) touch-down PCR which starts at high annealing temperature, then decreases annealing temperature in steps to reduce non-specific PCR product; iii) nested PCR which synthesizes more reliable product using an outer set of primers and an inner set of primers; iv) inverse PCR for amplification of regions flanking a known sequence. In this method, DNA is digested, the desired fragment is circularized by ligation, then PCR using primer complementary to the known sequence extending outwards; v) AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA). These methods create genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides; vi) RT-PCR which uses RNA-directed DNA polymerase (e.g., reverse transcriptase) to synthesize cDNAs which is then used for PCR. This method is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts; vii) RACE (rapid amplification of cDNA ends). This is used where information about DNA/protein sequence is limited. The method amplifies 3′ or 5′ ends of cDNAs generating fragments of cDNA with only one specific primer each (plus one adaptor primer). Overlapping RACE products can then be combined to produce full length cDNA; viii) DD-PCR (differential display PCR) which is used to identify differentially expressed genes in different tissues. First step in DD-PCR involves RT-PCR, then amplification is performed using short, intentionally nonspecific primers; ix) Multiplex-PCR in which two or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One DNA sequence can be use as control to verify the quality of PCR; x) Q/C-PCR (Quantitative comparative) which uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers; xi) Recusive PCR which is used to synthesize genes. Oligonucleotides used in this method are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (−20 bases); xii) Asymmetric PCR; xiii) In Situ PCR; xiv) Site-directed PCR Mutagenesis; xv) DOP-PCR that uses partially degenerate primers for whole-genome amplificationi; xvi) quantitative PCR using SYBR green or oligonucleotide probes to detect amplification; and xvii) error-prone PCR in which conditions are optimized to give an increased number of mutations in the PCR product.
It should be understood that this invention is not limited to any particular amplification system. As other systems are developed, those systems may benefit by practice of this invention.
Kits
The invention also contemplates kit formats which include a package unit having one or more containers containing modified DNA polymerases of the invention and compositions thereof. In some embodiments, the present invention provides kits further including containers of various reagents used for polynucleotide synthesis, including synthesis in PCR.
Inventive kits in accordance with the present invention may also contain one or more of the following items: polynucleotide precursors, primers, buffers, instructions, and controls. Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods.
EXAMPLES
Example 1
Directed Evolution Experiments Using a Chimeric Enzyme Kofu
To select mutated enzymes that would better be suited for recombinant DNA technologies, a directed evolution experiment is designed by simply mimicking the normal conditions under which the enzyme is usually used, or possibly under less than perfect conditions such as are expected in real-life applications. After conducting enough rounds of selection, an enzyme (or multiple enzymes) that is better suited for typical applications in recombinant DNA technologies should appear.
It is contemplated that this approach may be particularly suited to a chimeric enzyme that has been assembled from two different but similar enzymes, such as Kofu (see co-pending application entitled “Chimeric DNA polymerases” filed on even date, the disclosures of which are hereby incorporated by reference). The sequence of Kofu is shown in the Sequences section and in FIG. 1. The regions from KOD and Pfu are indicated in FIG. 1.
In this case, the component parts from different enzymes may not function well together in certain ways because they have evolved separately and been recently and artificially fused. Thus, small changes may be needed to bring the component parts into better conjunction. For example, mutations which bring the parts derived from KOD to more closely resemble the Pfu equivalent part (and vice versa) are likely to be selected for.
We have performed just such a selection on Kofu. In this particular experiment, the region that was mutated was limited to the polymerase domain of the enzyme. The PCR, in which the enrichment step manifests, was performed under near standard PCR conditions, without any hard, deliberately applied specific selective pressure. To varying degrees the reaction was made slightly suboptimal, as follows:
(1) Four different buffers were used; one was a standard PCR buffer, one contained known PCR enhancers, one contained higher than normal levels of the buffering component and the final was a combination of the previous two.
(2) The reaction contained impurities that may often be found in PCRs as they are commonly performed in real applications. The impurities included non-target DNA, RNA, proteins, lipids and other cellular components commonly found in biological samples.
(3) Primers were designed to have a propensity for primer-dimer formation (a common problem in PCR as it is performed in real applications).
(4) In some cases the extension time was marginally longer than required, or marginally shorter than ideal.
Several rounds of selection were conducted. During the course of the ongoing selection, it is likely that many different mutations will confer different types of advantage, to different degrees, either alone or in combination. Typically, during the first rounds of selection, there are no obvious dominant clones, while the huge numbers of neutral or disadvantageous mutants are likely to be eliminated. Thereafter, a large number of particular mutations typically appear in higher than expected numbers. These mutations are there because they have some advantages.
Typically, the selections are considered to have worked when the vast pool of mutants that are in the starting material have been eliminated and the pool is dominated by a remaining few types or families of mutants that have out-competed the other mutants and the wild type. At this stage, it is not necessary to define exactly the nature of the improvement that the mutations confer. The fact that it was selected for is sufficient proof, especially if the same mutation becomes dominant in independently run selections.
Further selection results in the number of some of these mutations increasing in the pool, while others may be eliminated possibly because they have some advantages but they are not sufficient enough to compete with better-adapted clones. At the same time, some previously unnoticed mutants may appear. The late appearance of these mutants might be due to the fact that these specific mutations were low in number in the starting pool, or that the mutation required another (or more than one) mutation in the same clone for the advantage to manifest. If selections continue even further, eventually, a few clones will likely to dominate substantially. Typically, it is important to isolate clones before this final point if it is desirable to isolate a wide range of beneficial mutations.
Example 2
Mutations Observed
Several rounds of selection were conducted as described in Example 1. During the course of the ongoing selections, many different mutations were observed either alone or in combination. Enzymes containing one or more of these mutations retain the enzymatic activity. Active enzymes with as many as 14 mutations were observed. An example is the highly active clone 5/7-F5 comprising the following mutations: E377K, L400P, N434S, K444M, P454S, A504V, C510S, F591I, V640I, K648R, V665M, 1697F, E734N and L742M. The mutations observed in all the clones that were sequenced are shown in Table 1.







TABLE 1







MUTATIONS OBSERVED








Position
Mutation











342
Q342L


343
P343A


343
P343R


343
P343S


344
L344P


344
L344Q


345
W345R


346
D346E


346
D346G


346
D346N


346
D346V


347
V347A


347
V347I


348
S348L


348
S348T


349
R349H


350
S350T


351
S351L


352
T352I


352
T352N


354
N354I


354
N354K


354
N354S


354
N354Y


355
L355F


355
L355I


355
L355V


356
V356A


356
V356L


356
V356M


357
E357G


358
W358R


361
L361M


361
L361V


362
R362G


362
R362K


363
K363R


365
Y365C


365
Y365F


365
Y365H


366
E366A


367
R367L


368
N368D


368
N368S


368
N368Y


369
E369D


369
E369V


370
V370A


370
V370I


370
V370L


371
A371T


371
A371V


372
P372L


373
N373S


374
K374R


375
P375L


376
S376G


376
S376N


376
S376R


376
S376T


377
E377D


377
E377G


377
E377K


377
E377R


377
E377V


378
E378D


378
E378G


378
E378K


378
E378V


379
E379K


379
E379V


381
Q381K


381
Q381R


382
R382H


382
R382L


384
L384H


384
L384R


384
L384V


385
R385H


386
E386G


386
E386K


386
E386V


387
S387T


389
T389I


389
T389S


392
F392Y


394
K394M


395
E395D


395
E395K


396
P396S


397
E397D


397
E397N


397
E397V


397
E397W


398
K398R


400
L400F


400
L400H


400
L400P


400
L400Y


402
E402G


403
N403K


404
I404V


410
R410H


410
R410L


414
P414S


415
S415T


416
I416T


416
I416V


422
V422I


424
P424L


424
P424S


425
D425G


425
D425N


426
T426M


427
L427F


427
L427P


427
L427R


428
N428D


428
N428S


428
N428Y


429
L429F


429
L429S


429
L429V


429
L429W


430
E430A


430
E430D


430
E430G


431
G431C


432
C432Y


433
K433E


434
N434D


434
N434I


434
N434K


434
N434S


434
N434Y


436
D436V


437
I437N


437
I437V


438
A438D


438
A438T


438
A438V


439
P439L


439
P439S


440
Q440H


440
Q440L


440
Q440R


441
V441I


442
G442E


443
H443D


443
H443R


443
H443Y


444
K444M


444
K444N


444
K444R


445
F445C


445
F445I


445
F445V


445
F445Y


447
K447E


447
K447N


447
K447R


448
D448Y


449
I449N


449
I449T


449
I449V


450
P450L


451
G451S


452
F452L


453
I453T


453
I453V


454
P454A


454
P454S


455
S455F


455
S455T


455
S455Y


456
L456M


456
L456V


458
G458A


458
G458D


458
G458S


458
G458V


459
H459N


459
H459Q


459
H459Y


460
L460S


461
L461P


461
L461Q


462
E462D


462
E462K


464
R464H


465
Q465H


465
Q465R


466
K466I


466
K466N


467
I467F


467
I467N


469
T469A


469
T469I


470
K470I


470
K470N


470
K470R


471
M471L


471
M471T


472
K472N


472
K472R


473
E473G


474
T474I


474
T474N


474
T474P


474
T474S


475
Q475H


475
Q475L


475
Q475R


476
D476E


476
D476G


476
D476N


477
P477H


477
P477L


477
P477S


477
P477T


478
I478F


478
I478L


478
I478N


478
I478T


479
E479D


479
E479K


479
E479V


480
K480E


480
K480M


480
K480T


481
I481F


481
I481L


481
I481N


481
I481T


481
I481V


483
L483F


483
L483H


484
D484E


486
R486L


488
K488I


488
K488N


488
K488R


489
A489P


492
L492S


493
L493I


494
A494S


494
A494T


494
A494V


494
A494V


496
S496P


496
S496T


497
F497Y


501
Y501C


501
Y501F


504
A504K


504
A504T


504
A504V


505
K505R


509
Y509C


509
Y509H


510
C510R


510
C510S


510
C510Y


511
K511N


512
E512D


512
E512G


512
E512K


512
E512Q


512
E512V


516
S516G


517
V517A


518
T518A


518
T518I


518
T518S


519
A519S


519
A519T


519
A519V


522
R522H


524
Y524H


525
I525M


526
E526V


527
L527S


528
V528A


529
W529R


530
K530G


530
K530M


530
K530N


530
K530R


531
E531K


532
L532Q


534
E534Q


534
E534V


535
K535I


536
F536L


536
F536S


536
F536Y


537
G537E


537
G537R


538
F538Y


539
K539I


539
K539R


540
V540L


541
L541I


541
L541P


550
A550V


551
T551I


552
I552T


552
I552V


553
P553H


553
P553L


554
G554D


555
G555R


555
G555W


556
E556D


556
E556K


556
E556Q


556
E556V


557
S557P


558
E558G


558
E558K


558
E558Q


559
E559D


560
I560L


560
I560T


560
I560V


561
K561E


561
K561M


561
K561R


562
K562I


562
K562R


563
K563E


563
K563I


563
K563N


563
K563R


563
K563T


564
A564T


564
A564V


565
L565M


566
E566D


566
E566G


567
F567I


567
F567S


567
F567Y


568
L568F


568
L568I


569
K569E


569
K569N


569
K569R


570
Y570F


570
Y570H


571
I571M


571
I571V


573
A573D


573
A573S


573
A573T


573
A573V


574
K574N


576
P576L


577
G577D


578
A578S


578
A578T


578
A578V


579
L579M


579
L579P


579
L579Q


580
E580G


580
E580K


582
E582D


582
E582G


582
E582K


582
E582V


584
E584K


585
G585R


586
F586L


588
K588E


588
K588R


589
R589C


591
F591I


591
F591L


591
F591Y


592
F592L


592
F592S


592
F592Y


593
V593A


593
V593L


594
T594A


594
T594S


595
K595R


597
K597R


599
A599T


599
A599V


600
V600A


600
V600L


601
I601F


602
D602N


604
E604D


605
G605D


605
G605S


606
K606E


606
K606N


606
K606R


608
T608A


608
T608K


608
T608M


609
T609A


609
T609I


609
T609S


610
R610K


614
I614M


614
I614S


614
I614T


614
I614V


615
V615A


615
V615I


617
R617G


619
W619R


620
S620G


620
S620N


621
E621D


621
E621G


621
E621V


622
I622F


622
I622V


623
A623T


623
A623V


625
E625D


626
T626A


626
T626S


628
A628T


628
A628V


629
R629H


629
R629S


630
V630A


630
V630I


630
V630L


631
L631S


632
E632G


632
E632V


633
A633T


633
A633V


634
L634V


635
L635M


635
L635P


636
K636I


636
K636R


637
D637G


638
G638E


638
G638R


638
G638V


638
G638W


640
V640A


640
V640D


640
V640F


640
V640I


641
E641G


641
E641K


642
K642E


642
K642M


642
K642N


642
K642R


643
A643T


643
A643V


644
V644A


644
V644L


644
V644M


645
R645L


645
R645P


645
R645Q


646
I646F


646
I646T


646
I646V


647
V647A


647
V647I


648
K648E


648
K648R


648
K648T


650
V650A


651
T651A


651
T651N


652
E652D


652
E652G


652
E652K


652
E652V


653
K653I


653
K653R


655
S655F


656
K656M


657
Y657C


658
E658G


658
E658K


659
V659I


660
P660L


660
P660Q


660
P660S


660
P660T


661
P661H


661
P661L


662
E662G


662
E662K


662
E662V


665
V665A


665
V665M


666
I666L


666
I666M


667
H667D


667
H667Y


668
E668G


668
E668K


668
E668V


669
Q669R


670
I670V


672
R672C


672
R672H


673
D673E


675
K675R


676
D676A


676
D676N


678
K678M


678
K678R


678
K678T


679
A679T


679
A679V


680
T680I


680
T680K


680
T680R


681
G681D


681
G681S


684
V684I


685
A685T


688
K688R


689
R689K


691
A691T


691
A691V


692
A692V


693
R693Q


693
R693W


694
G694D


695
V695A


695
V695D


695
V695G


695
V695I


695
V695L


696
K696E


697
I697F


698
R698H


698
R698P


701
T701A


701
T701S


702
V702M


703
I703M


703
I703T


704
S704G


704
S704N


706
I706T


706
I706V


707
V707I


708
L708S


709
K709E


709
K709M


709
K709N


709
K709Q


709
K709R


710
G710C


711
S711P


711
S711T


714
I714T


715
G715R


715
G715W


716
D716E


716
D716G


719
I719V


720
P720H


720
P720S


721
F721S


721
F721Y


722
D722G


723
E723D


723
E723V


724
F724Y


725
D725E


725
D725G


725
D725V


726
P726A


726
P726S


726
P726T


727
T727A


727
T727I


727
T727N


728
K728I


728
K728N


728
K728R


729
H729D


729
H729N


730
K730E


730
K730I


730
K730Q


731
Y731C


732
D732E


732
D732G


733
A733S


733
A733V


734
E734D


734
E734G


734
E734K


734
E734N


735
Y735F


737
I737N


737
I737T


738
E738D


738
E738G


738
E738K


739
N739H


739
N739K


740
Q740R


741
V741A


742
L742M


743
P743A


743
P743R


743
P743S


743
P743T


744
A744T


744
A744V


745
V745A


745
V745I


746
E746D


746
E746G


746
E746K


746
E746V


748
I748L


748
I748M


750
R750H


751
A751T


751
A751V


752
F752L


752
F752S


752
F752V


752
F752Y


754
Y754F


754
Y754H


756
K756E


757
E757D


757
E757G


758
D758G


758
D758N


758
D758Y


759
L759P


760
R760C


760
R760H


761
Y761C


761
Y761H


761
Y761N


762
Q762H


762
Q762L


762
Q762R


763
K763I


763
K763N


764
T764I


765
R765I


766
Q766R


767
V767A


767
V767L


768
G768D


768
G768R


768
G768S


769
L769M


769
L769P


769
L769R


770
S770P


770
S770Y


771
A771S


771
A771T


772
W772L


772
W772R


773
L773F


773
L773P


777
G777E


567
F567I


567
F567S


567
F567Y


568
L568F









Example 3
Types of Selective Advantage
There are a wide range of advantages that may have been selected for, some of which are listed and discussed below:
1) Expression Efficiency:
The clones that express higher levels of the enzyme will have an advantage over those that express less. The specific activity of the mutated enzyme may not have been improved but the total activity will have. This characteristics is particularly valuable to a manufacture of enzymes because this will allow increased production levels and/or reduced production costs.
2) Solubility and Folding Robustness:
When solubility increases, the probability of inclusion bodies forming decreases. Therefore, in these clones, a higher proportion of useful, correctly folded enzyme product is expressed.
3) Thermostability:
It is well known that, during the thermocycling required for PCR, a certain fraction of the enzyme is inactivated due to the heating. An enzyme that is resistant to heat-inactivation will maintain activity longer. Therefore, less enzyme can be used and/or more cycles can be conducted.
4) Activity:
Mutants with increased enzymatic activity provide more efficient polymerization.
5) Processivity:
Mutants with increased processivity are able to synthesize long PCR products. Mutant enzymes that can incorporate more nucleotides/extension step are likely to operate efficiently at lower concentrations.
6) Speed:
Mutants with increased elongation rate provide more efficient polymerization. Enzymes that are fast can also be used with shorter extension times. This is particularly valuable for a high-throughput system.
7) Concentration Robustness:
It is known that PCR reactions may not be carried out appropriately if too much or too little enzyme is used. Under the selection conditions we used, a polymerase that can generate appropriate products whether it is supplied in excess or at low levels will have an advantage and be selected for.
8) Resistance to Salts, PCR Additives and Other Impurities:
The selection was conducted in the presence of salts, PCR additives (e.g., intercalating dyes), and other impurities. The presence of slats may reduce the DNA binding affitnity of polymerases. The presence of impurities may interfere with formation of a desired PCR product. A polymerase that can resist to salts and impurities and synthesize desired products is advantageous and will be selected for. The characteristic is particularly suited for applications in which PCR is used in crude samples.
9) Fidelity:
All polymerases make mistakes during replication, either by incorporating the wrong dNTP or by stuttering which causes deletions and insertions. Such mistakes can eliminate functional genes during selection, so there is a pressure for mistakes not to be made. A polymerase with higher fidelity is advantageous and will be selected for.
10) Avoidance of Primer-Dimers:
As the selection PCR had a built-in propensity to produce primer-dimers, which compete with and so reduce the correct product, there is a selective pressure for polymerases that avoid primer-dimer formation. Polymerases that avoid primer-dimer formation are particularly valuable as primer-dimers are a common problem in PCR.
11) Strand-Displacement Activity:
Secondary structure in the DNA due to intramolecular self annealing may inhibit DNA strand-elongation catalyzed by the polymerase. Similarly, partial re-annealing of the complementary DNA in addition to the primer will inhibit PCR. Any enzyme with improved strand-displacement activity will have an advantage in the selection.
12) Knock-Out of Uracil Read-Ahead Function:
Type B polymerases have a read-ahead domain in the N-terminus that stalls the polymerase upon encountering a uracil residue in the template strand. Mutations that impede stalling at uracil residues may improve PCR efficiency and may therefore be selected for.
13) Increased Nucleotide Selectivity:
dUTP is formed during PCR as the deamination product of dCTP. As discussed above, incorporation of this nucleotide inhibits PCR. Any mutation that improves the selectivity for incorporating canonical nucleotides (dATP, dCTP, dGTP and dTTP) vs. modified nucleotides (e.g., dUTP), may improve PCR efficiency.
14) Pyrophosphate Tolerance:
Pyrophosphate is released during incorporation of nucleotides into the nascent strand by polymerases. Accumulation of pyrophosphate may lead to inhibition of the polymerase activity. Polymerases that were selected for in the Directed evolution example may have evolved to become less affected by product inhibition.
15) Unknown:
There many other factors involved in the process of PCR. Enzymes that are better adapted to PCR for any reason will be selected under our selection conditions.
Example 4
Mutations that were Selected for
The success of the selection were demonstrated if it was shown that (1) a variety of mutations have been selected for; (2) that these relatively few mutations have come to heavily dominate the pool; (3) that mutations appeared both singly and in combination; (4) that a final, dominating family was starting to appear; (5) that mutants displayed a variety of phenotypes; (6) that different profiles of mutants were selected with the different libraries; (7) that some mutations bring the KOD sections to more closely resemble the Pfu equivalent region, and vice versa; and/or (8) that at least some mutants have phenotypic characteristics that were predicted.
Exemplary mutations that were selected for are shown in Table 2. These mutations occurred at least once for every 40 clones sequenced (2.5%). Some mutations occurred in as many as 15% of the sequenced clones. All these mutations give the polymerase some kind of advantage in the selection. The list is prioritized. Highest priority is given to positions where mutations occur most frequently.







TABLE 2





Mutations that were selected for.
















 1)
F752Y


 2)
F591L


 3)
F591I


 4)
E668V


 5)
G638R


 6)
G638V


 7)
E734K


 8)
E377K


 9)
T609I


10)
T609A


11)
P454S


12)
E582K


13)
G715R


14)
E580K


15)
A691V


16)
E738G


17)
A494V


18)
K530R


19)
A550V


20)
E512K


21)
V615I


22)
V647A


23)
E652K


24)
V356M


25)
D346G


26)
S376G


27)
Q381R


28)
E386K


29)
R410H


30)
V441I


31)
K444R


32)
E462K


33)
T518A


34)
G555R


35)
K588R


36)
R589C


37)
K597R


38)
K606E


39)
K606N


40)
A633V


41)
A679T


42)
T680I


43)
K688R


44)
A733V


45)
A744V


46)
E746G


47)
A751T


48)
A751V


49)
Q766R


50)
W772R









Example 5
Mutant Phenotypes
Phenotypes of the selected mutants are closely related to the selective advantages described above. We have identified or expect to identify mutant polymerases that express well, are more soluble, that display higher activity, fidelity, processivity and/or speed, that are active over a wide range of concentrations, that are resistant to impurities, that work over a range of concentrations and/or have a higher fidelity. In addition, some mutant polymerases may have phenotypes that are not immediately measurable. Since many of these phenotypes may depend on the manner in which the DNA and polymerase interact, it is contemplated that the selected mutations may affect DNA-polymerase binding characteristics.
While the phenotypes of the mutants will usually be related to the advantages listed above, other phenotypes may be present. These phenotypes may confer no advantage, but merely be a side effect of the advantageous mutation. In addition some mutants may display phenotypes that could be considered disadvantageous. This is possible if the mutation confers an advantage (for example, high activity) but this comes at a cost (for example, high error-rate or lower DNA binding affinity). If the advantage outweighs the disadvantage, the mutation will still be selected for. Such mutations may have commercial uses, for example a low fidelity enzyme could be used in error-prone PCR (e.g., for mutagenesis). A polymerase with lower DNA-binding affinity may be useful in applications in which processive DNA synthesis is not required. An example of this is sequencing-by-synthesis where a single nucleotide is incorporated per cycle. The utility of an enzyme with lower DNA-binding affinity in sequencing is exemplified in US2006/0281109, which is incorporated herein by reference.
To demonstrate that a variety of phenotypes have been selected for, various clones were subjected to a number of phenotype tests. So far, we have conducted tests for a few different phenotypes: enzyme activity, binding affinity to DNA and fidelity. Exemplary mutations associated with these phenotypes are shown in Tables 3, 4, 5 and 6. Each list is prioritized. Priority rating is based on: strength of phenotype, the frequency with which this mutation occurs in the library pool after selection, and the confidence with which we can assign a phenotype.







TABLE 3







Mutations that increase enzyme activity








Priority



Ranking
Mutations











1.
F752Y


2.
F591L


3.
F591I


4.
G638V


5.
G638R


6.
E668V


7.
E734K


8.
V356M


9.
E738G


10.
E386K


11.
W772R


12.
E377K
















TABLE 4







Mutations that increase binding to DNA








Priority



Ranking
Mutations











1.
F591I


2.
F591L


3.
A550V


4.
E377K


5.
A494V


6.
E734K


7.
G638V


8.
G638R


9.
E668V


10.
D346G


11.
V356M


12.
E738G
















TABLE 5







Mutations that decrease binding to DNA








Priority



Ranking
Mutations





1.
R410H


2.
E582K


3.
E652K


4.
A679T


5.
S376G


6.
T680I
















TABLE 6







Mutations that decrease fidelity








Priority



Ranking
Mutations





1.
F591L


2.
F752Y


3.
F591I


4.
E668V


5.
V441I


6.
G638R


7.
S376G


8.
T680I









Example 6
Specific Examples of Phenotypes and Genotypes
The phenotypes associated with a particular mutation was assessed by expressing and purifying 49 clones. The binding affinity, enzyme activity and fidelity of each clone was determined as indicated in Examples 7-10 and compared to that of Kofu.
Specific examples of clones with altered phenotype compared to Kofu are shown in Table 7. Clones 6/7-D5, 10/7-D4 and 11/5Hi-E5 all contain the mutation F591L, in addition to other mutations. Sequencing of approximately 200 clones showed that the F591L mutation occurred in 15% of the clones. Thus, it is likely that this mutation gives the enzyme a selective advantage in the directed evolution experiment. The clones containing the F591L mutation are characterized by eluting from the heparin column at a higher salt concentration than Kofu, suggesting that they have high binding affinity for DNA. These clones also have higher activity than Kofu as measured both by the M13 activity assay and enzyme dilutions in PCR (see Examples 9 and 10). Furthermore, these clones have lower fidelity than Kofu. The other mutations that occur in these clones are not selected for, suggesting that they may not confer an advantage to the enzyme. These data indicate that the F591L mutation in Kofu increases the DNA-binding affinity, increases the enzyme activity, and decreases the fidelity of the enzyme.
Another example of a mutation that increases DNA-binding affinity is E377K. Clone 5/7-C4 contains only one other mutation and this mutation is not selected for, suggesting that the E377K is likely to be responsible for this phenotype.
Some mutations that were selected for usually occurred together with other mutations that were also selected for. One such example is R410H. This mutation occurs together with mutations that increase the DNA-binding affinity. Clones 11/5Hi-E3 and 5/7-A6 both contain the mutation R410H and each contains an additional mutation, F591L and A550V respectively, that increases the DNA-binding affinity. The presence of the R410H mutation reduces the DNA-binding affinity relative to clones that contain F591L or A550V but do not have the R410H mutation.
A similar approach was used to identify phenotypes for all the mutations shown in Tables 3-6. Examples of mutations in mutant clones obtained through directed evolution and associated phenotypes are shown in Table 7.







TABLE 7







Mutations in exemplary mutant clones and associated exemplary phenotypes














M13 activity
PCR activity
Fidelity
Binding affinity
Mutations
Mutations


Clone
Rel. to Kofu
Rel. to Kofu
10 × 10−6
mS/cm
Selected for
Other (not selected for)
















Kofu
1 
1 
1.5
37
n/a
n/a


6/7-D5
7x
2x
41
40.7
F591L
L400H, G458V, I706V


10/7-D4
7x
2x
64
39.3
F591L
K447E, K563T, K606E, E621G


11/5Hi-E5
4x
6x
12
40.8
F591L
F445I, K561E, K653R,


5/7-C4
1 
2x
4.8
41.9
E377K
A633T


11/5Hi-E3
3x
3x
3.7
38
R410H, F591L
E580G, K588R, K597R, A679V, R693Q, D725E


5/7-A6
3x
1 
7.6
33.8
R410H, A550V
P450L, K480M, K505R, K562R, P743T, V767A


5/7-H2
3x
1 
7
40.3
A550V
L427F, H459Q, K539R, V600A, I601F, F721S, T7









Example 7
Fidelity Assay
The fidelity of enzymes was determined by a method similar to that described by Cline et al. and references therein (Nucl. Acids Res., 1996, 24(18): 3546-3551). Lad was PCR amplified from E. coli and cloned into pUC19 to generate plasmid pKB-LacIQZalpha (SEQ ID NO:17). pKB-LacIQZalpha served both as template for PCR amplification of Lad in the fidelity assays and as vector for cloning the amplified Lad into for blue/white colony screening.
Specifically, 3×50 μl PCR reactions (for each enzyme) were set-up, using 2.4 ng of pKB-LacIQZalfa plasmid template (equivalent to 1 ng of lacI target), using varying amounts of each enzyme, to amplify the 1.386 Kb lacIQZalpha fragment. The amount of enzyme of each mutant to use in the fidelity assay was determined in an initial PCR using 2-fold dilutions of enzyme. The lowest concentrations of enzyme that gave specific PCR product in a sufficient yield for cloning were chosen for the fidelity assay (see Example 10). The PCR conditions for the fidelity assay were as follows: final concentrations of 1× KapaHifi Fidelity buffer, 2 mM MgCl2, 0.3 μM each of primers M13-40 (GTTTTCCCAGTCACGAC (SEQ ID NO:24)) and PKBlac-1R (GGTATCTTTATAGTCCTGTCG (SEQ ID NO:25)) and 0.3 mM each dNTP. Cycling parameters were: 95° C. 2 minutes, 25×(98° C. 25 seconds, 55° C. 15 seconds, 68° C. 1 minute), 68° C. 2 minutes.
PCR product yields were quantitated by means of gel electrophoresis and the numbers of template doublings were calculated. PCR products were digested with XbaI, NcoI and DpnI, gel-purified (without exposure to UV light) and ligated into XbaI-NcoI-digested pKB-LacIQZalpha. E. coli was transformed with the ligation mixtures and the cells were plated onto LB-Amp-X-gal plates. The number of blue colonies, white colonies and total number of colonies were recorded. The error rate f was calculated as f=−ln(F)/(d×(bp)), where F=fraction of white colonies ((total colonies minus blue colonies)/total colonies), d=number of template doublings and b=349 (only 349 bp of the lacI amplicon are scored).
The fidelities of Kofu and Kofu mutants ranged between 1.3 to 64×10−6 (see Table 7).
Example 8
DNA Binding Affinity of Kofu and Kofu Mutants
DNA binding affinity was measured based on heparin binding assays. Heparin is a naturally occurring sulphated glucosaminoglycan. Heparin consists of alternating units of various uronic acid residues and various D-glucosamine with most of these substituted with one or two sulphate groups. The three dimensional structure resembles a single helix. At physiological pH the sulphate groups are deprotonated. The negative charge and the helical structure mimic the structure and charge of DNA, enabling binding of DNA-binding proteins to heparin. DNA polymerases contain a number of positively charged amino acid residues that are involved in binding of the enzyme to DNA. This property can be utilized during purification of polymerases whereby the polymerase binds to heparin that is covalently coupled to agarose beads. The binding affinity of the polymerase is determined by the number and strength of binding interactions. The polymerase is eluted by increasing the amount of salt in the elution buffer. Ion-bonds between the polymerase and heparin will be disrupted by adding an increasing concentration of salt. The salt concentration at which the enzyme elutes is, therefore, indicative of the binding affinity of the polymerase for heparin and DNA.
Pellets of E. coli cells containing Kofu or mutants thereof were lysed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl (binding buffer). The lysates were incubated for 30 min at 75° C. to denature E. coli proteins, followed by centrifugation at 20 000 g for 20 min at 20° C. The supernatant was loaded onto a HiTrap Heparin column (GE Healthcare) and eluted on a 0.15 to 2 M NaCl gradient. The conductivity (mS/cm) at the elution peak was recorded as a measure of salt concentration of the eluate. A high conductivity indicates high affinity of the polymerase for heparin and DNA. The conductivity at the elution peak of Kofu was 37-38 mS/cm. The conductivity for low affinity polymerase mutants was between 34 and 37 mS/cm. The conductivity of high affinity polymerase mutants was between 38 and 51 mS/cm.
The conductivity is proportional to the amount of salt in a solution. We empirically determined the correlation between salt concentration and conductivity. We used the binding buffer and elution buffer at various ratios (final concentrations of 200 to 700 mM NaCl) and measured the conductivity. We plotted the conductivity vs. NaCl concentration. Linear regression analysis revealed that the conductivity (Cd) can be expressed as Cd=0.084×Cs+7.26, (R2=0.9995), where Cs is concentration of NaCl. From this we calculated that Kofu eluted at around 360 mM NaCl, and the mutants eluted at between around 320 and 520 mM NaCl.
Example 9
M13 Enzyme Activity Assay
Enzyme activity of Kofu and mutants of Kofu was measured either by M13 activity assay or by PCR using dilutions of enzyme. In the M13 activity assay, primed ssDNA was extended in an isothermal reaction using a range of dilutions of enzyme, and dsDNA was detected with SYBR green.
The following reactions were set up on ice: 50, 25, 12.5, 6.25, 3.1 or 1.6 ng enzyme was added per 25 μL reaction containing a final concentration of 1× KapaHifi Fidelity buffer, 2.5 mM MgCl2, 120 nM primer M13mp18-R (5′-AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACG-3′ (SEQ ID NO:26)), 0.3×SYBR Green, 0.3 mM dNTP and 200 ng M13mp18 ssDNA (NEB). Primer extension was performed in an MJ MiniOpticon (BioRad) with the following protocol: 100 cycles of (30 sec at 50° C., data acquisition). Primer extension was observed as an increase in fluorescence due to binding of SYBR green to dsDNA.
The rate of increase in fluorescence (the slope of the traces) for each mutant was compared to that of wild-type Kofu. A mutant requiring half as much enzyme to give the same slope (activity) as Kofu is scored as having twice the activity. Typically, mutants required between 8-fold less to 4-fold more enzyme to give the same activity as Kofu. This is equivalent to the mutants having between 8-fold more and 4-fold less activity than Kofu.
An increase in activity at a given protein concentration, may be due to changes in one or more of several factors. Some of these factors are: incorporation rate, off-rate and on-rate. The incorporation rate is the rate at which the enzyme incorporates nucleotides, i.e., nucleotides incorporated per unit of time. The on-rate (rate of association) is the rate at which the enzyme associates with the DNA template. The off-rate (rate of dissociation) is the rate at which the enzyme disassociates from the DNA template. The affinity of the polymerase for DNA is determined as ratio of the on-rate vs the off-rate. An increase in affinity can be due to either an increase in the on-rate or a decrease in the off-rate (at constant off-rate or on-rate, respectively).
An increase in processivity may be due to an increase in the incorporation rate or/and a decrease in the off-rate. An increase in the incorporation rate will enable incorporation of more nucleotides (assuming constant off-rate) before the enzyme and DNA disassociate. A decrease in the off-rate will increase the time the enzyme and DNA remain bound to each other, thus enabling incorporation of more nucleotides before they disassociate (assuming constant incorporation rate).
The elongation rate/enzyme activity (at a given protein concentration) is affected by the processivity and the affinity of the enzyme, and the underlying factors affecting affinity and processivity. Hence, an increase in the elongation rate/enzyme activity may be due to an increase in the incorporation rate, a decrease in the off-rate, an increase in the on-rate, or a combination thereof.
Exemplary results are shown in Table 7.
Example 10
PCR Enzyme Activity Assay
Another way of comparing enzyme activity is by using dilutions of enzyme in PCR. For each mutant and for Kofu, 25 μL reactions were set up with 2-fold dilutions of enzyme in the range 460-3.6 ng enzyme/reaction. The PCR reactions contained final concentrations of: 1× KapaHifi Fidelity buffer, 0.3 mM dNTP, 0.3 μM each of primers M13-40 (GTTTTCCCAGTCACGAC (SEQ ID NO:24)) and PKBlac-1R (GGTATCTTTATAGTCCTGTCG (SEQ ID NO:25)) and 1.4 ng/25 μL of pKB-LacIQZalfa. PCR cycling conditions were: 95° C. 2 minutes, 25×(98° C. 25 seconds, 55° C. 15 seconds, 68° C. 1 minute), 68° C. 2 minutes. The PCR products from the Kofu mutants were analyzed by gel electrophoresis and compared to that of wild-type Kofu. The highest dilution of enzyme that gave a specific product was scored. Enzymes that gave a PCR product with 2-fold less enzyme compared to Kofu were scored as having 2-fold higher activity. The activities of the mutants assayed ranged between 2-fold less active to 6-fold more active than Kofu.
Exemplary results are shown in Table 7.







TABLE 8





Sequences







Native DNA sequences of Pfu and KOD


Sequence 1


(SEQ ID NO: 1)


>Native Pfu nucleotide sequence from genomic sequence (Acc. No. AE010147)








   1
ATGATTTTAG ATGTGGATTA CATAACTGAA GAAGGAAAAC CTGTTATTAG GCTATTCAAA


 


  61
AAAGAGAACG GAAAATTTAA GATAGAGCAT GATAGAACTT TTAGACCATA CATTTACGCT


 


 121
CTTCTCAGGG ATGATTCAAA GATTGAAGAA GTTAAGAAAA TAACGGGGGA AAGGCATGGA


 


 181
AAGATTGTGA GAATTGTTGA TGTAGAGAAG GTTGAGAAAA AGTTTCTCGG CAAGCCTATT


 


 241
ACCGTGTGGA AACTTTATTT GGAACATCCC CAAGATGTTC CCACTATTAG AGAAAAAGTT


 


 301
AGAGAACATC CAGCAGTTGT GGACATCTTC GAATACGATA TTCCATTTGC AAAGAGATAC


 


 361
CTCATCGACA AAGGCCTAAT ACCAATGGAG GGGGAAGAAG AGCTAAAGAT TCTTGCCTTC


 


 421
GATATAGAAA CCCTCTATCA CGAAGGAGAA GAGTTTGGAA AAGGCCCAAT TATAATGATT


 


 481
AGTTATGCAG ATGAAAATGA AGCAAAGGTG ATTACTTGGA AAAACATAGA TCTTCCATAC


 


 541
GTTGAGGTTG TATCAAGCGA GAGAGAGATG ATAAAGAGAT TTCTCAGGAT TATCAGGGAG


 


 601
AAGGATCCTG ACATTATAGT TACTTATAAT GGAGACTCAT TCGACTTCCC ATATTTAGCG


 


 661
AAAAGGGCAG AAAAACTTGG GATTAAATTA ACCATTGGAA GAGATGGAAG CGAGCCCAAG


 


 721
ATGCAGAGAA TAGGCGATAT GACGGCTGTA GAAGTCAAGG GAAGAATACA TTTCGACTTG


 


 781
TATCATGTAA TAACAAGGAC AATAAATCTC CCAACATACA CACTAGAGGC TGTATATGAA


 


 841
GCAATTTTTG GAAAGCCAAA GGAGAAGGTA TACGCCGACG AGATAGCAAA AGCCTGGGAA


 


 901
AGTGGAGAGA ACCTTGAGAG AGTTGCCAAA TACTCGATGG AAGATGCAAA GGCAACTTAT


 


 961
GAACTCGGGA AAGAATTCCT TCCAATGGAA ATTCAGCTTT CAAGATTAGT TGGACAACCT


 


1021
TTATGGGATG TTTCAAGGTC AAGCACAGGG AACCTTGTAG AGTGGTTCTT ACTTAGGAAA


 


1081
GCCTACGAAA GAAACGAAGT AGCTCCAAAC AAGCCAAGTG AAGAGGAGTA TCAAAGAAGG


 


1141
CTCAGGGAGA GCTACACAGG TGGATTCGTT AAAGAGCCAG AAAAGGGGTT GTGGGAAAAC


 


1201
ATAGTATACC TAGATTTTAG AGCCCTATAT CCCTCGATTA TAATTACCCA CAATGTTTCT


 


1261
CCCGATACTC TAAATCTTGA GGGATGCAAG AACTATGATA TCGCTCCTCA AGTAGGCCAC


 


1321
AAGTTCTGCA AGGACATCCC TGGTTTTATA CCAAGTCTCT TGGGACATTT GTTAGAGGAA


 


1381
AGACAAAAGA TTAAGACAAA AATGAAGGAA ACTCAAGATC CTATAGAAAA AATACTCCTT


 


1441
GACTATAGAC AAAAAGCGAT AAAACTCTTA GCAAATTCTT TCTACGGATA TTATGGCTAT


 


1501
GCAAAAGCAA GATGGTACTG TAAGGAGTGT GCTGAGAGCG TTACTGCCTG GGGAAGAAAG


 


1561
TACATCGAGT TAGTATGGAA GGAGCTCGAA GAAAAGTTTG GATTTAAAGT CCTCTACATT


 


1621
GACACTGATG GTCTCTATGC AACTATCCCA GGAGGAGAAA GTGAGGAAAT AAAGAAAAAG


 


1681
GCTCTAGAAT TTGTAAAATA CATAAATTCA AAGCTCCCTG GACTGCTAGA GCTTGAATAT


 


1741
GAAGGGTTTT ATAAGAGGGG ATTCTTCGTT ACGAAGAAGA GGTATGCAGT AATAGATGAA


 


1801
GAAGGAAAAG TCATTACTCG TGGTTTAGAG ATAGTTAGGA GAGATTGGAG TGAAATTGCA


 


1861
AAAGAAACTC AAGCTAGAGT TTTGGAGACA ATACTAAAAC ACGGAGATGT TGAAGAAGCT


 


1921
GTGAGAATAG TAAAAGAAGT AATACAAAAG CTTGCCAATT ATGAAATTCC ACCAGAGAAG


 


1981
CTCGCAATAT ATGAGCAGAT AACAAGACCA TTACATGAGT ATAAGGCGAT AGGTCCTCAC


 


2041
GTAGCTGTTG CAAAGAAACT AGCTGCTAAA GGAGTTAAAA TAAAGCCAGG AATGGTAATT


 


2101
GGATACATAG TACTTAGAGG CGATGGTCCA ATTAGCAATA GGGCAATTCT AGCTGAGGAA


 


2161
TACGATCCCA AAAAGCACAA GTATGACGCA GAATATTACA TTGAGAACCA GGTTCTTCCA


 


2221
GCGGTACTTA GGATATTGGA GGGATTTGGA TACAGAAAGG AAGACCTCAG ATACCAAAAG


 


2281
ACAAGACAAG TCGGCCTAAC TTCCTGGCTT AACATTAAAA AATCCTAG


 







Sequence 2


(SEQ ID NO: 2)


>Native KOD nucleotide sequence (from genomic sequence, Acc. no. AP006878)








   1
ATGATCCTCG ACACTGACTA CATAACCGAG GATGGAAAGC CTGTCATAAG AATTTTCAAG


 


  61
AAGGAAAACG GCGAGTTTAA GATTGAGTAC GACCGGACTT TTGAACCCTA CTTCTACGCC


 


 121
CTCCTGAAGG ACGATTCTGC CATTGAGGAA GTCAAGAAGA TAACCGCCGA GAGGCACGGG


 


 181
ACGGTTGTAA CGGTTAAGCG GGTTGAAAAG GTTCAGAAGA AGTTCCTCGG GAGACCAGTT


 


 241
GAGGTCTGGA AACTCTACTT TACTCATCCG CAGGACGTCC CAGCGATAAG GGACAAGATA


 


 301
CGAGAGCATC CAGCAGTTAT TGACATCTAC GAGTACGACA TACCCTTCGC CAAGCGCTAC


 


 361
CTCATAGACA AGGGATTAGT GCCAATGGAA GGCGACGAGG AGCTGAAAAT GCTCGCCTTC


 


 421
GACATTGAAA CTCTCTACCA TGAGGGCGAG GAGTTCGCCG AGGGGCCAAT CCTTATGATA


 


 481
AGCTACGCCG ACGAGGAAGG GGCCAGGGTG ATAACTTGGA AGAACGTGGA TCTCCCCTAC


 


 541
GTTGACGTCG TCTCGACGGA GAGGGAGATG ATAAAGCGCT TCCTCCGTGT TGTGAAGGAG


 


 601
AAAGACCCGG ACGTTCTCAT AACCTACAAC GGCGACAACT TCGACTTCGC CTATCTGAAA


 


 661
AAGCGCTGTG AAAAGCTCGG AATAAACTTC GCCCTCGGAA GGGATGGAAG CGAGCCGAAG


 


 721
ATTCAGAGGA TGGGCGACAG GTTTGCCGTC GAAGTGAAGG GACGGATACA CTTCGATCTC


 


 781
TATCCTGTGA TAAGACGGAC GATAAACCTG CCCACATACA CGCTTGAGGC CGTTTATGAA


 


 841
GCCGTCTTCG GTCAGCCGAA GGAGAAGGTT TACGCTGAGG AAATAACCAC AGCCTGGGAA


 


 901
ACCGGCGAGA ACCTTGAGAG AGTCGCCCGC TACTCGATGG AAGATGCGAA GGTCACATAC


 


 961
GAGCTTGGGA AGGAGTTCCT TCCGATGGAG GCCCAGCTTT CTCGCTTAAT CGGCCAGTCC


 


1021
CTCTGGGACG TCTCCCGCTC CAGCACTGGC AACCTCGTTG AGTGGTTCCT CCTCAGGAAG


 


1081
GCCTATGAGA GGAATGAGCT GGCCCCGAAC AAGCCCGATG AAAAGGAGCT GGCCAGAAGA


 


1141
CGGCAGAGCT ATGAAGGAGG CTATGTAAAA GAGCCCGAGA GAGGGTTGTG GGAGAACATA


 


1201
GTGTACCTAG ATTTTAGATC CCTGTACCCC TCAATCATCA TCACCCACAA CGTCTCGCCG


 


1261
GATACGCTCA ACAGAGAAGG ATGCAAGGAA TATGACGTTG CCCCACAGGT CGGCCACCGC


 


1321
TTCTGCAAGG ACTTCCCAGG ATTTATCCCG AGCCTGCTTG GAGACCTCCT AGAGGAGAGG


 


1381
CAGAAGATAA AGAAGAAGAT GAAGGCCACG ATTGACCCGA TCGAGAGGAA GCTCCTCGAT


 


1441
TACAGGCAGA GGGCCATCAA GATCCTGGCA AACAGCTACT ACGGTTACTA CGGCTATGCA


 


1501
AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA CGGCCTGGGG AAGGGAGTAC


 


1561
ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT TTAAGGTAAT CTACAGCGAC


 


1621
ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG AAACCGTCAA AAAGAAGGCT


 


1681
ATGGAGTTCC TCAAGTATAT CAACGCCAAA CTTCCGGGCG CGCTTGAGCT CGAGTACGAG


 


1741
GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT ATGCGGTGAT AGACGAGGAA


 


1801
GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG ACTGGAGCGA GATAGCGAAA


 


1861
GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG GTGACGTCGA GAAGGCCGTG


 


1921
AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG AGGTTCCGCC GGAGAAGCTG


 


1981
GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA AGGCAACCGG TCCCCACGTT


 


2041
GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GTCAAAATAC GCCCTGGAAC GGTGATAAGC


 


2101
TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG CGATACCGTT CGACGAGTTC


 


2161
GACCCGACGA AGCACAAGTA CGACGCCGAG TACTACATTG AGAACCAGGT TCTCCCAGCC


 


2221
GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG ACCTGCGCTA CCAGAAGACG


 


2281
AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA CTTGA


 







Codon optimized sequences of Pfu and KOD


Sequence 3


(SEQ ID NO: 3)


>Pfu codon optimized nucleotide sequence








   1
ATGATTCTGG ATGTGGACTA TATCACCGAA GAGGGCAAAC CGGTTATACG TTTATTTAAG


 


  61
AAAGAGAATG GTAAATTCAA GATCGAGCAT GACCGCACGT TCCGTCCATA CATTTACGCG


 


 121
TTGCTTCGGG ATGATAGCAA AATTGAGGAA GTCAAAAAGA TCACCGGGGA ACGTCATGGA


 


 181
AAAATAGTAA GAATTGTGGA CGTTGAAAAA GTCGAAAAGA AATTTCTGGG CAAACCGATC


 


 241
ACTGTATGGA AGCTCTATCT GGAACATCCT CAGGATGTGC CCACAATTCG AGAAAAAGTT


 


 301
CGTGAGCACC CAGCCGTCGT GGATATATTT GAATATGACA TCCCTTTTGC AAAACGCTAC


 


 361
TTAATTGATA AAGGCCTGAT CCCGATGGAG GGGGAAGAAG AACTTAAAAT TCTGGCTTTT


 


 421
GACATAGAAA CGCTCTATCA TGAGGGAGAA GAATTTGGCA AAGGTCCCAT CATTATGATT


 


 481
TCTTACGCGG ATGAGAACGA AGCCAAGGTA ATCACTTGGA AAAATATTGA CCTGCCGTAC


 


 541
GTTGAAGTGG TCAGTTCAGA GCGGGAAATG ATTAAACGTT TTTTACGCAT CATTAGAGAG


 


 601
AAAGATCCAG ATATAATCGT TACATATAAC GGCGACTCCT TCGATTTTCC TTACCTGGCA


 


 661
AAACGAGCTG AAAAATTGGG TATTAAACTT ACCATCGGGC GTGACGGATC GGAACCGAAA


 


 721
ATGCAACGCA TTGGCGATAT GACGGCGGTA GAGGTGAAAG GTCGGATACA CTTTGATCTG


 


 781
TATCATGTCA TCACCCGTAC TATTAATCTC CCCACATACA CGTTAGAAGC CGTTTATGAG


 


 841
GCAATATTCG GCAAGCCGAA AGAAAAAGTG TACGCTGACG AAATCGCGAA GGCATGGGAG


 


 901
AGCGGCGAAA ACCTGGAGCG CGTAGCAAAA TATTCTATGG AAGATGCTAA AGCGACCTAC


 


 961
GAATTGGGGA AAGAATTTCT TCCAATGGAA ATTCAGCTGA GTCGTTTAGT CGGACAACCT


 


1021
CTGTGGGACG TTTCACGCTC CTCGACTGGC AATCTCGTGG AGTGGTTCCT GTTGAGAAAA


 


1081
GCCTATGAAC GAAACGAAGT AGCACCGAAT AAACCAAGCG AGGAAGAATA TCAGCGTCGC


 


1141
CTTCGCGAGT CTTACACAGG TGGGTTTGTT AAGGAACCGG AGAAAGGTCT TTGGGAAAAC


 


1201
ATCGTGTATT TAGATTTCCG TGCGCTGTAC CCCAGTATTA TAATCACCCA CAATGTCTCA


 


1261
CCTGACACGC TCAACTTGGA AGGTTGCAAA AATTATGATA TTGCTCCGCA AGTTGGACAT


 


1321
AAGTTTTGTA AAGATATTCC GGGCTTCATC CCGTCCCTGC TTGGTCACTT ACTGGAAGAG


 


1381
CGCCAAAAAA TTAAGACCAA AATGAAAGAG ACTCAGGATC CCATTGAAAA GATCCTGCTC


 


1441
GATTACCGGC AAAAAGCCAT TAAATTGCTT GCAAACTCGT TTTATGGGTA CTATGGCTAT


 


1501
GCGAAGGCTC GTTGGTACTG CAAAGAATGT GCCGAGAGCG TGACAGCATG GGGTCGCAAA


 


1561
TATATAGAAT TAGTATGGAA GGAGCTGGAA GAAAAATTCG GATTCAAAGT CCTGTACATC


 


1621
GATACGGATG GCCTCTATGC GACCATTCCT GGTGGGGAGT CTGAAGAAAT CAAGAAAAAA


 


1681
GCCTTGGAAT TCGTTAAGTA CATTAATAGT AAATTACCGG GACTGCTTGA ACTGGAGTAT


 


1741
GAAGGCTTCT ACAAAAGAGG TTTTTTCGTT ACTAAGAAAC GATATGCCGT AATAGATGAA


 


1801
GAGGGGAAAG TCATCACACG TGGCCTCGAG ATTGTTCGCC GGGACTGGTC AGAGATAGCA


 


1861
AAGGAAACGC AGGCGCGCGT GCTCGAAACC ATCTTGAAAC ATGGTGATGT AGAGGAAGCC


 


1921
GTCCGCATTG TTAAAGAGGT GATCCAGAAG TTAGCAAACT ATGAAATTCC ACCGGAAAAA


 


1981
CTGGCGATAT ACGAGCAAAT CACTCGTCCC CTTCACGAAT ATAAAGCTAT TGGACCTCAT


 


2041
GTAGCCGTCG CGAAGAAACT GGCTGCAAAA GGCGTTAAGA TAAAACCAGG TATGGTGATC


 


2101
GGGTACATTG TACTCCGCGG CGACGGTCCG ATTTCCAATA GAGCCATCTT GGCGGAGGAA


 


2161
TATGATCCTA AAAAGCATAA ATACGACGCT GAATATTACA TTGAGAACCA GGTCTTGCCG


 


2221
GCAGTTCTGC GGATACTTGA AGGATTTGGC TATCGTAAAG AAGATCTGCG CTATCAAAAG


 


2281
ACGCGACAGG TGGGTCTGAC TAGCTGGTTG AATATCAAAA AATCGTAA


 







Sequence 4


(SEQ ID NO: 4)


>Pfu codon optimized nucleotide sequence, extra 9 nt in 5′ area.








   1
ATGGCTAGCG CCATTCTGGA TGTGGACTAT ATCACCGAAG AGGGCAAACC GGTTATACGT


 


  61
TTATTTAAGA AAGAGAATGG TAAATTCAAG ATCGAGCATG ACCGCACGTT CCGTCCATAC


 


 121
ATTTACGCGT TGCTTCGGGA TGATAGCAAA ATTGAGGAAG TCAAAAAGAT CACCGGGGAA


 


 181
CGTCATGGAA AAATAGTAAG AATTGTGGAC GTTGAAAAAG TCGAAAAGAA ATTTCTGGGC


 


 241
AAACCGATCA CTGTATGGAA GCTCTATCTG GAACATCCTC AGGATGTGCC CACAATTCGA


 


 301
GAAAAAGTTC GTGAGCACCC AGCCGTCGTG GATATATTTG AATATGACAT CCCTTTTGCA


 


 361
AAACGCTACT TAATTGATAA AGGCCTGATC CCGATGGAGG GGGAAGAAGA ACTTAAAATT


 


 421
CTGGCTTTTG ACATAGAAAC GCTCTATCAT GAGGGAGAAG AATTTGGCAA AGGTCCCATC


 


 481
ATTATGATTT CTTACGCGGA TGAGAACGAA GCCAAGGTAA TCACTTGGAA AAATATTGAC


 


 541
CTGCCGTACG TTGAAGTGGT CAGTTCAGAG CGGGAAATGA TTAAACGTTT TTTACGCATC


 


 601
ATTAGAGAGA AAGATCCAGA TATAATCGTT ACATATAACG GCGACTCCTT CGATTTTCCT


 


 661
TACCTGGCAA AACGAGCTGA AAAATTGGGT ATTAAACTTA CCATCGGGCG TGACGGATCG


 


 721
GAACCGAAAA TGCAACGCAT TGGCGATATG ACGGCGGTAG AGGTGAAAGG TCGGATACAC


 


 781
TTTGATCTGT ATCATGTCAT CACCCGTACT ATTAATCTCC CCACATACAC GTTAGAAGCC


 


 841
GTTTATGAGG CAATATTCGG CAAGCCGAAA GAAAAAGTGT ACGCTGACGA AATCGCGAAG


 


 901
GCATGGGAGA GCGGCGAAAA CCTGGAGCGC GTAGCAAAAT ATTCTATGGA AGATGCTAAA


 


 961
GCGACCTACG AATTGGGGAA AGAATTTCTT CCAATGGAAA TTCAGCTGAG TCGTTTAGTC


 


1021
GGACAACCTC TGTGGGACGT TTCACGCTCC TCGACTGGCA ATCTCGTGGA GTGGTTCCTG


 


1081
TTGAGAAAAG CCTATGAACG AAACGAAGTA GCACCGAATA AACCAAGCGA GGAAGAATAT


 


1141
CAGCGTCGCC TTCGCGAGTC TTACACAGGT GGGTTTGTTA AGGAACCGGA GAAAGGTCTT


 


1201
TGGGAAAACA TCGTGTATTT AGATTTCCGT GCGCTGTACC CCAGTATTAT AATCACCCAC


 


1261
AATGTCTCAC CTGACACGCT CAACTTGGAA GGTTGCAAAA ATTATGATAT TGCTCCGCAA


 


1321
GTTGGACATA AGTTTTGTAA AGATATTCCG GGCTTCATCC CGTCCCTGCT TGGTCACTTA


 


1381
CTGGAAGAGC GCCAAAAAAT TAAGACCAAA ATGAAAGAGA CTCAGGATCC CATTGAAAAG


 


1441
ATCCTGCTCG ATTACCGGCA AAAAGCCATT AAATTGCTTG CAAACTCGTT TTATGGGTAC


 


1501
TATGGCTATG CGAAGGCTCG TTGGTACTGC AAAGAATGTG CCGAGAGCGT GACAGCATGG


 


1561
GGTCGCAAAT ATATAGAATT AGTATGGAAG GAGCTGGAAG AAAAATTCGG ATTCAAAGTC


 


1621
CTGTACATCG ATACGGATGG CCTCTATGCG ACCATTCCTG GTGGGGAGTC TGAAGAAATC


 


1681
AAGAAAAAAG CCTTGGAATT CGTTAAGTAC ATTAATAGTA AATTACCGGG ACTGCTTGAA


 


1741
CTGGAGTATG AAGGCTTCTA CAAAAGAGGT TTTTTCGTTA CTAAGAAACG ATATGCCGTA


 


1801
ATAGATGAAG AGGGGAAAGT CATCACACGT GGCCTCGAGA TTGTTCGCCG GGACTGGTCA


 


1861
GAGATAGCAA AGGAAACGCA GGCGCGCGTG CTCGAAACCA TCTTGAAACA TGGTGATGTA


 


1921
GAGGAAGCCG TCCGCATTGT TAAAGAGGTG ATCCAGAAGT TAGCAAACTA TGAAATTCCA


 


1981
CCGGAAAAAC TGGCGATATA CGAGCAAATC ACTCGTCCCC TTCACGAATA TAAAGCTATT


 


2041
GGACCTCATG TAGCCGTCGC GAAGAAACTG GCTGCAAAAG GCGTTAAGAT AAAACCAGGT


 


2101
ATGGTGATCG GGTACATTGT ACTCCGCGGC GACGGTCCGA TTTCCAATAG AGCCATCTTG


 


2161
GCGGAGGAAT ATGATCCTAA AAAGCATAAA TACGACGCTG AATATTACAT TGAGAACCAG


 


2221
GTCTTGCCGG CAGTTCTGCG GATACTTGAA GGATTTGGCT ATCGTAAAGA AGATCTGCGC


 


2281
TATCAAAAGA CGCGACAGGT GGGTCTGACT AGCTGGTTGA ATATCAAAAA ATCGTAA


 







Sequence 5


(SEQ ID NO: 5)


>KOD codon optimized nucleotide sequence








   1
ATGATTCTGG ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG


 


  61
AAAGAGAATG GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG


 


 121
TTACTGAAGG ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG


 


 181
ACAGTGGTAA CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA


 


 241
GAAGTGTGGA AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT


 


 301
CGCGAACATC CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC


 


 361
CTCATCGACA AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC


 


 421
GACATTGAAA CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT


 


 481
TCTTATGCGG ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC


 


 541
GTGGACGTCG TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG


 


 601
AAAGATCCAG ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA


 


 661
AAAAGATGCG AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA


 


 721
ATCCAGCGTA TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG


 


 781
TATCCGGTAA TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG


 


 841
GCAGTTTTTG GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA


 


 901
ACAGGCGAGA ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT


 


 961
GAATTGGGTA AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC


 


1021
CTGTGGGACG TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA


 


1081
GCATACGAGC GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT


 


1141
CGCCAATCCT ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC


 


1201
GTGTATCTGG ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC


 


1261
GACACTTTGA ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA


 


1321
TTTTGCAAGG ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA


 


1381
CAAAAAATCA AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC


 


1441
TACCGCCAGC GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG


 


1501
CGTGCTCGCT GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC


 


1561
ATCACCATGA CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT


 


1621
ACAGACGGAT TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG


 


1681
ATGGAATTCC TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA


 


1741
GGGTTTTACA AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA


 


1801
GGCAAGATTA CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA


 


1861
GAAACACAGG CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG


 


1921
CGAATTGTAA AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG


 


1981
GTGATACACG AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA


 


2041
GCAGTCGCCA AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT


 


2101
TACATTGTAT TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT


 


2161
GATCCAACCA AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG


 


2221
GTTGAGCGTA TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT


 


2281
AGACAGGTGG GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAA


 







Sequence 6


(SEQ ID NO: 6)


>KOD codon optimized nucleotide sequence, extra 9 nt in 5′ area.








   1
ATGGCTAGCG CCATTCTGGA TACCGACTAT ATCACGGAAG ATGGCAAACC GGTGATACGT


 


  61
ATTTTTAAGA AAGAGAATGG TGAGTTCAAA ATCGAGTACG ACCGCACTTT TGAGCCATAT


 


 121
TTCTACGCGT TACTGAAGGA CGATAGCGCC ATTGAAGAAG TTAAAAAAAT CACCGCAGAG


 


 181
CGGCATGGGA CAGTGGTAAC CGTGAAGAGA GTTGAAAAAG TCCAGAAAAA ATTTTTGGGA


 


 241
CGACCTGTAG AAGTGTGGAA ACTTTATTTC ACTCACCCCC AAGATGTTCC GGCTATACGT


 


 301
GATAAAATTC GCGAACATCC AGCGGTCATT GATATTTACG AATATGATAT ACCTTTTGCC


 


 361
AAGCGTTACC TCATCGACAA AGGCCTGGTG CCGATGGAAG GTGATGAAGA ATTAAAAATG


 


 421
TTGGCATTCG ACATTGAAAC ACTTTATCAC GAGGGGGAAG AGTTTGCTGA GGGTCCCATC


 


 481
CTGATGATTT CTTATGCGGA TGAAGAGGGT GCCCGCGTAA TAACCTGGAA GAACGTTGAT


 


 541
CTCCCGTACG TGGACGTCGT TAGTACGGAA CGGGAAATGA TCAAACGTTT CCTGCGCGTA


 


 601
GTGAAAGAGA AAGATCCAGA CGTCTTAATT ACCTATAATG GTGATAACTT TGATTTTGCA


 


 661
TACCTGAAAA AAAGATGCGA AAAGTTGGGC ATAAATTTCG CTCTTGGTCG AGACGGGTCA


 


 721
GAGCCTAAAA TCCAGCGTAT GGGAGATCGC TTTGCGGTTG AAGTGAAAGG CCGGATTCAT


 


 781
TTCGACCTGT ATCCGGTAAT TCGTCGCACT ATCAACCTCC CCACATACAC GTTAGAAGCC


 


 841
GTCTATGAGG CAGTTTTTGG TCAACCGAAG GAAAAAGTTT ACGCTGAGGA AATTACCACT


 


 901
GCGTGGGAAA CAGGCGAGAA TCTGGAACGT GTAGCCCGCT ATTCTATGGA GGATGCAAAA


 


 961
GTTACCTATG AATTGGGTAA GGAATTTCTT CCAATGGAGG CGCAGCTGTC GAGATTAATA


 


1021
GGGCAGAGCC TGTGGGACGT GTCTCGAAGT TCAACGGGAA ACCTCGTCGA ATGGTTTCTG


 


1081
TTGCGGAAAG CATACGAGCG TAATGAACTT GCCCCTAACA AACCGGATGA AAAGGAGCTG


 


1141
GCACGCCGTC GCCAATCCTA TGAAGGCGGT TACGTTAAAG AACCAGAGCG GGGGTTATGG


 


1201
GAAAATATCG TGTATCTGGA TTTCCGTTCG CTCTACCCGA GCATTATCAT TACCCACAAC


 


1261
GTATCTCCCG ACACTTTGAA TCGCGAGGGC TGTAAAGAAT ATGATGTCGC GCCGCAGGTT


 


1321
GGTCATAGAT TTTGCAAGGA CTTCCCGGGA TTTATACCAA GTCTGCTTGG CGATTTACTG


 


1381
GAAGAGCGAC AAAAAATCAA AAAGAAAATG AAAGCTACAA TCGATCCGAT AGAACGTAAG


 


1441
CTGCTCGACT ACCGCCAGCG GGCCATCAAA ATTTTGGCAA ACTCATATTA TGGTTACTAT


 


1501
GGGTACGCGC GTGCTCGCTG GTATTGTAAA GAGTGCGCCG AATCCGTGAC GGCATGGGGC


 


1561
CGTGAATACA TCACCATGAC TATTAAGGAG ATAGAAGAGA AATATGGTTT CAAAGTAATC


 


1621
TACTCGGATA CAGACGGATT CTTTGCGACG ATTCCCGGTG CCGATGCAGA AACCGTCAAG


 


1681
AAAAAAGCGA TGGAATTCCT TAAGTATATA AATGCTAAAT TACCTGGTGC CCTGGAGCTG


 


1741
GAATACGAAG GGTTTTACAA ACGCGGATTC TTTGTTACTA AGAAAAAATA TGCGGTGATC


 


1801
GACGAGGAAG GCAAGATTAC GACCAGAGGC CTCGAGATTG TACGGCGTGA TTGGAGCGAA


 


1861
ATCGCTAAAG AAACACAGGC ACGTGTCTTG GAGGCATTAC TGAAAGATGG GGACGTTGAA


 


1921
AAGGCGGTGC GAATTGTAAA AGAAGTCACC GAAAAACTTT CTAAGTACGA AGTTCCGCCA


 


1981
GAGAAACTGG TGATACACGA ACAAATCACT CGTGATCTGA AAGACTATAA GGCTACAGGC


 


2041
CCGCATGTAG CAGTCGCCAA ACGCCTCGCG GCTCGGGGTG TTAAAATTCG TCCCGGAACG


 


2101
GTGATCAGTT ACATTGTATT GAAGGGCTCA GGTCGCATAG GGGATAGAGC AATCCCTTTC


 


2161
GACGAGTTTG ATCCAACCAA ACACAAATAT GATGCCGAAT ACTATATTGA AAACCAGGTC


 


2221
TTGCCGGCGG TTGAGCGTAT ACTGCGCGCT TTCGGCTATC GAAAGGAAGA TCTTCGTTAC


 


2281
CAAAAAACTA GACAGGTGGG TCTGTCCGCA TGGCTCAAAC CTAAGGGAAC GTAA


 







Sequence 7


(SEQ ID NO: 7)


>pKB13-Pfu codon optimized nucleotide sequence in pUC19 vector








   1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA


 


  61
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG


 


 121
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC


 


 181
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC


 


 241
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT


 


 301
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT


 


 361
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGGTCTCAGC GCCATTCTGG


 


 421
ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG AAAGAGAATG


 


 481
GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG TTACTGAAGG


 


 541
ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG ACAGTGGTAA


 


 601
CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA GAAGTGTGGA


 


 661
AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT CGCGAACATC


 


 721
CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC CTCATCGACA


 


 781
AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC GACATTGAAA


 


 841
CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT TCTTATGCGG


 


 901
ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC GTGGACGTCG


 


 961
TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG AAAGATCCAG


 


1021
ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA AAAAGATGCG


 


1081
AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA ATCCAGCGTA


 


1141
TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG TATCCGGTAA


 


1201
TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG GCAGTTTTTG


 


1261
GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA ACAGGCGAGA


 


1321
ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT GAATTGGGTA


 


1381
AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC CTGTGGGACG


 


1441
TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA GCATACGAGC


 


1501
GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT CGCCAATCCT


 


1561
ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC GTGTATCTGG


 


1621
ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC GACACTTTGA


 


1681
ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA TTTTGCAAGG


 


1741
ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA CAAAAAATCA


 


1801
AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC TACCGCCAGC


 


1861
GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG CGTGCTCGCT


 


1921
GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC ATCACCATGA


 


1981
CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT ACAGACGGAT


 


2041
TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG ATGGAATTCC


 


2101
TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA GGGTTTTACA


 


2161
AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA GGCAAGATTA


 


2221
CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA GAAACACAGG


 


2281
CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG CGAATTGTAA


 


2341
AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG GTGATACACG


 


2401
AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA GCAGTCGCCA


 


2461
AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT TACATTGTAT


 


2521
TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT GATCCAACCA


 


2581
AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG GTTGAGCGTA


 


2641
TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT AGACAGGTGG


 


2701
GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAATGATA TGAGACCGGA TCCTCTAGAG


 


2761
TCGACCTGCA GGCATGCAAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT


 


2821
TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG


 


2881
GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG


 


2941
TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT


 


3001
TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG


 


3061
CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG


 


3121
GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG


 


3181
GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA


 


3241
CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT


 


3301
GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC


 


3361
TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG


 


3421
GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC


 


3481
TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA


 


3541
CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG


 


3601
TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT


 


3661
CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC


 


3721
ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA


 


3781
TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA


 


3841
CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT


 


3901
TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC


 


3961
CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT


 


4021
GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT


 


4081
GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG


 


4141
CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT


 


4201
ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT


 


4261
GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC


 


4321
TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT


 


4381
AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG


 


4441
GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG


 


4501
ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT


 


4561
TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC


 


4621
ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT


 


4681
TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT


 


4741
TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG


 


4801
AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT


 


4861
TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG


 


4921
CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA


 


4981
ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTC


 







Sequence 8


(SEQ ID NO: 8)


>pKB8-KOD codon optimized nucleotide sequence in pUC19 vector








   1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA


 


  61
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG


 


 121
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC


 


 181
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC


 


 241
ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT


 


 301
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT


 


 361
TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGGTCTCAGC GCCATTCTGG


 


 421
ATACCGACTA TATCACGGAA GATGGCAAAC CGGTGATACG TATTTTTAAG AAAGAGAATG


 


 481
GTGAGTTCAA AATCGAGTAC GACCGCACTT TTGAGCCATA TTTCTACGCG TTACTGAAGG


 


 541
ACGATAGCGC CATTGAAGAA GTTAAAAAAA TCACCGCAGA GCGGCATGGG ACAGTGGTAA


 


 601
CCGTGAAGAG AGTTGAAAAA GTCCAGAAAA AATTTTTGGG ACGACCTGTA GAAGTGTGGA


 


 661
AACTTTATTT CACTCACCCC CAAGATGTTC CGGCTATACG TGATAAAATT CGCGAACATC


 


 721
CAGCGGTCAT TGATATTTAC GAATATGATA TACCTTTTGC CAAGCGTTAC CTCATCGACA


 


 781
AAGGCCTGGT GCCGATGGAA GGTGATGAAG AATTAAAAAT GTTGGCATTC GACATTGAAA


 


 841
CACTTTATCA CGAGGGGGAA GAGTTTGCTG AGGGTCCCAT CCTGATGATT TCTTATGCGG


 


 901
ATGAAGAGGG TGCCCGCGTA ATAACCTGGA AGAACGTTGA TCTCCCGTAC GTGGACGTCG


 


 961
TTAGTACGGA ACGGGAAATG ATCAAACGTT TCCTGCGCGT AGTGAAAGAG AAAGATCCAG


 


1021
ACGTCTTAAT TACCTATAAT GGTGATAACT TTGATTTTGC ATACCTGAAA AAAAGATGCG


 


1081
AAAAGTTGGG CATAAATTTC GCTCTTGGTC GAGACGGGTC AGAGCCTAAA ATCCAGCGTA


 


1141
TGGGAGATCG CTTTGCGGTT GAAGTGAAAG GCCGGATTCA TTTCGACCTG TATCCGGTAA


 


1201
TTCGTCGCAC TATCAACCTC CCCACATACA CGTTAGAAGC CGTCTATGAG GCAGTTTTTG


 


1261
GTCAACCGAA GGAAAAAGTT TACGCTGAGG AAATTACCAC TGCGTGGGAA ACAGGCGAGA


 


1321
ATCTGGAACG TGTAGCCCGC TATTCTATGG AGGATGCAAA AGTTACCTAT GAATTGGGTA


 


1381
AGGAATTTCT TCCAATGGAG GCGCAGCTGT CGAGATTAAT AGGGCAGAGC CTGTGGGACG


 


1441
TGTCTCGAAG TTCAACGGGA AACCTCGTCG AATGGTTTCT GTTGCGGAAA GCATACGAGC


 


1501
GTAATGAACT TGCCCCTAAC AAACCGGATG AAAAGGAGCT GGCACGCCGT CGCCAATCCT


 


1561
ATGAAGGCGG TTACGTTAAA GAACCAGAGC GGGGGTTATG GGAAAATATC GTGTATCTGG


 


1621
ATTTCCGTTC GCTCTACCCG AGCATTATCA TTACCCACAA CGTATCTCCC GACACTTTGA


 


1681
ATCGCGAGGG CTGTAAAGAA TATGATGTCG CGCCGCAGGT TGGTCATAGA TTTTGCAAGG


 


1741
ACTTCCCGGG ATTTATACCA AGTCTGCTTG GCGATTTACT GGAAGAGCGA CAAAAAATCA


 


1801
AAAAGAAAAT GAAAGCTACA ATCGATCCGA TAGAACGTAA GCTGCTCGAC TACCGCCAGC


 


1861
GGGCCATCAA AATTTTGGCA AACTCATATT ATGGTTACTA TGGGTACGCG CGTGCTCGCT


 


1921
GGTATTGTAA AGAGTGCGCC GAATCCGTGA CGGCATGGGG CCGTGAATAC ATCACCATGA


 


1981
CTATTAAGGA GATAGAAGAG AAATATGGTT TCAAAGTAAT CTACTCGGAT ACAGACGGAT


 


2041
TCTTTGCGAC GATTCCCGGT GCCGATGCAG AAACCGTCAA GAAAAAAGCG ATGGAATTCC


 


2101
TTAAGTATAT AAATGCTAAA TTACCTGGTG CCCTGGAGCT GGAATACGAA GGGTTTTACA


 


2161
AACGCGGATT CTTTGTTACT AAGAAAAAAT ATGCGGTGAT CGACGAGGAA GGCAAGATTA


 


2221
CGACCAGAGG CCTCGAGATT GTACGGCGTG ATTGGAGCGA AATCGCTAAA GAAACACAGG


 


2281
CACGTGTCTT GGAGGCATTA CTGAAAGATG GGGACGTTGA AAAGGCGGTG CGAATTGTAA


 


2341
AAGAAGTCAC CGAAAAACTT TCTAAGTACG AAGTTCCGCC AGAGAAACTG GTGATACACG


 


2401
AACAAATCAC TCGTGATCTG AAAGACTATA AGGCTACAGG CCCGCATGTA GCAGTCGCCA


 


2461
AACGCCTCGC GGCTCGGGGT GTTAAAATTC GTCCCGGAAC GGTGATCAGT TACATTGTAT


 


2521
TGAAGGGCTC AGGTCGCATA GGGGATAGAG CAATCCCTTT CGACGAGTTT GATCCAACCA


 


2581
AACACAAATA TGATGCCGAA TACTATATTG AAAACCAGGT CTTGCCGGCG GTTGAGCGTA


 


2641
TACTGCGCGC TTTCGGCTAT CGAAAGGAAG ATCTTCGTTA CCAAAAAACT AGACAGGTGG


 


2701
GTCTGTCCGC ATGGCTCAAA CCTAAGGGAA CGTAATGATA TGAGACCGGA TCCTCTAGAG


 


2761
TCGACCTGCA GGCATGCAAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT


 


2821
TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG


 


2881
GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG


 


2941
TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT


 


3001
TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG


 


3061
CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG


 


3121
GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG


 


3181
GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA


 


3241
CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT


 


3301
GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC


 


3361
TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG


 


3421
GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC


 


3481
TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA


 


3541
CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG


 


3601
TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT


 


3661
CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC


 


3721
ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA


 


3781
TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA


 


3841
CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT


 


3901
TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC


 


3961
CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT


 


4021
GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT


 


4081
GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG


 


4141
CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT


 


4201
ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT


 


4261
GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC


 


4321
TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT


 


4381
AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG


 


4441
GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG


 


4501
ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT


 


4561
TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC


 


4621
ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT


 


4681
TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT


 


4741
TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG


 


4801
AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT


 


4861
TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG


 


4921
CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA


 


4981
ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTC


 







Amino acid sequences of Pfu and KOD


Sequence 9


(SEQ ID NO: 9)


>Pfu amino acid sequence








   1
MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG


 


  61
KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY


 


 121
LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY


 


 181
VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK


 


 241
MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE


 


 301
SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK


 


 361
AYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVS


 


 421
PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL


 


 481
DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI


 


 541
DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE


 


 601
EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK


 


 661
LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE


 


 721
YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGLTSWL NIKKS*


 







Sequence 10


(SEQ ID NO: 10)


>Pfu amino acid sequence, extra 3 aa in 5′ area.








   1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE


 


  61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA


 


 121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID


 


 181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLKKRCEKLG IKLTIGRDGS


 


 241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK


 


 301
AWESGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLV GQPLWDVSRS STGNLVEWFL


 


 361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPEKGL WENIVYLDFR ALYPSIIITH


 


 421
NVSPDTLNLE GCKNYDIAPQ VGHKFCKDIP GFIPSLLGHL LEERQKIKTK MKETQDPIEK


 


 481
ILLDYRQKAI KLLANSFYGY YGYAKARWYC KECAESVTAW GRKYIELVWK ELEEKFGFKV


 


 541
LYIDTDGLYA TIPGGESEEI KKKALEFVKY INSKLPGLLE LEYEGFYKRG FFVTKKRYAV


 


 601
IDEEGKVITR GLEIVRRDWS EIAKETQARV LETILKHGDV EEAVRIVKEV IQKLANYEIP


 


 661
PEKLAIYEQI TRPLHEYKAI GPHVAVAKKL AAKGVKIKPG MVIGYIVLRG DGPISNRAIL


 


 721
AEEYDPKKHK YDAEYYIENQ VLPAVLRILE GFGYRKEDLR YQKTRQVGLT SWLNIKKS*


 







Sequence 11


(SEQ ID NO: 11)


>KOD amino acid sequence








   1
MILDTDYITE DGKPVIRIFK KENGEFKIEY DRTFEPYFYA LLKDDSAIEE VKKITAERHG


 


  61
TVVTVKRVEK VQKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVIDIY EYDIPFAKRY


 


 121
LIDKGLVPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNVDLPY


 


 181
VDVVSTEREM IKRFLRVVKE KDPDVLITYN GDNFDFAYLK KRCEKLGINF ALGRDGSEPK


 


 241
IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGQPKEKV YAEEITTAWE


 


 301
TGENLERVAR YSMEDAKVTY ELGKEFLPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRK


 


 361
AYERNELAPN KPDEKELARR RQSYEGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP


 


 421
DTLNREGCKE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKIKKKMKAT IDPIERKLLD


 


 481
YRQRAIKILA NSYYGYYGYA RARWYCKECA ESVTAWGREY ITMTIKEIEE KYGFKVIYSD


 


 541
TDGFFATIPG ADAETVKKKA MEFLKYINAK LPGALELEYE GFYKRGFFVT KKKYAVIDEE


 


 601
GKITTRGLEI VRRDWSEIAK ETQARVLEAL LKDGDVEKAV RIVKEVTEKL SKYEVPPEKL


 


 661
VIHEQITRDL KDYKATGPHV AVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPFDEF


 


 721
DPTKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLSAWLK PKGT


 







Sequence 12


(SEQ ID NO: 12)


>KOD amino acid sequence, extra 3 aa in 5′ area.








   1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE


 


  61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA


 


 121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD


 


 181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS


 


 241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT


 


 301
AWETGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLI GQSLWDVSRS STGNLVEWFL


 


 361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPERGLW ENIVYLDFRS LYPSIIITHN


 


 421
VSPDTLNREG CKEYDVAPQV GHRFCKDFPG FIPSLLGDLL EERQKIKKKM KATIDPIERK


 


 481
LLDYRQRAIK ILANSYYGYY GYARARWYCK ECAESVTAWG REYITMTIKE IEEKYGFKVI


 


 541
YSDTDGFFAT IPGADAETVK KKAMEFLKYI NAKLPGALEL EYEGFYKRGF FVTKKKYAVI


 


 601
DEEGKITTRG LEIVRRDWSE IAKETQARVL EALLKDGDVE KAVRIVKEVT EKLSKYEVPP


 


 661
EKLVIHEQIT RDLKDYKATG PHVAVAKRLA ARGVKIRPGT VISYIVLKGS GRIGDRAIPF


 


 721
DEFDPTKHKY DAEYYIENQV LPAVERILRA FGYRKEDLRY QKTRQVGLSA WLKPKGT*


 







DNA sequences of chimeras Pod and Kofu


Sequence 13


(SEQ ID NO: 13)


>Pod codon optimized nucleotide sequence








   1
ATGGCTAGCG CCATTCTGGA TGTGGACTAT ATCACCGAAG AGGGCAAACC GGTTATACGT


 


  61
TTATTTAAGA AAGAGAATGG TAAATTCAAG ATCGAGCATG ACCGCACGTT CCGTCCATAC


 


 121
ATTTACGCGT TGCTTCGGGA TGATAGCAAA ATTGAGGAAG TCAAAAAGAT CACCGGGGAA


 


 181
CGTCATGGAA AAATAGTAAG AATTGTGGAC GTTGAAAAAG TCGAAAAGAA ATTTCTGGGC


 


 241
AAACCGATCA CTGTATGGAA GCTCTATCTG GAACATCCTC AGGATGTGCC CACAATTCGA


 


 301
GAAAAAGTTC GTGAGCACCC AGCCGTCGTG GATATATTTG AATATGACAT CCCTTTTGCA


 


 361
AAACGCTACT TAATTGATAA AGGCCTGATC CCGATGGAGG GGGAAGAAGA ACTTAAAATT


 


 421
CTGGCTTTTG ACATAGAAAC GCTCTATCAT GAGGGAGAAG AATTTGGCAA AGGTCCCATC


 


 481
ATTATGATTT CTTACGCGGA TGAGAACGAA GCCAAGGTAA TCACTTGGAA AAATATTGAC


 


 541
CTGCCGTACG TTGAAGTGGT CAGTTCAGAG CGGGAAATGA TTAAACGTTT TTTACGCATC


 


 601
ATTAGAGAGA AAGATCCAGA TATAATCGTT ACATATAACG GCGACTCCTT CGATTTTCCT


 


 661
TACCTGGCAA AACGAGCTGA AAAATTGGGT ATTAAACTTA CCATCGGGCG TGACGGATCG


 


 721
GAACCGAAAA TGCAACGCAT TGGCGATATG ACGGCGGTAG AGGTGAAAGG TCGGATACAC


 


 781
TTTGATCTGT ATCATGTCAT CACCCGTACT ATTAATCTCC CCACATACAC GTTAGAAGCC


 


 841
GTTTATGAGG CAATATTCGG CAAGCCGAAA GAAAAAGTGT ACGCTGACGA AATCGCGAAG


 


 901
GCATGGGAGA GCGGCGAAAA CCTGGAGCGC GTAGCAAAAT ATTCTATGGA AGATGCTAAA


 


 961
GCGACCTACG AATTGGGGAA AGAATTTCTT CCAATGGAAA TTCAGCTGTC GAGATTAATA


 


1021
GGGCAGAGCC TGTGGGACGT GTCTCGAAGT TCAACGGGAA ACCTCGTCGA ATGGTTTCTG


 


1081
TTGCGGAAAG CATACGAGCG TAATGAACTT GCCCCTAACA AACCGGATGA AAAGGAGCTG


 


1141
GCACGCCGTC GCCAATCCTA TGAAGGCGGT TACGTTAAAG AACCAGAGCG GGGGTTATGG


 


1201
GAAAATATCG TGTATCTGGA TTTCCGTTCG CTCTACCCGA GCATTATCAT TACCCACAAC


 


1261
GTATCTCCCG ACACTTTGAA TCGCGAGGGC TGTAAAGAAT ATGATGTCGC GCCGCAGGTT


 


1321
GGTCATAGAT TTTGCAAGGA CTTCCCGGGA TTTATACCAA GTCTGCTTGG CGATTTACTG


 


1381
GAAGAGCGAC AAAAAATCAA AAAGAAAATG AAAGCTACAA TCGATCCGAT AGAACGTAAG


 


1441
CTGCTCGACT ACCGCCAGCG GGCCATCAAA ATTTTGGCAA ACTCATATTA TGGTTACTAT


 


1501
GGGTACGCGC GTGCTCGCTG GTATTGTAAA GAGTGCGCCG AATCCGTGAC GGCATGGGGC


 


1561
CGTGAATACA TCACCATGAC TATTAAGGAG ATAGAAGAGA AATATGGTTT CAAAGTAATC


 


1621
TACTCGGATA CAGACGGATT CTTTGCGACG ATTCCCGGTG CCGATGCAGA AACCGTCAAG


 


1681
AAAAAAGCGA TGGAATTCGT TAAGTACATT AATAGTAAAT TACCGGGACT GCTTGAACTG


 


1741
GAGTATGAAG GCTTCTACAA AAGAGGTTTT TTCGTTACTA AGAAACGATA TGCCGTAATA


 


1801
GATGAAGAGG GGAAAGTCAT CACACGTGGC CTCGAGATTG TTCGCCGGGA CTGGTCAGAG


 


1861
ATAGCAAAGG AAACGCAGGC GCGCGTGCTC GAAACCATCT TGAAACATGG TGATGTAGAG


 


1921
GAAGCCGTCC GCATTGTTAA AGAGGTGATC CAGAAGTTAG CAAACTATGA AATTCCACCG


 


1981
GAAAAACTGG CGATATACGA GCAAATCACT CGTCCCCTTC ACGAATATAA AGCTATTGGA


 


2041
CCTCATGTAG CCGTCGCGAA GAAACTGGCT GCAAAAGGCG TTAAGATAAA ACCAGGTATG


 


2101
GTGATCGGGT ACATTGTACT CCGCGGCGAC GGTCCGATTT CCAATAGAGC CATCTTGGCG


 


2161
GAGGAATATG ATCCTAAAAA GCATAAATAC GACGCTGAAT ATTACATTGA GAACCAGGTC


 


2221
TTGCCGGCAG TTCTGCGGAT ACTTGAAGGA TTTGGCTATC GTAAAGAAGA TCTGCGCTAT


 


2281
CAAAAGACGC GACAGGTGGG TCTGACTAGC TGGTTGAATA TCAAAAAATC GTAA


 







Sequence 14


(SEQ ID NO: 14)


>Kofu codon optimized nucleotide sequence








   1
ATGGCTAGCG CCATTCTGGA TACCGACTAT ATCACGGAAG ATGGCAAACC GGTGATACGT


 


  61
ATTTTTAAGA AAGAGAATGG TGAGTTCAAA ATCGAGTACG ACCGCACTTT TGAGCCATAT


 


 121
TTCTACGCGT TACTGAAGGA CGATAGCGCC ATTGAAGAAG TTAAAAAAAT CACCGCAGAG


 


 181
CGGCATGGGA CAGTGGTAAC CGTGAAGAGA GTTGAAAAAG TCCAGAAAAA ATTTTTGGGA


 


 241
CGACCTGTAG AAGTGTGGAA ACTTTATTTC ACTCACCCCC AAGATGTTCC GGCTATACGT


 


 301
GATAAAATTC GCGAACATCC AGCGGTCATT GATATTTACG AATATGATAT ACCTTTTGCC


 


 361
AAGCGTTACC TCATCGACAA AGGCCTGGTG CCGATGGAAG GTGATGAAGA ATTAAAAATG


 


 421
TTGGCATTCG ACATTGAAAC ACTTTATCAC GAGGGGGAAG AGTTTGCTGA GGGTCCCATC


 


 481
CTGATGATTT CTTATGCGGA TGAAGAGGGT GCCCGCGTAA TAACCTGGAA GAACGTTGAT


 


 541
CTCCCGTACG TGGACGTCGT TAGTACGGAA CGGGAAATGA TCAAACGTTT CCTGCGCGTA


 


 601
GTGAAAGAGA AAGATCCAGA CGTCTTAATT ACCTATAATG GTGATAACTT TGATTTTGCA


 


 661
TACCTGAAAA AAAGATGCGA AAAGTTGGGC ATAAATTTCG CTCTTGGTCG AGACGGGTCA


 


 721
GAGCCTAAAA TCCAGCGTAT GGGAGATCGC TTTGCGGTTG AAGTGAAAGG CCGGATTCAT


 


 781
TTCGACCTGT ATCCGGTAAT TCGTCGCACT ATCAACCTCC CCACATACAC GTTAGAAGCC


 


 841
GTCTATGAGG CAGTTTTTGG TCAACCGAAG GAAAAAGTTT ACGCTGAGGA AATTACCACT


 


 901
GCGTGGGAAA CAGGCGAGAA TCTGGAACGT GTAGCCCGCT ATTCTATGGA GGATGCAAAA


 


 961
GTTACCTATG AATTGGGTAA GGAATTTCTT CCAATGGAGG CGCAGCTGAG TCGTTTAGTC


 


1021
GGACAACCTC TGTGGGACGT TTCACGCTCC TCGACTGGCA ATCTCGTGGA GTGGTTCCTG


 


1081
TTGAGAAAAG CCTATGAACG AAACGAAGTA GCACCGAATA AACCAAGCGA GGAAGAATAT


 


1141
CAGCGTCGCC TTCGCGAGTC TTACACAGGT GGGTTTGTTA AGGAACCGGA GAAAGGTCTT


 


1201
TGGGAAAACA TCGTGTATTT AGATTTCCGT GCGCTGTACC CCAGTATTAT AATCACCCAC


 


1261
AATGTCTCAC CTGACACGCT CAACTTGGAA GGTTGCAAAA ATTATGATAT TGCTCCGCAA


 


1321
GTTGGACATA AGTTTTGTAA AGATATTCCG GGCTTCATCC CGTCCCTGCT TGGTCACTTA


 


1381
CTGGAAGAGC GCCAAAAAAT TAAGACCAAA ATGAAAGAGA CTCAGGATCC CATTGAAAAG


 


1441
ATCCTGCTCG ATTACCGGCA AAAAGCCATT AAATTGCTTG CAAACTCGTT TTATGGGTAC


 


1501
TATGGCTATG CGAAGGCTCG TTGGTACTGC AAAGAATGTG CCGAGAGCGT GACAGCATGG


 


1561
GGTCGCAAAT ATATAGAATT AGTATGGAAG GAGCTGGAAG AAAAATTCGG ATTCAAAGTC


 


1621
CTGTACATCG ATACGGATGG CCTCTATGCG ACCATTCCTG GTGGGGAGTC TGAAGAAATC


 


1681
AAGAAAAAAG CCTTGGAATT CCTTAAGTAT ATAAATGCTA AATTACCTGG TGCCCTGGAG


 


1741
CTGGAATACG AAGGGTTTTA CAAACGCGGA TTCTTTGTTA CTAAGAAAAA ATATGCGGTG


 


1801
ATCGACGAGG AAGGCAAGAT TACGACCAGA GGCCTCGAGA TTGTACGGCG TGATTGGAGC


 


1861
GAAATCGCTA AAGAAACACA GGCACGTGTC TTGGAGGCAT TACTGAAAGA TGGGGACGTT


 


1921
GAAAAGGCGG TGCGAATTGT AAAAGAAGTC ACCGAAAAAC TTTCTAAGTA CGAAGTTCCG


 


1981
CCAGAGAAAC TGGTGATACA CGAACAAATC ACTCGTGATC TGAAAGACTA TAAGGCTACA


 


2041
GGCCCGCATG TAGCAGTCGC CAAACGCCTC GCGGCTCGGG GTGTTAAAAT TCGTCCCGGA


 


2101
ACGGTGATCA GTTACATTGT ATTGAAGGGC TCAGGTCGCA TAGGGGATAG AGCAATCCCT


 


2161
TTCGACGAGT TTGATCCAAC CAAACACAAA TATGATGCCG AATACTATAT TGAAAACCAG


 


2221
GTCTTGCCGG CGGTTGAGCG TATACTGCGC GCTTTCGGCT ATCGAAAGGA AGATCTTCGT


 


2281
TACCAAAAAA CTAGACAGGT GGGTCTGTCC GCATGGCTCA AACCTAAGGG AACGTAA


 







Amino acid sequences of chimeras Pod and Kofu


Sequence 15


(SEQ ID NO: 15)


>Pod amino acid sequence








   1
MASAILDVDY ITEEGKPVIR LFKKENGKFK IEHDRTFRPY IYALLRDDSK IEEVKKITGE


 


  61
RHGKIVRIVD VEKVEKKFLG KPITVWKLYL EHPQDVPTIR EKVREHPAVV DIFEYDIPFA


 


 121
KRYLIDKGLI PMEGEEELKI LAFDIETLYH EGEEFGKGPI IMISYADENE AKVITWKNID


 


 181
LPYVEVVSSE REMIKRFLRI IREKDPDIIV TYNGDSFDFP YLAKRAEKLG IKLTIGRDGS


 


 241
EPKMQRIGDM TAVEVKGRIH FDLYHVITRT INLPTYTLEA VYEAIFGKPK EKVYADEIAK


 


 301
AWESGENLER VAKYSMEDAK ATYELGKEFL PMEIQLSRLI GQSLWDVSRS STGNLVEWFL


 


 361
LRKAYERNEL APNKPDEKEL ARRRQSYEGG YVKEPERGLW ENIVYLDFRS LYPSIIITHN


 


 421
VSPDTLNREG CKEYDVAPQV GHRFCKDFPG FIPSLLGDLL EERQKIKKKM KATIDPIERK


 


 481
LLDYRQRAIK ILANSYYGYY GYARARWYCK ECAESVTAWG REYITMTIKE IEEKYGFKVI


 


 541
YSDTDGFFAT IPGADAETVK KKAMEFVKYI NSKLPGLLEL EYEGFYKRGF FVTKKRYAVI


 


 601
DEEGKVITRG LEIVRRDWSE IAKETQARVL ETILKHGDVE EAVRIVKEVI QKLANYEIPP


 


 661
EKLAIYEQIT RPLHEYKAIG PHVAVAKKLA AKGVKIKPGM VIGYIVLRGD GPISNRAILA


 


 721
EEYDPKKHKY DAEYYIENQV LPAVLRILEG FGYRKEDLRY QKTRQVGLTS WLNIKKS*


 







Sequence 16


(SEQ ID NO: 16)


>Kofu amino acid sequence








   1
MASAILDTDY ITEDGKPVIR IFKKENGEFK IEYDRTFEPY FYALLKDDSA IEEVKKITAE


 


  61
RHGTVVTVKR VEKVQKKFLG RPVEVWKLYF THPQDVPAIR DKIREHPAVI DIYEYDIPFA


 


 121
KRYLIDKGLV PMEGDEELKM LAFDIETLYH EGEEFAEGPI LMISYADEEG ARVITWKNVD


 


 181
LPYVDVVSTE REMIKRFLRV VKEKDPDVLI TYNGDNFDFA YLKKRCEKLG INFALGRDGS


 


 241
EPKIQRMGDR FAVEVKGRIH FDLYPVIRRT INLPTYTLEA VYEAVFGQPK EKVYAEEITT


 


 301
AWETGENLER VARYSMEDAK VTYELGKEFL PMEAQLSRLV GQPLWDVSRS STGNLVEWFL


 


 361
LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPEKGL WENIVYLDFR ALYPSIIITH


 


 421
NVSPDTLNLE GCKNYDIAPQ VGHKFCKDIP GFIPSLLGHL LEERQKIKTK MKETQDPIEK


 


 481
ILLDYRQKAI KLLANSFYGY YGYAKARWYC KECAESVTAW GRKYIELVWK ELEEKFGFKV


 


 541
LYIDTDGLYA TIPGGESEEI KKKALEFLKY INAKLPGALE LEYEGFYKRG FFVTKKKYAV


 


 601
IDEEGKITTR GLEIVRRDWS EIAKETQARV LEALLKDGDV EKAVRIVKEV TEKLSKYEVP


 


 661
PEKLVIHEQI TRDLKDYKAT GPHVAVAKRL AARGVKIRPG TVISYIVLKG SGRIGDRAIP


 


 721
FDEFDPTKHK YDAEYYIENQ VLPAVERILR AFGYRKEDLR YQKTRQVGLS AWLKPKGT*


 







Sequence 17


(SEQ ID NO: 17)


>pLACIQZa








   1
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA


 


  61
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG


 


 121
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC


 


 181
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC


 


 241
ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT


 



                                                          GT


 301
TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT


 



TTTCCCAGTCACGAC >>> Primer M13-40 (SEQ ID NO: 24)


 361
TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGAT


 



  XbaI


 421
CCTCTAGAGCCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA


 


 481
ATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG


 


 541
AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG


 


 601
TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC


 


 661
CAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTG


 


 721
GCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTG


 


 781
TTTGATGGTGGTTGACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCAC


 


 841
TACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAG


 


 901
CGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTG


 


 961
CATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTG


 


1021
AATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGA


 


1081
ACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCAC


 


1141
GCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGA


 


1201
GACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTG


 


1261
GTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCAC


 


1321
CGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACC


 


1381
CAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAG


 


1441
ACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCG


 


1501
GTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGA


 


1561
AACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTC


 


1621
TGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG


 


1681
GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCAACGTAAATGCA


 



                                    NcoI


1741
TGCCGCTTCGCCTTCCGGCCACCAGAATAGCCTGCGCCATGGGCTTCCTCGCTCACTGAC


 


1801
TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA


 


1861
CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA


 


1921
AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT


 


1981
GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA


 



                          PRIMER PKBLACIR <<< GCTGTCCTGATATT



TCTATGG (SEQ ID NO: 25)


 


2041
AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG


 


2101
CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA


 


2161
CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA


 


2221
CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG


 


2281
GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG


 


2341
TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA


 


2401
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC


 


2461
TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG


 


2521
ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC


 


2581
GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC


 


2641
TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG


 


2701
TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT


 


2761
CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG


 


2821
GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA


 


2881
GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT


 


2941
TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA


 


3001
GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG


 


3061
TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC


 


3121
ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG


 


3181
GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA


 


3241
TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT


 


3301
ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC


 


3361
AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC


 


3421
TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA


 


3481
TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA


 


3541
AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT


 


3601
TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA


 


3661
AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAA


 


3721
ACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC









EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. The articles “a”, “an”, and “the” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth herein. It should also be understood that any embodiment of the invention, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. Furthermore, where the claims recite a composition, the invention encompasses methods of using the composition and methods of making the composition.
INCORPORATION OF REFERENCES
All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.

## Claims

1. A modified DNA polymerase having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16, or the amino acid sequence as set forth in, which amino acid sequence includes one or more amino acid substitutions relative to SEQ ID NO:16, such substitutions being selected from the group consisting of F752Y, F591L, F591I, E668V, G638R, G638V, E734K, E377K, T609I, T609A, P454S, E582K or G715R of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions alter enzyme activity, fidelity, processivity, elongation rate, stability, or solubility.

2. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F752Y, F591L, F591I, G638V, G638R, E668V, E734K, V356M, E738G, E386K, W772R, or E377K of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions increase the enzyme activity of the DNA polymerase.

3. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F591I, F591L, A550V, E377K, A494V, E734K, G638V, G638R, E668V, D346G, V356M or E738G of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions increase the DNA binding affinity of the DNA polymerase.

4. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of R410H, E582K, E652K, A679T, S376G, or T680I of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions decreases the DNA binding affinity of the DNA polymerase.

5. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F591L, F752Y, F591I, E668V, V441I, G638R, S376G or T6801 of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions decreases the fidelity of the DNA polymerase.

6. The modified DNA polymerase of any one of claim 1, 2, 3, 4 or 5, wherein the DNA polymerase is a fusion polymerase.

7. A kit comprising the modified DNA polymerase of any one of claim 1, 2, 3, 4 or 5.