Chimeric DNA polymerases

[0001] The present application is a national phase entry of international application Ser. No. PCT/US2009/063166, filed Nov. 3, 2009 which claims priority to U.S. Provisional patent application Ser. No. 61/110,862, filed Nov. 3, 2008, the entire disclosure of which is incorporated herein by reference.

[0002] 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 235 kb in size. The entire contents of the Sequence Listing are herein incorporated by reference.

[0003] DNA polymerases are 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. Some DNA polymerases can correct mistakes in newly-synthesized DNA. This process is known as error correction. 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 base excision, the polymerase can re-insert the correct base and replication can continue. The proofreading function gives the DNA replication much higher fidelity than it would have if synthesis were the result of only a base-pairing selection step. Brutlag, D. and Kornberg, A., J. Biol. Chem., 247:241-248 (1972). DNA polymerases with 3′-5′ proofreading exonuclease activity have a substantially lower error rate when compared with a non-proofreading exonuclease-possessing polymerase. Chang, L. M. S., J. Biol. Chem., 252:1873-1880 (1977). However, sometimes, the advantage of these polymerases is offset by its relatively low processivity that reduces the yield of DNA amplification products.

[0004] The present invention encompasses the discovery that domain swapping can combine desired functional characteristics (e.g., high processivity, high elongation rate, thermostability, resistance to salt, PCR additives (e.g., PCR enhancers) and other impurities, and high fidelity) of different DNA polymerases in a chimeric enzyme. Thus, the present invention provides, among other things, robust, fast and accurate DNA polymerases for DNA amplification, synthesis, detection, sequencing and other important recombinant DNA techniques.

[0005] In one aspect, the present invention provides chimeric polymerases containing a first domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in a first DNA polymerase characterized with high processivity, elongation rate, salt resistance, thermostability or TMAC tolerance; and a second domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in a second DNA polymerase characterized with high fidelity, wherein the chimeric polymerases are characterized with both high fidelity and high processivity, elongation rate, or salt resistance. 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. As used herein, the term “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 herein, the term “high salt resistance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a salt concentration higher than 30 mM (e.g., higher than 35 mM, 40 mM, 45 mM, or 50 mM). As used herein, 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. As used herein, the term “high TMAC tolerance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a TMAC (tetra-methyl ammonium chloride) concentration higher than 10 mM (e.g., higher than 15 mM, 20 mM, 25 mM, 30 mM). As used herein, the term “high thermostability” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity after more than 30 minutes incubation at 98° C. (e.g., 45 min, 60 min, 90 min, 180 min, 210 min, 240 min). The terms of “processivity,” “elongation rate,” “fidelity,” “salt resistance,” “TMAC tolerance,” and “thermostability” are further defined in the Definitions section.

[0006] In some embodiments, exemplary first DNA polymerases suitable for the present invention include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. In some embodiments, the first DNA polymerase is KOD polymerase. In some embodiments, exemplary second DNA polymerases suitable for the invention include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D. In some embodiments, the second DNA polymerase is Pfu polymerase. In particular embodiments, the first DNA polymerase is KOD polymerase and the second DNA polymerase is Pfu polymerase.

[0007] In some embodiments, suitable first domain is an exonuclease domain, N-terminal domain, and/or a thumb domain. In some embodiments, suitable second domain is palm and/or fingers domain.

[0008] In some embodiments, amino acid sequences found in the first DNA polymerase correspond to amino acid residues 26 to 105 of KOD polymerase (SEQ ID NO:11), amino acid residues 156 to 301 of KOD polymerase (SEQ ID NO:11), and/or amino acid residues 612 to 749 of KOD polymerase (SEQ ID NO:11).

[0009] In some embodiments, amino acid sequences found in the second DNA polymerase correspond to amino acid residues 394 to 563 of Pfu polymerase (SEQ ID NO:9).

[0010] In some embodiments, a chimeric polymerase in accordance with the present invention include a first domain having a consensus sequence selected from the group consisting of

• [0011] XXLXXXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIX XXXXNXXXAXXKXXCXXXXXNFALXXXXXXXXXXXXIXXMXXRFXXXXXXXXX XXXXPXXRXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXT (SEQ ID NO:30), wherein X is any amino acid or a peptide bond;
• [0012] XXEXXXXYXXXXEXXFXXXXKXXXAXXXXXXXXAXXXXTVXTVKRXXXXQXXX XXRXVEXXXXXFTXXXXXXAXXDXIXXXXX (SEQ ID NO:31), wherein X is any amino acid or a peptide bond;
• [0013] XXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXVXXXXXVXHX XXXXDXKDXXXTXXXXXXXXRXXXRXXXRXXTXXSXXXXKXSXRXGDXXXPF DXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXX (SEQ ID NO:32), wherein X is any amino acid or a peptide bond;
• [0014] NGX₁FKIEX₂DRTFX₃PYX₄YALLX₅DDSX₆IEEVKKITX₇ERHGX₈X₉VX₁₀X₁₁X₁₂X₁₃VEKVX₁₄KKFLGX₁₅PX₁₆X₁₇VWKLYX₁₈X₁₉HPQDVPX₂₀IRX₂₁KX₂₂REHPA (SEQ ID NO:33), wherein X₁ is not K; X₂ is not H; X₃ is not R; X₄ is not I; X₅ is not R; X₆ is not K; X₇ is not G; X₈ is not K; X₉ is not I; X₁₀ is not R; X₁₁ is not I; X₁₂ is not V; X₁₃ is not D; X₁₄ is not E; X₁₅ is not K; X₁₆ is not I; X₁₇ is not T; X₁₈ is not L; X₁₉ is not E; X₂₀ is not T; X₂₁ is not E; and X₂₂ is not V;
• [0015] PIX₁MISYADEX₂X₃AX₄VITWKNX₅DLPYVX₆VVSX₇EREMIKRFLRX₈X₉X₁₀EKDPDX₁₁X₁₂X₁₃TYNGDX₁₄FDFX₁₅YLX₁₆KRX₁₇EKLGIX₁₈X₁₉X₂₀X₂₁GRDGSEPXX₂₂QRX₂₃GDX₂₄X₂₅AVEVKGRIHFDLYX₂₆VIX₂₇RTINLPTYTLEAVYEAX₂₈FGX₂₉PKEKVYAX₃₀EIX₃₁X₃₂AWEX₃₃ (SEQ ID NO:34), wherein X₁ is not I; X₂ is not N; X₃ is not E; X₄ is not K; X₅ is not I; X₆ is not E; X₇ is not S; X₈ is not I; X₉ is not I; X₁₀ is not R; X₁₁ is not I; X₁₂ is not I; X₁₃ is not V; X₁₄ is not S; X₁₅ is not P; X₁₆ is not A; X₁₇ is not A; X₁₈ is not K; X₁₉ is not L; X₂₀ is not T; X₂₁ is not I; X₂₂ is not M; X₂₃ is not I; X₂₄ is not M; X₂₅ is not T; X₂₆ is not H; X₂₇ is not T; X₂₈ is not I; X₂₉ is not K; X₃₀ is not D; X₃₁ is not A; X₃₂ is not K; and X₃₃ is not S;
• [0016] RDWSEIAKETQARVLEX₁X₂LKX₃GDVEX₄AVRIVKEVX₅X₆KLX₇X₈YEX₉PPEKLX₁₀IX₁₁EQITRX₁₂LX₁₃X₁₄YKAX₁₅GPHVAVAKX₁₆LAAX₁₇GVKIX₁₈PGX₁₉VIX₂₀YIVLX₂₁GX₂₂GX₂₃IX₂₄X₂₅RAIX₂₆X₂₇X₂₈EX₂₉DPX₃₀KHKYDAEYYIENQVLPAVX₃₁RILX₃₂X₃₃FG (SEQ ID NO:35), wherein X₁ is not T; X₂ is not I; X₃ is not H; X₄ is not E; X₅ is not I; X₆ is not Q; X₇ is not A; X₈ is not N; X₉ is not I; X₁₀ is not A; X₁₁ is not Y; X₁₂ is not P; X₁₃ is not H; X₁₄ is not E; X₁₅ is not I; X₁₆ is not K; X₁₇ is not K; X₁₈ is not K; X₁₉ is not M; X₂₀ is not G; X₂₁ is not R; X₂₂ is not D; X₂₃ is not P; X₂₄ is not S; X₂₅ is not N; X₂₆ is not L; X₂₇ is not A; X₂₈ is not E; X₂₉ is not Y; X₃₀ is not K; X₃₁ is not L; X₃₂ is not E; and X₃₃ is not G;
  and combinations thereof;
  and a second domain having a consensus sequence selected from the group consisting of
• [0017] XKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXXNXXIXXXXXXKXXXXI XXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXXXXFXXXXX XXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXIXXXXLYXXXXXG ESXEIXXXXLX (SEQ ID NO:36), wherein X is any amino acid or a peptide bond;
• [0018] EX₁GLWENIVYLDFRX₂LYPSIIITHNVSPDTLNX₃EGCKX₄YDX₅APQVGHX₆FCKDX₇PGFIPSLLGX₈LLEERQKIKX₉KMKX₁₀TX₁₁DPIEX₁₂X₁₃LLDYRQX₁₄AIKX₁₅LANSX₁₆YGYYGYAX₁₇ARWYCKECAESVTAWGRX₁₈YIX₁₉X₂₀X₂₁X₂₂KEX₂₃EEKX₂₄GFKVX₂₅YX₂₆DTDGX₂₇X₂₈ATIPGX₂₉X₃₀X₃₁EX₃₂X₃₃KKKAX₃₄E (SEQ ID NO:37), wherein X₁ is not R; X₂ is not S; X₃ is not R; X₄ is not E; X₅ is not V; X₆ is not R; X₇ is not F; X₈ is not D; X₉ is not K; X₁₀ is not A; X₁₁ is not I; X₁₂ is not R; X₁₃ is not K; X₁₄ is not R; X₁₅ is not I; X₁₆ is not Y; X₁₇ is not R; X₁₈ is not E; X₁₉ is not T; X₂₀ is not M; X₂₁ is not T; X₂₂ is not I; X₂₃ is not I; X₂₄ is not Y; X₂₅ is not I; X₂₆ is not S; X₂₇ is not F; X₂₈ is not F; X₂₉ is not A; X₃₀ is not D; X₃₁ is not A; X₃₂ is not T; X₃₃ is not V; X₃₄ is not M,
  and combinations thereof,
  wherein the chimeric polymerase is characterized with high fidelity and high processivity, elongation rate, salt resistance, TMAC or other PCR enhancer tolerance or thermostability.

[0019] In some embodiments, chimeric polymerases in accordance with the present invention are defined by consensus sequence XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXKXXXAXXXXXX XXAXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXI XXYXXXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLX XXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIXXXXX NXXXAXXKXXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXPX XRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXTXXXXXXXXRXXXXX XXVXXXXXXXXXXXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXV XXXXXSXEXYQXXXXEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXX XXXXXLXXXXNXXIXXXXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQX XXXKIXXXXXXKXXXLXXXXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVW XXLXXXFXXXXLXIXXXXLYXXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXX XXXXXXXXXXXXXKXXXXXXXXXITXXXXXXXXXXXXXXXXXXXXXXXXALX XKXXXXKXXXXXXXXTEXXSKXXVXXXXXVXHXXXXXDXKDXXXTXXXXXXX XRXXXRXXXXRXXTXXTXXSXXXXKXSXRXGDXXXPFDXFXXTXXXXXXXXXXXXX XXXXXEXXXRAXXXXXXXXXXXXXXXXXXSAXXKPXGT (SEQ ID NO:38), wherein X is any amino acid or a peptide bond, and wherein the chimeric polymerase has a fidelity higher than that of KOD and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of Pfu.

[0020] In some embodiments, chimeric polymerases in accordance with the present invention are defined by consensus sequence XIXDTDYXTXDGXPXXRIFXKXXGEFXXXYDXXEPYFYALLKDDSAIXXXXXXXA XRHGTVXTVKRXXXXQXKFLXRXVEVWXLXFTHPQDVPAXXDXIXXHXXVIDIYE YDIPFAKRYLIDXGLVPMEGDEXLXMXXXDIETXYHEGXEFAEGXXLMISYADXEG ARVITWKXVDLPYVDVVSTEXEMIKRXXXVVKEKDPDVLIXYXGDNEDXAYLKXR CEXLGXNFALXRXXXXXEPKIXXMGXRFAVEXXGRXHFDLXPXXRXTXNLPTYXL XXVYEXVSGQXKXKXXXXEEITTXWETXXXXXXXARYSMEDAXYTXELGXEFXPM EAXLXXLVGXPXWDVXRSSTGNLVEWXLLXXAYXRNEVAPNKPSXEEYQXRXXE XYTGXFVXEPEKGLWXXXXXLDXXALYPSIIIXXHNVSPDTLXLEXCXNYDIAPXVG XKFCKDIPGFIPSXLXHLXXXRQXXKTXMXEXQDPXEKIXLDYRQKAXKLLXNSFY GYXGYXKARWYXXECAESVTXWGRKYIELVWXELEXXFGFKXLYIDTDGLYATIP GGESXEIKXXXLXFLXYINAXLPGALELEYEXFYXRGFFVXKKKYAXIDEEXXITTR GLEXVRRDWSXXAKETXAXVLEALLXDXXVXKAVXXVXXXTEXXSKYXVPXEKL VIHEQITRDXKDYXATGPHVAXAKRLXXGXXXRPGTXISYXXLKGSGRXGDRXIPF DEFXXTKHXYDXXYYIENQVLPAVERXLRAFGYXXXXLXXQXXXQXGLSAWXKP XGT (SEQ ID NO:39), wherein X is any amino acid or a peptide bond.

[0021] In some embodiments, the present invention further provides chimeric polymerases containing a first domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in an exonuclease domain, an N-terminal domain, and/or a thumb domain of a first DNA polymerase; and a second domain having a sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to an amino acid sequence found in palm and/or fingers domain of a second DNA polymerase. In some embodiments, the chimeric polymerase has a fidelity higher than that of the second DNA polymerase and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of the first DNA polymerase.

[0022] In another aspect, the present invention provides methods of engineering chimeric polymerases. Inventive methods in accordance with the present invention include steps of: (a) providing an N-terminal domain, an exonuclease domain, and/or a thumb domain based on a first DNA polymerase; (b) providing a palm and/or fingers domain based on a second DNA polymerase; (c) combining the domains from step (a) and step (b) to form a chimeric polymerase; wherein the chimeric polymerase has a fidelity higher than that of the first DNA polymerase and a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability higher than that of the second DNA polymerase. In some embodiments, a chimeric polymerase engineered according to the present invention has a processivity, an elongation rate, a salt resistance, a TMAC or other PCR enhancer tolerance or a thermostability substantially similar to that of the first DNA polymerase and a fidelity substantially similar to that of the second DNA polymerase.

[0023] In some embodiments, exemplary first DNA polymerases suitable for the present invention include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. In some embodiments, the first DNA polymerase is KOD polymerase. In some embodiments, exemplary second DNA polymerases suitable for the invention include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D. In some embodiments, the second DNA polymerase is Pfu polymerase.

[0024] In some embodiments, the first DNA polymerase is KOD polymerase and the second DNA polymerase is Pfu polymerase. In some embodiments, the first DNA polymerase is Pfu polymerase and the second DNA polymerase is KOD polymerase.

[0025] In some embodiments, the present invention provides methods of improving the fidelity of a DNA polymerase. In particular embodiments, inventive methods in accordance with the invention include a step of replacing a sequence within the palm and/or fingers domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher fidelity relative to the DNA polymerase of interest.

[0026] In some embodiments, the present invention provides methods of improving the processivity, elongation rate, salt resistance, TMAC or other PCR enhancer tolerance or thermostability of a DNA polymerase. In particular embodiments, inventive methods in accordance with the present invention include a step of replacing a sequence within the N-terminal domain, the exonuclease domain and/or the thumb domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher processivity, elongation rate, salt resistance, TMAC or other PCR enhancer tolerance or thermostability relative to the DNA polymerase of interest.

[0027] The present invention provides various chimeric polymerases described herein including chimeric polymerases engineered and/or improved using inventive methods as described herein. In some embodiments, chimeric polymerases in accordance with the present invention contain an amino acid sequence at least 80% identical to SEQ ID NO:16 (the Kofu amino acid sequence as shown in the Sequences section). In particular embodiments, a chimeric polymerase in accordance with the present invention contains the amino acid sequence of SEQ ID NO:16. In some embodiments, chimeric polymerases in accordance with the present invention contain an amino acid sequence at least 80% identical to SEQ ID NO:15 (the Pod amino acid sequence as shown in the Sequences section). In particular embodiments, a chimeric polymerase in accordance with the present invention contains the amino acid sequence of SEQ ID NO:15.

[0028] The present invention also provides kits and compositions containing various chimeric polymerases described herein and uses thereof (e.g., methods of amplifying DNA fragments using chimeric DNA polymerases of the invention). In addition, the present invention provides nucleotide sequences encoding various chimeric polymerases described herein and vectors and/or cells containing the nucleotide sequences according to the invention.

[0035] 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 groups. 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.

[0036] 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.

[0037] Chimeric polymerase: As used herein, the term “chimeric polymerase” (also referred to as “chimera”) refers to any polymerase containing two or more heterologous domains, amino acid sequences, peptides, and/or proteins joined either covalently or non-covalently to produce a polymerase that does not occur in nature. Typically, a chimeric polymerase contains a first domain joined to a second domain, wherein the first and second domains are not found in the same relationship in nature. Typically, the first domain is derived from a first DNA polymerase and a second domain is 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, salt tolerance, tolerance to PCR additives or thermostability). 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.

[0038] 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.

[0039] 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 (3^rd 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).

[0040] Domain: As used herein, the term “Domain” as used herein refers to an amino acid sequence of a polypeptide (e.g., polymerase) comprising one or more defined functions or properties.

[0041] 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).

[0042] 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^rd ed., Cold Spring Harbor Laboratory Press, NY)).

[0043] 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 complementary to the template nucleotide). 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. (1996) 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 lad-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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 towards the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides.

[0048] 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.

[0049] 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 appropriate 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 lower temperatures to form sufficiently stable hybrid complexes with template.

[0050] Salt resistance: As used herein, the term “salt resistance” (also referred to as salt tolerance) refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity in the presence of salt or PCR additives (e.g., TMAC). In some embodiments, resistance to salt or PCR additives is measured by the maximum salt concentration at which a DNA polymerase is still active. The maximum salt concentration differs for each polymerase and is known in the art, or can be experimentally determined according to methods in the art. For example, Pfu is inhibited at 30 mM salt (in a PCR reaction).

[0051] 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.

[0052] 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.

[0053] 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)).

[0054] 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 98° C. for a time depending mainly on the temperature and the nucleic acid length, typically about 0.2 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.).

[0055] TMAC or other PCR enhancer tolerance: As used herein, the term “TMAC or other PCR enhancer tolerance” (also referred to as TMAC or other PCR enhancer resistance) refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity in the presence of TMAC or other PCR enhancers (e.g., glycerol, DMSO, betaine, amides, other tetramethyl ammonium salts).

[0056] The present invention provides, among other things, chimeric DNA polymerases containing heterologous domains having sequences derived from at least two DNA polymerases that have at least one distinct functional characteristics (e.g., elongation rate, processivity, error rate or fidelity, salt tolerance or resistance) and methods of making and using the same.

DNA Polymerases

[0057] Chimeric DNA polymerases in accordance with the present invention may be engineered from any DNA polymerases, in particular, thermostable 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).

[0058] Suitable exemplary archaeal polymerases include, but are not limited to, DNA polymerases from archaea (e.g., Thermococcus litoralis (Vent™, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent™, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), 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 sp. GE8 (GenBank: CAC12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), 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: Q58295), 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., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus.

[0059] DNA polymerases suitable for the present invention include DNA polymerases that have not yet been isolated. Suitable polymerases for the present invention include fusion polymerases. Fusion polymerases generally contain an additional protein domain at the N- or C-terminus that changes the phenotype of the fusion polymerase compared to the polymerase without the extra domain. Exemplary polymerases include, but are not limited to, polymerases with double-stranded DNA-binding domains fused at the C- or N-terminus. Further examples of fusion polymerases include those with dUTPase fused to the N-or C-terminus (U.S. patent application 20070190538).

[0060] In some embodiments, chimeric DNA polymerases according to the invention contain sequences derived from two or more DNA polymerases that have at least one distinct functional characteristic. Exemplary functional characteristics include, but are not limited to, processivity, elongation rate, fidelity, resistance to salt or PCR additive (e.g., PCR enhancers), thermostability, strand displacement activity, exonuclease activity, uracil read-ahead function, nucleotide selectivity, ability to incorporate modified analogs, and reverse transcriptase activity. For example, some DNA polymerases are characterized with high fidelity. As used herein, 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. Some DNA polymerases are characterized with high processivity. 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. Some DNA polymerases are characterized with high elongation rate. As used herein, the term “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). Some enzymes are characterized with high resistance to salt (also referred to as salt tolerance). As used herein, the term “high resistance to salt” (also referred to as high salt tolerance) refers to the ability of a DNA polymerase to substantially maintain its activity at a salt concentration higher than 30 mM (e.g., higher than 35 mM, 40 mM, 45 mM, 50 mM). In addition, some enzymes are characterized with resistance to PCR additives. Certain PCR additives are PCR enhancers. For example, Kovarova et al. showed that TMA salts, DMSO, betaine and formamide act as PCR enhancers (Kovarova and Draber. (2000) Nucl. Acids. Res. 28(13), e70). Another example of PCR enhancers is glycerol. Some enzymes are characterized with resistance to PCR enhancers, in particular, TMAC (also referred to as TMAC tolerance). As used herein, the term “high TMAC tolerance” refers to the ability of a DNA polymerase to substantially maintain its enzymatic activity at a TMAC (tetra-methyl ammonium chloride) concentration higher than 10 mM (e.g., higher than 15 mM, 20 mM). Certain characteristics of exemplary DNA polymerases are shown in Table 1.

[0061]

TABLE 1
Characteristics of exemplary DNA polymerases
			Elongation
	Fidelity/	Processivity	rate	Salt
Polymerases	Error rate	(nts)	(nts/s)	tolerance
Pfu	2.0 × 10−6	>20	25	30 mM
KOD	4.45 × 10−6	~300	106-138
TNA1		150
T. zilligii	2.0 × 10−6
P. abyssi	0.66 × 10−6
T. gorgonarius	2.2-3.4 × 10−6

[0062] Typically, enzymes with high salt tolerance are also characterized with high processivity and/or elongation rate. Without wishing to be bound by any theories, it is thought that salt tolerance affects the binding affinity between polymerase and DNA which, in turn, affects processivity or elongation rate. Typically, binding of polymerases to DNA involves binding interaction between positively charged amino acid residues and negatively charged DNA. At high salt concentrations, competition from the anions of the salt for the positively charged amino acid residues on the polymerases lead to diminished DNA binding affinity. See, Pavlov et al. (2002) Proc. Natl. Acad. Sci. 99(21): 13510-13515, which is incorporated by reference herein. On the other hand, increasing the contact points between DNA and polymerase may increase the salt resistance of the polymerase as well as the processivity or elongation rate because the additional contact points between DNA and polymerase may increase binding affinity of the polymerase for DNA and decrease the rate of dissociation so that the polymerase will remain associated with DNA longer, which will in turn lead to an increase in processivity. For example, Pavlov et al. added helix-hairpin-helix (HhH) motifs from topoisomerase V to Taq and Pfu. These motifs are involved in DNA binding in topoisomerase V. Pavlov et al. showed that both Pfu and Taq become more salt resistant when fused to the HhH motifs. Pavlov et al. also showed that HhH fusion to both Taq and Pfu increased the processivity of the polymerases. As another example, dsDNA binding proteins, e.g., Sso7d, can be fused to DNA polymerases to increase the number of contact points between DNA and polymerases (Wang et al. (2004) Nucl. Acids Res. 32(3): 1197-1207, which is incorporated by reference herein). Sso7d is a sequence non-specific dsDNA binding protein involved in ensuring DNA stability and/or DNA packing in Sulfolobus solfataricus. Fusion of Sso7d to both Taq and Pfu increased the salt resistance and processivity of the polymerases.

[0063] Exemplary DNA polymerases characterized with high processivity, elongation rate, thermostability, salt or PCR enhancer tolerance include, but are not limited to, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, or phi29. Exemplary DNA polymerases characterized with high fidelity include, but are not limited to, polymerases isolated from Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, or P. sp. GB-D.

[0064] As non-limiting examples, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases are used to engineer chimeric DNA polymerases (see the Example sections).

Domains of DNA Polymerases

[0065] Typically, archaeal DNA polymerases include at least the following domains: N-terminal domain, exonuclease domain (e.g., 3′->5′ exonuclease domain), palm, fingers, and thumb domain (see FIG. 1). Knowledge of domain structure, function and coordination is primary based on crystal structure studies and site-directed mutagenesis of various DNA polymerases, in particular, archaeal DNA polymerases. For example, among the first crystal structures of family B DNA polymerases obtained was that of bacteriophage RB69 DNA polymerase (Wang et al. (1997) Cell, 89:1087-1099, which is incorporated by reference herein). Among the first crystal structures of archaeal DNA polymerases solved was Tgo DNA polymerase (see, Hopfner et al. 1999 Proc. Natl. Acad. Sci. 96(7), 3600-3605, which is incorporated by reference herein). Recently, crystal structures of the following archaeal family B DNA polymerases have been reported: DNA polymerase from Thermococcus sp. 9° N-7 (Rodriguez et al. (2000) J. Mol. Biol. 299:447-462, which is incorporated by reference herein), KOD1 DNA polymerase (Hashimoto et al. 2001 J. Mol. Biol. 306(3), 469-477, which is incorporated by reference herein), Pfu DNA polymerase (see, U.S. Pat. Nos. 5,948, 663; 5,866, 395; 5,545, 552; 5,556, 772 and Kim et al. (2008) Int. J. Biol. Macromol. 42(4), 356-61, all of which are hereby incorporated by reference).

[0066] Various functions, such as substrate binding, nucleotide transfer, catalytic activity, proofreading, have been assigned to various domains based on the structural-functional analysis of DNA polymerases. It has also been suggested that the domains tightly coordinate with each other to complete the DNA replication process.

[0067] For example, the polymerase activity has been associated with palm, fingers and thumb domains. In particular, the palm subdomain is thought to be the catalytic site of the polymerase. The polymerase catalyzes a phosphoryl transfer reaction in which the alpha phosphate of the incoming dNTP undergoes nucleophilic attack from the OH primer terminus. Typically, three carboxylate side chains are important to this active site. These residues may bind two metal ions (Mg++) which may facilitate deprotonation of the OH terminus and formation of a transition state at the alpha phosphate of the dNTP. The thumb domain is believed to interact with the minor grove of the newly synthesized dsDNA and also with the incoming nucleotide. The thumb domain is less conserved but typically has a largely helical structure. The fingers domain may play a role in template fixation and nucleotide specificity. Like the thumb domain, it is likely to interact with the incoming nucleotide. The thumb domain may contain α helices, and/or β strands. It is thought that unbound DNA polymerases form open conformations of the fingers and thumb domains, and when the DNA is bound, the two domains move towards the palm domain to hold the DNA template and primer more tightly and to probe for Watson-Crick base pairing between the incoming nucleotide and the template nucleotide. The presence of a nucleotide that forms a Watson-Crick base pair with the template facilitates formation of an appropriate conformation of the active site of the polymerase and subsequent incorporation of this nucleotide. For review see Hamilton et al. (2001) BioTechniques 31:370-383. It was reported that mutagenesis in the palm/fingers domain may affects the nucleotide selectivity and affinity and mutagenesis in the thumb domain may affect the binding affinity to dsDNA. Important amino acids in the palm, fingers and thumb domain are described in U.S. Application Publication No. 20060281109, which is hereby incorporated by reference.

[0068] The uracil read-ahead function has been associated with the N-terminal domain. For example, archaeal family B DNA polymerases are able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion to prevent an A-T mutation. A “pocket” in the N-terminal domains of archaeal DNA polymerases was identified to be positioned to interact with the template strand and provide this uracil read-ahead function (Fogg et al. (2002) Nature Structural Biology 9(12), 922-927).

[0069] The exonuclease domain is associated with either 5′->3′ exonuclease activity, 3′->5″ exonuclease activity or both, which is required to remove incorrectly inserted nucleotide. When a mismatched nucleotide is incorporated, the template/primer strand binds to the polymerase more weakly and/or is misaligned with respect to the polymerase active site causing the mismatched nucleotide to be moved to the active site of the exonuclease domain and excised.

[0070] It is thought that the fidelity is affected by the ratio of the polymerase and the exonuclease activity, which may be influenced by the rate of dissociation, conformational change, and the rate of nucleotide incorporation in the presence of mismatched nucleotides. It has also been suggested that the balance between the 3′->5′ exonuclease activity and the polymerase activity is mediated by a flexible loop containing the Y-GG/A motif located between the N-terminal and exonuclease domains and the C-terminal polymerase domains (i.e., the palm, fingers and thumb domains). See, Bohlke et al. (2000) Nucl. Acids Res. 28(20), 3910-3917. A unique loop of the exonuclease domain, and the tip of the thumb are important for the coordination of proofreading and polymerase activities in DNA polymerases. Site-directed mutagenesis in this loop, especially at H147 in KOD DNA polymerase, suggested that electrostatic and hydrophobic interactions between this loop and the thumb affect the ratio between exonuclease activity and polymerase activity and hence fidelity. See, Kuroita et al. J. Mol. Biol. (2005) 351, 291-298.

Domain Swapping

[0071] According to the present invention, heterologous domains from different DNA polymerases (e.g., polymerases with at least one distinct functional characteristic) may be combined to form a chimeric polymerase. Suitable domains include naturally-occurring N-terminal domains, exonuclease domains, palm, fingers, and/or thumb domains found in various DNA polymerases. Naturally-occurring N-terminal domains, exonuclease domains, palm, fingers, and/or thumb domains in various DNA polymerases are well defined. For example, an N-terminal domain may include a sequence corresponding to amino acid residues 26 to 105 of KOD polymerase (SEQ ID NO:11); an exonuclease domain may include a region corresponding to amino acid residues 156 to 301 of KOD polymerase (SEQ ID NO:11); a thumb domain may include a region corresponding to amino acid residues 612 to 749 of KOD polymerase (SEQ ID NO:11); and palm and fingers domain may include a region corresponding to amino acid residues 394 to 563 of Pfu polymerase (SEQ ID NO:9).

[0072] Corresponding domains or 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.

[0073] In some embodiments, a suitable domain may be a variant (e.g., mutant or fragment) of a naturally-occurring domain sequence. For example, a suitable domain may have a 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 of a naturally-occurring domain found in a DNA polymerase of interest.

[0074] It is further contemplated that sequences defining the N-terminal domain, exonuclease domain, palm, fingers, and/or thumb domains may correlate with certain enzymatic characteristics of DNA polymerases, such as, fidelity or error rate, elongation rate, processivity, and salt resistance. For example, as described in the Examples section, the present inventors have demonstrated that sequences defining the N-terminal, exonuclease, and/or thumb domain may correlate with the characteristics associated with elongation rate, processivity, thermostability, TMAC tolerance and/or salt resistance; and that sequences defining the palm and/or fingers domain may correlate with the characteristics associated with fidelity or error rate of DNA polymerases.

[0075] In addition, based on sequence alignments between various DNA polymerases (see, e.g., FIG. 1), it is further contemplated that domains correlative with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance may be defined by one or more of the following positive consensus sequences:

Positive Consensus Sequence 1 (Defining an N-Terminal Domain)

• [0076] XXLXXXXXXXEGXRXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIX XXXXNXXXAXXKXXCXXXXXNFALXXXXXXXXXXXXIXXMXXRFXXXXXXXXX XXXXPXXRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTTXXXT (SEQ ID NO:30), wherein X is any amino acid or a peptide bond;
  Positive Consensus Sequence 2 (Defining an Exonuclease Domain)
• [0077] XXEXXXXYXXXXEXXFXXXXKXXXAXXXXXXXXAXXXXTVXTVKRXXXXQXXX XXRXVEXXXXXFTXXXXXXAXXDXIXXXXX (SEQ ID NO:3 1), wherein X is any amino acid or a peptide bond; and
  Positive Consensus Sequence 3 (Defining a Thumb Domain)
• [0078] XXXXXXXXXXXXXXXXALXXDXXXXKXXXXXXXXTEXXSKXXVXXXXXVXHX XXXXDXKDXXXTXXXXXXXXRXXXRXXXXRXXTXXSXXXXKXSXRXGDXXXPF DXFXXTXXXXXXXXXXXXXXXXXXEXXXRAXX (SEQ ID NO:32), wherein X is any amino acid or a peptide bond.

[0079] Additionally or alternatively, a domain or domains correlative with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance may be defined by one or more of the following negative consensus sequences:

Negative Consensus Sequence 1 (Defining an N-Terminal Domain)

• [0080] NGX₁FKIEX₂DRTFX₃PYX₄YALLX₅DDSX₆IEEVKKITX₇ERHGX₈X₉VX₁₀X₁₁X₁₂X₁₃VEKVX₁₄KKFLGX₁₅PX₁₆X₁₇VWKLYX₁₈X₁₉HPQDVPX₂₀IRX₂₁KX₂₂REHPA (SEQ ID NO:33), wherein X₁ is not K; X₂ is not H; X₃ is not R; X₄ is not I; X₅ is not R; X₆ is not K; X₇ is not G; X₈ is not K; X₉ is not I; X₁₀ is not R; X₁₁ is not I; X₁₂ is not V; X₁₃ is not D; X₁₄ is not E; X₁₅ is not K; X₁₆ is not I; X₁₇ is not T; X₁₈ is not L; X₁₉ is not E; X₂₀ is not T; X₂₁ is not E; and X₂₂ is not V;
  Negative Consensus Sequence 2 (Defining an Exonuclease Domain)
• [0081] PIX MISYADEX₂X₃AX₄VITWKNX₅DLPYVX₆VV SX₇EREMIKRFLRX₈X₉X₁₀EKDPDX₁₁X₁₂X₁₃TYNGDX₁₄FDFX₁₅YLX₁₆KRX₁₇EKLGIX₁₈X₁₉X₂₀X₂₁ GRDGSEPKX₂₂QRX₂₃GDX₂₄X₂₅AVEVKGRIHFDLYX₂₆VIX₂₇RTINLPTYTLEAVYEAX₂₈FGX₂₉PKEKVYAX₃₀EIX₃₁X₃₂AWEX₃₃ (SEQ ID NO:34), wherein X₁ is not I; X₂ is not N; X₃ is not E; X₄ is not K; X₅ is not I; X₆ is not E; X₇ is not S; X₈ is not I; X₉ is not I; X₁₀ is not R; X₁₁ is not I; X₁₂ is not I; X₁₃ is not V; X₁₄ is not S; X₁₅ is not P; X₁₆ is not A; X₁₇ is not A; X₁₈ is not K; X₁₉ is not L; X₂₀ is not T; X₂₁ is not I; X₂₂ is not M; X₂₃ is not I; X₂₄ is not M; X₂₅ is not T; X₂₆ is not H; X₂₇ is not T; X₂₈ is not I; X₂₉ is not K; X₃₀ is not D; X₃₁ is not A; X₃₂ is not K; and X₃₃ is not S; and
  Negative Consensus Sequence 3 (Defining a Thumb Domain)
• [0082] RDWSEIAKETQARVLEX₁X₂LKX₃GDVEX₄AVRIVKEVX₅X₆KLX₇X₈YEX₉PPEKLX₁₀IX₁₁EQITRX₁₂LX₁₃X₁₄YKAX₁₅GPHVAVAKX₁₆LAAX₁₇GVKIX₁₈PGX₁₉VIX₂₀YIVLX₂₁GX₂₂GX₂₃IX₂₄X₂₅RAIX₂₆X₂₇X₂₈EX₂₉DPX₃₀KHKYDAEYYIENQVLPAVX₃₁RILX₃₂X₃₃FG (SEQ ID NO:35), wherein X₁ is not T; X₂ is not I; X₃ is not H; X₄ is not E; X₅ is not I; X₆ is not Q; X₇ is not A; X₈ is not N; X₉ is not I; X₁₀ is not A; X₁₁ is not Y; X₁₂ is not P; X₁₃ is not H; X₁₄ is not E; X₁₅ is not I; X₁₆ is not K; X₁₇ is not K; X₁₈ is not K; X₁₉ is not M; X₂₀ is not G; X₂₁ is not R; X₂₂ is not D; X₂₃ is not P; X₂₄ is not S; X₂₅ is not N; X₂₆ is not L; X₂₇ is not A; X₂₈ is not E; X₂₉ is not Y; X₃₀ is not K; X₃₁ is not L; X₃₂ is not E; and X₃₃ is not G.

[0083] In some embodiments, a domain correlative with high fidelity may be defined by the following positive consensus sequence (defining palm and fingers domain):

• [0084] XKXXXXXXXXXXXXAXXXXXXXXXXXXXXXXXLXXXNXXIXXXXXXKXXXXI XXXXXXXXXHXXXXXXXXXTXXXEXQXXXXKIXXXXXXKXXXLXXXXFXXXXX XXKXXXXXXXXXXXXXXXXXKXXELVWXXLXXXFXXXXLXEXXXXLYXXXXXG ESXEIXXXXLX (SEQ ID NO:36), wherein X is any amino acid or a peptide bond.

[0085] Additionally or alternatively, a domain correlative with high fidelity may be defined by the following negative consensus sequence (defining palm and fingers domain):

• [0086] EX₁GLWENIVYLDFRX₂LYPSIIITHNVSPDTLNX₃EGCKX₄YDX₅APQVGHX₆FCKDX₇PGFIPSLLGX₈LLEERQKIKX₉KMKX₁₀TX₁₁DPIEX₁₂X₁₃LLDYRQX₁₄AIKX₁₅LANSX₁₆YGYYGYAX₁₇ARWYCKECAESVTAWGRX₁₈YIX₁₉X₂₀X₂₁X₂₂KEX₂₃EEKX₂₄GFKVX₂₅YX₂₆DTDGX₂₇X₂₈ATIPGX₂₉X₃₀X₃₁EX₃₂X₃₃KKKAX₃₄E (SEQ ID NO:37), wherein X₁ is not R; X₂ is not S; X₃ is not R; X₄ is not E; X₅ is not V; X₆ is not R; X₇ is not F; X₈ is not D; X₉ is not K; X₁₀ is not A; X₁₁ is not I; X₁₂ is not R; X₁₃ is not K; X₁₄ is not R; X₁₅ is not I; X₁₆ is not Y; X₁₇ is not R; X₁₈ is not E; X₁₉ is not T; X₂₀ is not M; X₂₁ is not T; X₂₂ is not I; X₂₃ is not I; X₂₄ is not Y; X₂₅ is not I; X₂₆ is not S; X₂₇ is not F; X₂₈ is not F; X₂₉ is not A; X₃₀ is not D; X₃₁ is not A; X₃₂ is not T; X₃₃ is not V; X₃₄ is not M.

[0087] Therefore, appropriate domains may be taken or derived from DNA polymerases with distinct functional characteristics to engineer a chimeric DNA polymerase with desirable combinations of functional features. In some embodiments, inventive methods in accordance with the present invention include steps of: (a) providing an N-terminal domain, an exonuclease domain, and/or a thumb domain based on a first DNA polymerase; (b) providing palm and/or fingers domain based on a second DNA polymerase; (c) combining the domains from step (a) and step (b) to form a chimeric polymerase. In some embodiments, the first and the second DNA polymerases are characterized with at least one distinct characteristic. For example, the first DNA polymerase may be characterized with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance and the second DNA polymerase may be characterized with high fidelity. In some embodiments, the first DNA polymerase may be characterized with high fidelity and the second DNA polymerase may be characterized with high processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance. In some embodiments, a chimeric polymerase engineered according to the invention has a processivity, elongation rate, thermostability, TMAC tolerance or salt resistance substantially similar to that of the first DNA polymerase and a fidelity substantially similar to that of the second DNA polymerase. In some embodiments, a chimeric polymerases engineered according to the present invention has the fidelity higher than that of the first DNA polymerase and the processivity, elongation rate or salt resistance higher than that of the second DNA polymerase.

[0088] The present invention further contemplates methods of improving the fidelity, processivity, elongation rate, thermostability, TMAC tolerance and/or salt resistance of a DNA polymerase. In some embodiments, inventive methods in accordance with the invention include a step of replacing a sequence within the palm-fingers domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher fidelity relative to the DNA polymerase of interest.

[0089] Additionally or alternatively, in some embodiments, inventive methods in accordance with the present invention include a step of replacing a sequence within the N-terminal domain, the exonuclease domain and/or the thumb domain of the DNA polymerase of interest with a corresponding sequence from a different DNA polymerase that is characterized with higher processivity, elongation rate, thermostability, TMAC tolerance or salt resistance relative to the DNA polymerase of interest.

[0090] As a non-limiting example, the present inventors have engineered a chimeric DNA polymerase Kofu and its reciprocal chimera POD based on KOD polymerase and Pfu polymerase (see the Examples section). As discussed in the example section, Kofu contains the N-terminal domain, the exonuclease domain and the thumb domain from KOD polymerase and the palm-fingers domain from Pfu polymerase. The sequence of Kofu polymerase is provided in SEQ ID NO:16. The reciprocal chimera POD contains the N-terminal domain, the exonuclease domain and the thumb domain from Pfu polymerase and the palm-fingers domain from KOD polymerase. The sequence of POD polymerase is provided in SEQ ID NO:15.

[0091] As discussed in the examples section, the Kofu chimeric polymerase displays the approximate replication fidelity of Pfu but the elongation speed, processivity, thermostability, TMAC tolerance and PCR performance similar to KOD. Alternatively, the Pod chimeric polymerase displays the approximate replication fidelity of KOD but the elongation speed, processivity, thermostability, TMAC tolerance and PCR performance similar to Pfu.

[0092] In some embodiments, the present invention provides variants of Kofu chimeric polymerase that contain an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:16 (Kofu amino acid sequence). In particular embodiments, variants of Kofu chimeric polymerase in accordance with the invention have processivity, elongation rate, thermostability, TMAC tolerance and/or fidelity substantially similar to Kofu.

[0093] In some embodiments, variants of Kofu chimeric polymerases in accordance with the present invention are defined by consensus sequence XXXXTXXXXXDXXXXXXIXXXXXXEXXXXYXXXXEXXFXXXXKXXXAXXXXXX XXAXXXXTVXTVKRXXXXQXXXXXRXVEXXXXXFTXXXXXXAXXDXIXXXXXXI XXYXXXXXXXXXXXXXXXXVXXXXDXXXXMXXXXXXXXXXXXXXXAEXXXLX XXXXXXEGXRXXXXXXVXXXXXDXXXTXXXXXXXXXXVVKXXXXXVLIXXXXX NXXXAXXKXXCXXXXXNFALXXXXXXXXXXIXXMXXRFXXXXXXXXXXXXXPX XRXXXXXXXXXXXXXXXXVXXQXXXXXXXEXXTXXXTXXXXXXXXRXXXXX XXVXXXXXXXXXXXXAXXXXXVXXPXXXXXXXXXXXXXXXXXXXXXXXXXXV XXXXSXEXYQXXXXEXXTXXFXXXXXKXXXXXXXXXXXXAXXXXXXXXXXXX XXXXXLXXXXNXXIXXXXXXKXXXXIXXXXXXXXXHXXXXXXXXXTXXXEXQX XXXKIXXXXXXKXXXLXXXXFXXXXXXXKXXXXXXXXXXXXXXXXXKXXELVW XXLXXXFXXXXLXIXXXXLYXXXXXGESXEIXXXXLXXLXXXXAXXXXAXXXXX XXXXXXXXXXXXXKXXXXXXXXXITXXXXXXXXXXXXXXXXXXXXXXXXALX XDXXXXKXXXXXXXXTEXXSKXXVXXXXXVXHXXXXXDXKDXXXTXXXXXXX XRXXXRXXXXRXXTXXSXXXXKXSXRXGDXXXPFDXFXXTXXXXXXXXXXXXX XXXXXEXXXRAXXXXXXXXXXXXXXXXXXSZXXKPXGT (SEQ ID NO:38), wherein X is any amino acid or a peptide bond.

[0094] In some embodiments, variants of Kofu chimeric polymerases in accordance with the present invention are defined by consensus sequence

• [0095] XIXDTDYXTXDGXPXXRIFXKXXGEFXXXYDXXFEPYFYALLKDDSAIXXXXXXXA XRHGTVXTVKRXXXXQXKFLXRXVEVWXLXFTHPQDVPAXXDXIXXHXXVIDIYE YDIPFAKRYLIDXGLVPMEGDEXLXMXXXDIETXYHEGXEFAEGXXLMISYADXEG ARVITWKXVDLPYVDVVSTEXEMIKRXXXVVKEKDPDVLIXYXGDNFDXAYLKXR CEXLGXNFALXRXXXXXEPKIXXMGXRFAVEXXGRXHFDLXPXXRXTXNLPTYXL XXVYEXVXGQXKXKXXXEEITTXWETXXXXXXXARYSMEDAXVTXELGXEFXPM EAXLXXLVGPXWDVXRSSTGNLVEWXLLXXAYXRNEVAPNKPSXEEYQXRXXE XYTGXFVXEPEKGLWXXXXXLDXXALYPSIIXXHNVSPDTLXLEXCXNYDIAPXVG XKFCKDIPGFIPSXLXHLXXXRQXXKTXMXEXQDPXEXIXLDYRQKAXKLLXNSFY GYXGYXKARWYXXECAESVTXWGRKYIELVWXELEXXFGFKXLYIDTDGLYATIP GGESXEIKXXXLXFLXYINAXLPGALELEYEXFYXRGFFVXKKKYAXIDEEXXITTR GLEXVRRDWSXXAKETXAXVLEALLXDXXVXKAVXXVXXXTEXXSKYXVPXEKL VIHEQITRDXKDYXATGPHVAXAKRLXXRGXXXRPGTXISYXXLKGSGRXGDRXIPF DEFXXTKHXYDXXYYIENQVLPAVERXLRAFGYXXXXLXXQXXXQXGLSAWXKP XGT (SEQ ID NO:39), wherein X is any amino acid or a peptide bond

[0096] In some embodiments, the present invention provide variants of POD chimeric polymerases that contain an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:15 (Pod amino acid sequence). In particular embodiments, variants of POD chimeric polymerases in accordance with the present invention have processivity, elongation rate, thermostability, TMAC tolerance and/or fidelity substantially similar to POD.

Expression of Chimeric DNA Polymerases of the Invention

[0097] Standard recombinant DNA techniques (e.g., restriction enzyme digestion, ligation, PCR) can be used to engineer chimeric DNA polymerases in accordance with the present invention. Methods well known in the art may be applied to express and isolate chimeric 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. Expression vectors are commercially available from, for example, Novagen (http://www.emdbiosciences.com/html/NVG/AllTables.html#).

[0098] In addition, bacteria expressing an integrated inducible form of the T7 RNA polymerase gene may be transformed with an expression vector bearing a chimeric 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.

[0099] 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 PLUS™ 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.

[0100] There are many methods known to those of skill in the art that are suitable for the purification of a chimeric 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.

[0101] Further, DNA polymerase mutants 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.

Uses of Chimeric DNA Polymerases of the Invention

[0102] Chimeric 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, chimeric 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, and amplifying, detecting, and/or cloning nucleic acid sequences using polymerase chain reaction (PCR).

[0103] In some embodiments, the invention provides robust, fast, and accurate enzymes for PCR. 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.

[0104] Chimeric DNA polymerases with higher processivity, elongation rate and/or fidelity are expected to reduce error rate, improve efficiency and success rate of long-range amplification (higher yield, longer targets amplified), and/or reduce the amount of required DNA template.

[0105] 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).

[0106] 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) Reclusive 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 amplification; xvi) quantitative PCR using SYBR green or oligonucleotide probes to detect amplification; xvii) whole-genome amplification using adaptor-ligated DNA fragment libraries as template, and xviii) error-prone PCR in which conditions are optimized to give an increased number of mutations in the PCR product.

[0107] 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

[0108] The invention also contemplates kit formats which include a package unit having one or more containers containing chimeric 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.

[0109] 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.

[0110] The two enzymes we chose to include in this experiment were Pyroccocus furiosus DNA polymerase (Pfu) and Thermococcus Kodarensis (KOD) DNA polymerases. The two enzymes have similar domain structure and have a 79% identity at the amino acid level using blastP alignments (see Table 2). The domain structures of Pfu and KOD are illustrated in FIG. 1.

[0111]

TABLE 2
ClustalW alignment of Pfu and KOD
PFU	1	MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG	60
KOD	1	....T.....D......I......E....Y....E..F....K...A........A....	60
PFU	61	KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY	120
KOD	61	TV.TVKR....Q.....R.VE.....FT......A..D.I......I..Y..........	120
PFU	121	LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY	180
KOD	121	......V....D....M...............AE...L.......EG.R......V....	180
PFU	181	VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPK	240
KOD	181	.D...T..........VVK.....VLI.....N...A..K..C.....NFAL........	240
PFU	241	MQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWE	300
KOD	241	I..M..RF.............P..R................V..Q.......E..TT...	300
PFU	301	SGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRK	360
KOD	301	T........R.......V............A.....I..S....................	360
PFU	361	AYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVS	420
KOD	361	......L.....D.K.LA..~.Q..E.. Y.....R............S...........	419
PFU	421	PDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILL	480
KOD	420	.....R....E..V......R....F.........D.........K...A.I....RK..	479
PFU	481	DYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYI	540
KOD	480	....R...I....Y.......R.................E..TMTI..I...Y....I.S	539
PFU	541	DTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDE	600
KOD	540	....FF.....ADA.TV....M..L....A....A..................K......	599
PFU	601	EGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEK	660
KOD	600	...IT........................AL..D....K........TE..SK..V....	659
PFU	661	LAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEE	720
KOD	660	.V.H.....D.KD...T........R...R....R..T..S....K.S.R.GD...PFD.	719
PFU	721	YDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS*	776
KOD	720	F..T..................E...RA..................SA..KP.GT*	775
PFU	(SEQ ID NO: 9)
KOD	(SEQ ID NO: 11)

[0112] Pfu and KOD have very distinct phenotypic characteristics, in particular, with respect to elongation rate, processivity and error rate (See Table 3):

[0113]

	TABLE 3
	Pfu	KOD
Elongation Rate:	25 nt/s	106-138 nt/s (Takagi et al. 1997)
Processivity:	>20 nt	~300 nt (Takagi et al. 1997)
Error Rate	1.5 × 10−6	4.45 × 10−6 (internal data)
(mutations/nt/doubling):

[0114] Thus, the goal was to find chimeric combinations of these two enzymes which exhibited the error rate comparable to Pfu (2.0×10⁻⁶) with the processivity and/or elongation rate comparable to KOD (˜300nt/s and 106-138nt/s respectively). An enzyme with the above mentioned characteristics has utility as a robust, fast, and accurate enzyme for PCR.

[0115] Restriction sites were inserted into the codon-optimized nucleotide sequence of KOD and Pfu polymerases at positions that approximately flank the polymerase domain of the enzymes (see Example 2). For example, PvuII and EcoRI sites flanking the polymerase domain (the palm and fingers domain) were used to replace the polymerase domain of Pfu with that of KOD to generate the chimera deemed Pod (FIG. 2). This chimera contains the N-terminal domain, the 3′-5′ exonuclease domain and the thumb domain of Pfu and the palm and fingers domain of KOD. The reciprocal swap, yielding the chimera Kofu, was generated by replacing the polymerase domain (palm and fingers) of KOD with that of Pfu.

[0116] Native DNA sequences for Pyrococcus furiosus polymerase I (SEQ ID NO:1) and Thermococcus kodakarensis polymerase I (SEQ ID NO:2) were retrieved from Genbank. These two DNA sequences were in silico codon optimized by Codon Devices (Cambridge, Mass.) for expression in E. Coli resulting in SEQ ID NO:3 for the Pfu polymerase I codon optimized gene DNA sequence and SEQ ID NO:4 for the KOD polymerase I codon optimized gene DNA sequence. The two codon optimized genes were chemically synthesized and cloned into pUC19 by Codon Devices (Cambridge, Mass.) resulting in SEQ ID NO:7 for Pfu polymerase I and SEQ ID NO:8 for KOD polymerase I.

[0117] KOD (SEQ ID NO:8) and Pfu (SEQ ID NO:7) polymerase codon optimized pUC 19 constructs were cloned into the pKBexp vector as follows:

[0118] The pKBexp vector contains two Eco31I sites with non-complementary overhangs enabling directional cloning of inserts using a single restriction enzyme. KOD and Pfu polymerase genes were designed with two flanking Eco31I sites that enabled directional and in-frame cloning into pKBexp.

[0119] Purified DNA from the pKBexp vector was digested with Eco31I and purified from an agarose gel. KOD and Pfu codon optimized pUC DNA constructs (SEQ ID NO.8 and SEQ ID NO.7) were likewise digested with Eco31I and the roughly 2.3 kilobase insert fragments were cut out from an agarose gel and purified. 30 ng of KOD or Pfu polymerase genes were ligated with 15 ng of digested pKBexp using T4 DNA ligase. The ligation reactions were purified and used to transform competent E. coli DH10B. DNA minipreps were made of ampicillin resistant clones. The presence of inserts was confirmed by digestion of the minipreps with XbaI and HindIII, two enzymes that flank the insert. The cloning of the KOD polymerase gene sequence in pKBexp deemed pKB11 and the Pfu polymerase gene in pKBexp deemed pKB14 were confirmed by DNA sequencing.

[0120] The codon-optimized sequences of KOD (SEQ ID NO:5) and Pfu (SEQ ID NO:3) polymerase I genes were designed with restriction sites that approximately flank the finger and palm domains of KOD and Pfu polymerases. The KOD codon optimized sequence contains a PvuII restriction site and an EcoRI restriction site. The Pfu codon optimized sequence contains a PvuII restriction site and an EcoRI restriction site.

[0121] Purified DNA from pKB11 and pKB14 were each digested the restriction enzymes EcoRI and PvuII. The large fragment (4.7 kb) and small fragment (0.7 kb) from each digest were separately extracted and purified from an agarose gel. The small fragments from each restriction digest contained the finger and palm domains of KOD and Pfu respectively. The digested and purified large fragments (containing the expression vector and remaining polymerase fragments) were dephosphorylated using Shrimp Alkaline Phosphate. The construct deemed POD was created by ligation of 30 ng of the 4.7 kb Pfu large fragment (aa residues 1 to 335 and 567 to 778 of Pfu DNA polymerase with 10 ng of the 0.7 kb KOD small fragment (corresponding to amino acid residues 336 to 565 of KOD DNA polymerase SEQ ID NO:11). POD thus includes N-terminal, exonuclease and thumb domains from Pfu DNA polymerase and palm and finger domains from KOD. The construct deemed Kofu was made by ligation of 30 ng of the 4.7 kb KOD large fragment (corresponding to amino acid residues 1 to 335 and 566 to 777 of KOD DNA polymerase SEQ ID NO:11) with 10 ng of the 0.7 kb Pfu small fragment (corresponding to amino acid residues 336 to 566 of Pfu DNA polymerase SEQ ID NO:11). Kofu thus includes N-terminal, exonuclease and thumb domains from KOD DNA polymerase and palm and finger domains from Pfu. The ligation reactions were used to transform E. coli DH10B. The construction of Pod (SEQ ID NO:13) and Kofu (SEQ ID NO:14) was confirmed by DNA sequencing. The domain structures of POD and Kofu are illustrated in FIG. 1. Expression and purification of chimeric polymerases are done using methods known in the art, for example, as reviewed in “Detailed description of the invention.”

[0122] 10 ng of each enzyme was incubated at 98° C. for 240, 120, 60, 30, 15, 8, 4, 2, 1 or 0 min in a 10 μl volume containing the following: 20 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 6 mM (NH4)₂SO₄, 25 or 50 mM KCl (25 mM for Pfu and Pod, 50 mM for KOD and Kofu). 10 μl of primer/template mix was added to each tube after the heat incubation. The primer template mix contained the following: 20 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 6 mM (NH4)₂SO₄, 0.6 mM dNTP, 0.6 04 each of primers HPRT1-F1 (5′-tttggaaacatctggagtcct -3′ (SEQ ID NO:40)) and HPRT1-R1 (5′-gcccaaagggaactgatagtc -3′ (SEQ ID NO:41)), 2 ng human genomic DNA per μl, and 25 or 50 mM KCl (25 mM for Pfu and Pod, 50 mM for KOD and Kofu). The amplifications were performed with the following cycling protocol: 3 minutes at 95° C., 35×(20 seconds at 98° C., 20 seconds at 60° C., 20 seconds at 72° C.), 20 seconds at 72° C. The PCR products were analysed on an agarose gel (see FIG. 3). As illustrated in FIG. 3, no amplification was observed for Pfu after pre-incubation of the enzyme for 4 hours at 98° C. In contrast, KOD, Kofu and Pod were able to amplify a PCR product for all time points tested.

[0123] 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). LacI was PCR amplified from E. coli and cloned into pUC19 to degenerate plasmid pKB-LacIQZalpha (SEQ ID NO:17). pKB-LacIQZalpha served both as template for PCR amplification of LacI in the fidelity assays and as vector for cloning the amplified LacI into for blue/white colony screening.

[0124] 2×50 μl PCR reactions (for each enzyme) were set-up, using 70 ng of pKB-LacIQZalfa plasmid template (equivalent to 25 ng of lad target) and 2.5U of each enzyme to amplify the 1.386 Kb lacIOZalpha fragment. The PCR conditions were as follows: amplification with Pfu and Pod were done in Pfu buffer (Fermentas); KOD and Kofu in Novagen KOD buffer 1. Final concentrations of 2 mM MgCl2, 0.4 μM each of primers M13-40 (GTTTTCCCAGTCACGAC (SEQ ID NO:42)) and PKBLac-1R (GGTATCTTTATAGTCCTGTCG (SEQ ID NO:43)) and 0.2 mM each dNTP. Cycling parameters for Pfu and Pod were: 94° C. 4 minutes, 30×(94° C. 15 seconds, 55° C. 15 seconds, 72° C. 3 minutes), 72° C. 6 minutes. Cycling parameters for KOD and Kofu were: 94° C. 2 minutes, 30×(98° C. 15 seconds, 55° C. 2 seconds, 72° C. 20 seconds), 72° C. 30 seconds.

[0125] PCR product yields were quantitated by means of gel electrophoresis and the number 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 lad amplicon are scored). Exemplary results are summarized in Table 4. As shown in Table 4, Pfu and Kofu have similar fidelity and that their fidelity is higher than that of KOD and Pod.

[0126]

TABLE 4
Fidelity of KOD, Pfu, Kofu and Pod
	White		Blue	Total	Fidelity
	colonies	Doublings d	colonies	colonies	f (×10−6)
KOD	21130	7.77	246	21376	4.27
Pfu	19270	7.76	77	19347	1.47
Kofu	12817	5.8	39	12856	1.50
Pod	22039	7.19	221	22260	3.98

[0127] Processivity can be determined and calculated using assays described in (Wang et al. Nucl. Acids Res, 2004, 32(3): 1197-1207; and Von Hippel et al. NY Acad Sci 1994; 726:118-131). Briefly, 0.8 pmoles of a 5′FAM-labelled primer (−40M13LFF, 5′FAM-GTTTTCCCAGTCACGACGTTGTAAAACGACGGCC-3′ (SEQ ID NO:44)) is added to 1.6 pmoles of ssM13mp18 DNA in the presence of 20 mM Tris-HCl pH 8.0, 25 mM KCl, 2.5 mM MgCl2, 0.3 mM dNTP in a 16 microL volume. The primer is annealed to the template by heating to 95° C. for 2 minutes followed by slow cooling to 72° C. in a thermocycler at a rate of 0.1° C/second, incubation for 10 minutes at 72° C. and further cooling at 0.1° C./second to 4° C. The polymerases are diluted in 20 mM Tris-HCl pH 8.0, 25 mM KCl. The primed template and the diluted polymerases are heated to 72° C. and the reaction is started by adding 4 μl diluted polymerase to 16 μl of primed template. The polymerases are diluted to give polymerase:template ratios of 1:10-1:10000. The reactions are terminated after various timepoints by adding EDTA to a final concentration of 10 mM.

[0128] The extension reactions are analyzed on an ABI 3130XL Genetic Analyzer. The median product length is determined for each reaction. The median product length is defined as the length of the product at which the total fluorescence intensity of all products up to that length equals 50% of the sum of fluorescence intensities of all detectable products. The traces for those samples where the median product length does not change with a change in polymerase concentration or incubation time are used to calculate the processivity according to Von Hippel et al. (Von Hippel et al. NY Acad Sci 1994; 726:118-131). Each peak (I) with a fluorescence level significantly above background level is integrated to give the fluorescence intensity of that peak (ni). The total fluorescence intensity (nT) is the sum of the fluorescence of all peaks. The integration data are plotted as log(ni/nT) vs n−1, where n is the number of nucleotides incorporated. The data is fitted to the following equation: log(ni/nT)=(n−1)log Pi+log(1−Pi). Pi, the microscopic processivity factor, is defined as the probability of not terminating extension at position i. The average primer extension length is determined from 1/(1−Pi).

[0129] Previous studies (Pavlov et al. (2002) Proc Natl Acad Sci. 99(21), 13510-13515; Wang et al. (2004) Nucl Acids Res. 32(3), 1197-1207) have shown that there is a direct correlation between increased tolerance of polymerases to salt and the processivity of polymerases. For all polymerases tested (from family A or family B), it was found that polymerases with increased salt tolerance also have increased processivity. We therefore compared the salt tolerance of our chimeras with that of the parental polymerases as a proxy for processivity.

[0130] The protein concentration of the purified KOD, Pfu, Kofu and Pod where determined using a Bioanalyzer 2100 (Agilent, Santa Clara, Calif., USA) with the Protein 230 Kit from the same supplier. The polymerases were tested in real-time PCR with increasing amounts of KCl added. The reactions were performed in a 20 μl volume containing 20 mM Tris-HCl pH 8.0, 6 mM (NH₄)₂SO₄, 2 mM MgCl₂, 3% DMSO, 10 ng polymerase, 20 ng human genomic DNA, 0.3 mM each dNTP, 0.25×SYBR Green (Invitrogen, Carlsbad, Calif., USA). A diluted stock 20×SYBR Green in DMSO was made), 0.3 04 forward primer HPRT1-F1 (5′-tttggaaacatctggagtcct-3′ (SEQ ID NO:40)) and 0.3 μM reverse primer HPRT1-R1(5′-gcccaaagggaactgatagtc-3′ (SEQ ID NO:41)). KCl was added to final concentrations of 10, 25, 50, 75, 100 or 125 mM. PCR amplification was performed in a Corbett 6000 HRM real-time thermocycler (Corbett Life Science, Sidney, Australia) with the following cycling protocol: 3 minutes at 95° C., 40 cycles of (10 seconds at 95° C., 20 seconds at 60° C., 20 seconds at 72° C., data acquisition), followed by a melting curve analysis step of: ramp from 72° C. to 95° C. in 1° C. steps, wait for 5 seconds before data acquisition at the end of each step. 8 μl of each sample was analysed on a 1.5% agarose gel. 5 μl of Fermentas GeneRuler™ Mix, cat no. SM0333 (Fermentas, Vilnius, Lithuania) was loaded onto the gel as a DNA marker. Exemplary results are shown in FIG. 4.

[0131] Tetra-methyl ammonium-containing salts enhance PCR reactions as shown by Kovarova et al. (Kovarova, M. and Draber, P.; Nucl. Acids Res. (2000) 28(13) e70-). One such salt is tetra-methyl ammonium chloride (TMAC). We therefore compared the TMAC tolerance of our chimeras with that of the parental polymerases.

[0132] The polymerases were tested in real-time PCR with increasing amounts of TMAC added. The reactions were performed in a 20 μl volume containing 20 mM Tris-HCl pH 8.0, 6 mM (NH₄)₂SO₄, 2 mM MgCl₂, 25 mM KCl, 10 ng polymerase, 20 ng human genomic DNA, 0.3 mM each dNTP, 0.25×SYBR Green (Invitrogen, Carlsbad, Calif., USA. A diluted stock 20×SYBR Green in DMSO was made), 0.3 μM forward primer HPRT1-F1 (5′-tttggaaacatctggagtcct-3′ (SEQ ID NO:40)) and 0.3 μM reverse primer HPRT1-R1(5′-gcccaaagggaactgatagtc-3′ (SEQ ID NO:41)). TMAC was added to final concentrations of 0, 10, 20, 40, 60, 80, 100 or 120 mM. PCR amplification was performed in a Corbett 6000 HRM real-time thermocycler (Corbett Life Science, Sidney, Australia) with the following cycling protocol: 3 minutes at 95° C., 40 cycles of (10 seconds at 95° C., 20 seconds at 50° C., 20 seconds at 72° C., data acquisition), followed by a melting curve analysis step of: ramp from 72° C. to 95° C. in 1° C. steps, wait for 5 seconds before data acquisition at the end of each step. 8 μl of each sample was analysed on a 1.5% agarose gel. 5 μl of Fermentas GeneRuler™ Mix, cat no. SM0333 (Fermentas, Vilnius, Lithuania) was loaded onto the gel as a DNA marker. Exemplary results are shown in FIG. 5.

[0133] This example is designed to show that the positions where the swapping between domains take place may vary.

[0134] Additional chimeras are made by swapping the palm and finger domains of KOD and Pfu polymerases where the exact position of the swap varies slightly compared to positions for Kofu and Pod. Kofu-II (SEQ ID NO:26) is made by replacing amino acid residues 305 to 615 of KOD (SEQ ID NO: 12) with amino acids 305 to 616 of Pfu (SEQ ID NO:10). Pod-II (SEQ ID NO:27) is made by replacing amino acids 305 to 616 of Pfu (SEQ ID NO:10) with amino acids 305 to 615 of KOD (SEQ ID NO:12).

[0135] Kofu-III (SEQ ID NO:28) is made by replacing amino acid residues 396 to 564 of KOD (SEQ ID NO: 12) with amino acids 397 to 565 of Pfu (SEQ ID NO:10). Pod-III (SEQ ID NO:29) is made by replacing amino acids 397 to 565 of Pfu (SEQ ID NO:10) with amino acids 396 to 564 of KOD (SEQ ID NO:12).

[0136] The amino acid sequence of chimeras Kofu-II, Pod-II, Kofu-III and Pod-III are reverse translated and codon-optimized for expression in E. coli. Additional nucleotide sequences containing Eco31I restriction sites are added to the 5′ and 3′ ends of the construct to facilitate cloning into an expression vector. More specifically, the 5′ and 3′ sequences can be designed so that the overhangs, after digestion of the DNA with Eco31I, are complementary to the overhangs in a particular expression vector (e.g., pKB). Codon optimization and gene synthesis is performed by GeneArt Gmbh. Expression and purification of chimeric polymerases are done using methods known in the art, for example, as reviewed in “Detailed description of the invention”. The thermostability, fidelity, processivity, salt resistance and TMAC resistance of the chimeric polymerases are determined as described in Examples 5 through 9.

[0137] Chimeras 9Nli and Li9N are designed based on the alignment in FIG. 1. They are made by swapping the palm and finger domains between the DNA polymerases of T. litoralis and Thermococcus sp. 9 degrees N-7. The overall sequence identity between these two polymerases are 77% on the amino acid level.

[0138] Chimera 9Nli can be made by replacing the palm and finger region of the 9N polymerase with the palm and finger region of the T. litoralis polymerase. In this particular example, 9Nli is made by replacing amino acids 347 to 580 of 9N polymerase (SEQ ID NO:18) with amino acids 349 to 583 of T. litoralis polymerase (SEQ ID NO:19). The sequence of the coding region of 9Nli is provided as SEQ ID NO:20.

[0139] Chimera LiN9 can be made by replacing the palm and finger domain of the DNA polymerase of T. litoralis with the finger domain of the DNA polymerase of 9 degrees North. In this particular example, LiN9 is made by replacing amino acids 349 to 583 of T. litoralis polymerase (SEQ ID NO:19) with amino acids 347 to 580 of 9 degrees N-7 polymerase (SEQ ID NO:18). The sequence of the coding region of LiN9 is provided as SEQ ID NO:21.

[0140] Chimerase GoZi and ZiGo are designed based on the alignment in FIG. 1. They are made by swapping the palm and finger domains between the DNA polymerases of T. gorgonarius and T. zilligii. The overall sequence identity between these two polymerases are 94% on the amino acid level.

[0141] Chimera GoZi can be made by replacing the palm and finger region of the T. gorgonarius polymerase with the palm and finger region of the T. zilligii polymerase. In this particular example, GoZi is made by replacing amino acids 391 to 559 of T. gorgonarius polymerase (SEQ ID NO:22) with amino acids 391 to 559 of T. zilligii polymerase (SEQ ID NO:23). The sequence of the resulting chimera GoZi is provided as SEQ ID NO:24.

[0142] Chimera ZiGo can be made by replacing the palm and finger domain of the DNA polymerase of T. zilligii with the finger domain of the DNA polymerase of T. gorgonarius. In this particular example, ZiGo is made by replacing amino acids 391 to 559 of T. zilligii polymerase (SEQ ID NO:23) with amino acids 391 to 559 of T. gorgonarius polymerase (SEQ ID NO:22). The sequence of the coding region of ZiGo is provided as SEQ ID NO:25.

[0143]

TABLE 5
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 YLAKRAEKLG 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: 42)
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: 43)
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
Amino acid sequences of DNA polymerases from T. litoralis, Thermococcus sp. 9 degrees
N-7 and chimeras thereof.
Sequence 18 (SEQ ID NO: 18)
Thermococcus sp. 9 degrees N-7 DNA polymerase amino acid sequence (acc no. U47108)
1	MILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYA LLKDDSAIED VKKVTAKRHG
61	TVVKVKRAEK VQKKFLGRPI EVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMI SYADGSEARV ITWKKIDLPY
181	VDVVSTEKEM IKRFLRVVRE KDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGKPKEKV YAEEIAQAWE
301	SGEGLERVAR YSMEDAKVTY ELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWFLLRK
361	AYKRNELAPN KPDERELARR RGGYAGGYVK EPERGLWDNI VYLDFRSLYP SIIITHNVSP
421	DTLNREGCKE YDVAPEVGHK FCKDFPGFIP SLLGDLLEER QKIKRKMKAT VDPLEKKLLD
481	YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGREY IEMVIRELEE KFGFKVLYAD
541	TDGLHATIPG ADAETVKKKA KEFLKYINPK LPGLLELEYE GFYVRGFFVT KKKYAVIDEE
601	GKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661	VIHEQITRDL RDYKATGPHV AVAKRLAARG VKIRPGTVIS YIVLKGSGRI GDRAIPADEF
721	DPTKHRYDAE YYIENQVLPA VERILKAFGY RKEDLRYQKT KQVGLGAWLK VKGKK
Sequence 19 (SEQ ID NO: 19)
T. litoralis DNA polymerase amino acid sequence (acc no. M74198.1)
1	MILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA LLKDDSAIEE IKAIKGERHG
61	KTVRVLDAVK VRKKFLGREV EVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMI SYADEEEARV ITWKNIDLPY
181	VDVVSNEREM IKRFVQVVKE KDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPE
241	PKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAV YEAVLGKTKS KLGAEEIAAI
301	WETEESMKKL AQYSMEDARA TYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWYLL
361	RVAYARNELA PNKPDEEEYK RRLRTTYLGG YVKEPEKGLW ENIIYLDFRS LYPSIIVTHN
421	VSPDTLEKEG CKNYDVAPIV GYRFCKDFPG FIPSILGDLI AMRQDIKKKM KSTIDPIEKK
481	MLDYRQRAIK LLANSYYGYM GYPKARWYSK ECAESVTAWG RHYIEMTIRE IEEKFGFKVL
541	YADTDGFYAT IPGEKPELIK KKAKEFLNYI NSKLPGLLEL EYEGFYLRGF FVTKKRYAVI
601	DEEGRITTRG LEVVRRDWSE IAKETQAKVL EAILKEGSVE KAVEVVRDVV EKIAKYRVPL
661	EKLVIHEQIT RDLKDYKAIG PHVAIAKRLA ARGIKVKPGT IISYIVLKGS GKISDRVILL
721	TEYDPRKHKY DPDYYIENQV LPAVLRILEA FGYRKEDLRY QSSKQTGLDA WLKR
Sequence 20 (SEQ ID NO: 20)
Amino acid sequence of chimeric DNA polymerase 9Nli
1	MILDTDYITE NGKPVIRVFK KENGEFKIEY DRTFEPYFYA LLKDDSAIED VKKVTAKRHG
61	TVVKVKRAEK VQKKFLGRPI EVWKLYFNHP QDVPAIRDRI RAHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELTMLAF DIETLYHEGE EFGTGPILMI SYADGSEARV ITWKKIDLPY
181	VDVVSTEKEM IKRFLRVVRE KDPDVLITYN GDNFDFAYLK KRCEELGIKF TLGRDGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AVFGKPKEKV YAEEIAQAWE
301	SGEGLERVAR YSMEDAKVTY ELGREFFPME AQLSRLIGQS LWDVSRSSTG NLVEWYLLRV
361	AYARNELAPN KPDEEEYKRR LRTTYLGGYV KEPEKGLWEN IIYLDFRSLY PSIIVTHNVS
421	PDTLEKEGCK NYDVAPIVGY RFCKDFPGFI PSILGDLIAM RQDIKKKMKS TIDPIEKKML
481	DYRQRAIKLL ANSYYGYMGY PKARWYSKEC AESVTAWGRH YIEMTIREIE EKFGFKVLYA
541	DTDGFYATIP GEKPELIKKK AKEFLNYINS KLPGLLELEY EGFYVRGFFV TKKKYAVIDE
601	EGKITTRGLE IVRRDWSEIA KETQARVLEA ILKHGDVEEA VRIVKEVTEK LSKYEVPPEK
661	LVIHEQITRD LRDYKATGPH VAVAKRLAAR GVKIRPGTVI SYIVLKGSGR IGDRAIPADE
721	FDPTKHRYDA EYYIENQVLP AVERILKAFG YRKEDLRYQK TKQVGLGAWL KVKGKK
Sequence 21 (SEQ ID NO: 21)
Amino acid sequence of chimeric DNA polymerase Li9N
1	MILDTDYITK DGKPIIRIFK KENGEFKIEL DPHFQPYIYA LLKDDSAIEE IKAIKGERHG
61	KTVRVLDAVK VRKKFLGREV EVWKLIFEHP QDVPAMRGKI REHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELKLLAF DIETFYHEGD EFGKGEIIMI SYADEEEARV ITWKNIDLPY
181	VDVVSNEREM IKRFVQVVKE KDPDVIITYN GDNFDLPYLI KRAEKLGVRL VLGRDKEHPE
241	PKIQRMGDSF AVEIKGRIHF DLFPVVRRTI NLPTYTLEAV YEAVLGKTKS KLGAEEIAAI
301	WETEESMKKL AQYSMEDARA TYELGKEFFP MEAELAKLIG QSVWDVSRSS TGNLVEWFLL
361	RKAYKRNELA PNKPDERELA RRRGGYAGGY VKEPERGLWD NIVYLDFRSL YPSIIITHNV
421	SPDTLNREGC KEYDVAPEVG HKFCKDFPGF IPSLLGDLLE ERQKIKRKMK ATVDPLEKKL
481	LDYRQRAIKI LANSFYGYYG YAKARWYCKE CAESVTAWGR EYIEMVIREL EEKFGFKVLY
541	ADTDGLHATI PGADAETVKK KAKEFLKYIN PKLPGLLELE YEGFYLRGFF VTKKRYAVID
601	EEGRITTRGL EVVRRDWSEI AKETQAKVLE AILKEGSVEK AVEVVRDVVE KIAKYRVPLE
661	KLVIHEQITR DLKDYKAIGP HVAIAKRLAA RGIKVKPGTI ISYIVLKGSG KISDRVILLT
721	EYDPRKHKYD PDYYIENQVL PAVLRILEAF GYRKEDLRYQ SSKQTGLDAW LKR
Amino acid sequences of DNA polymerases from T. gorgonarius, T. zilligii and chimeras
thereof.
Sequence 22 (SEQ ID NO: 22)
T. gorgonarius DNA polymerase amino acid sequence (acc no. 4699806)
1	MILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA LLKDDSAIED VKKITAERHG
61	TTVRVVRAEK VKKKFLGRPI EVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNIDLPY
181	VDVVSTEKEM IKRFLKVVKE KDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AIFGQPKEKV YAEEIAQAWE
301	TGEGLERVAR YSMEDAKVTY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361	AYERNELAPN KPDERELARR RESYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP
421	DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLD
481	YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYAD
541	TDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEE
601	DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661	VIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEF
721	DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLGAWLK PKT
Sequence 23 (SEQ ID NO: 23)
T. zilligii DNA polymerase amino acid sequence
1	MILDADYITE DGKPVIRVFK KEKGEFKIDY DRDFEPYIYA LLKDDSAIED IKKITAERHG
61	TTVRVTRAER VKKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVVDIY EYDIPFAKRY
121	LIDRGLIPME GDEELRMLAF DIETLYHEGE EFGEGPILMI SYADEEGARV ITWKNIDLPY
181	VESVSTEKEM IKRFLKVIQE KDPDVLITYN GDNFDFAYLK KRSETLGVKF ILGRDGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLETVYE AIFGQPKEKV YAEEIARAWE
301	SGEGLERVAR YSMEDAKATY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361	AYERNELAPN KPDERELARR AESYAGGYVK EPEKGLWENI VYLDYKSLYP SIIITHNVSP
421	DTLNREGCRE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKVKKKMKAT VDPIERKLLD
481	YRQRAIKILA NSYYGYYGYA NARWYCRECA ESVTAWGRQY IETTMREIEE KFGFKVLYAD
541	TDGFFATIPG ADAETVKKKA KEFLNYINPR LPGLLELEYE GFYRRGFFVT KKKYAVIDEE
601	DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SRYEVPPEKL
661	VIYEQITRDL RDYRATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGPGRV GDRAIPFDEF
721	DPAKHRYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT KQAGLGAWLK PKT
Sequence 24 (SEQ ID NO: 24)
Amino acid sequence of chimeric DNA polymerase GoZi
1	MILDTDYITE DGKPVIRIFK KENGEFKIDY DRNFEPYIYA LLKDDSAIED VKKITAERHG
61	TTVRVVRAEK VKKKFLGRPI EVWKLYFTHP QDVPAIRDKI KEHPAVVDIY EYDIPFAKRY
121	LIDKGLIPME GDEELKMLAF DIETLYHEGE EFAEGPILMI SYADEEGARV ITWKNIDLPY
181	VDVVSTEKEM IKRFLKVVKE KDPDVLITYN GDNFDFAYLK KRSEKLGVKF ILGREGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLEAVYE AIFGQPKEKV YAEEIAQAWE
301	TGEGLERVAR YSMEDAKVTY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361	AYERNELAPN KPDERELARR RESYAGGYVK EPEKGLWENI VYLDYKSLYP SIIITHNVSP
421	DTLNREGCRE YDVAPQVGHR FCKDFPGFIP SLLGDLLEER QKVKKKMKAT VDPIERKLLD
481	YRQRAIKILA NSYYGYYGYA NARWYCRECA ESVTAWGRQY IETTMREIEE KFGFKVLYAD
541	TDGFFATIPG ADAETVKKKA KEFLDYINAK LPGLLELEYE GFYKRGFFVT KKKYAVIDEE
601	DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SKYEVPPEKL
661	VIYEQITRDL KDYKATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGSGRI GDRAIPFDEF
721	DPAKHKYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT RQVGLGAWLK PKT
Sequence 25 (SEQ ID NO: 25)
Amino acid sequence of chimeric DNA polymerase ZiGo
1	MILDADYITE DGKPVIRVFK KEKGEFKIDY DRDFEPYIYA LLKDDSAIED IKKITAERHG
61	TTVRVTRAER VKKKFLGRPV EVWKLYFTHP QDVPAIRDKI REHPAVVDIY EYDIPFAKRY
121	LIDRGLIPME GDEELRMLAF DIETLYHEGE EFGEGPILMI SYADEEGARV ITWKNIDLPY
181	VESVSTEKEM IKRFLKVIQE KDPDVLITYN GDNFDFAYLK KRSETLGVKF ILGRDGSEPK
241	IQRMGDRFAV EVKGRIHFDL YPVIRRTINL PTYTLETVYE AIFGQPKEKV YAEEIARAWE
301	SGEGLERVAR YSMEDAKATY ELGKEFFPME AQLSRLVGQS LWDVSRSSTG NLVEWFLLRK
361	AYERNELAPN KPDERELARR AESYAGGYVK EPERGLWENI VYLDFRSLYP SIIITHNVSP
421	DTLNREGCEE YDVAPQVGHK FCKDFPGFIP SLLGDLLEER QKVKKKMKAT IDPIEKKLLD
481	YRQRAIKILA NSFYGYYGYA KARWYCKECA ESVTAWGRQY IETTIREIEE KFGFKVLYAD
541	TDGFFATIPG ADAETVKKKA KEFLNYINPR LPGLLELEYE GFYRRGFFVT KKKYAVIDEE
601	DKITTRGLEI VRRDWSEIAK ETQARVLEAI LKHGDVEEAV RIVKEVTEKL SRYEVPPEKL
661	VIYEQITRDL RDYRATGPHV AVAKRLAARG IKIRPGTVIS YIVLKGPGRV GDRAIPFDEF
721	DPAKHRYDAE YYIENQVLPA VERILRAFGY RKEDLRYQKT KQAGLGAWLK PKT
Amino acid sequences of additional chimeras of KOD and Pfu DNA polymerases.
Sequence 26 (SEQ ID NO: 26)
Amino acid sequence of chimeric DNA polymerase Kofu-II.
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 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 LEALLKDGDV EKAVRIVKEV TEKLSKYEVP
661	PEKLVIHEQI TRDLKDYKAT GPHVAVAKRL AARGVKIRPG TVISYIVLKG SGRIGDRAIP
721	FDEFDPTKHK YDAEYYIENQ VLPAVERILR AFGYRKEDLR YQKTRQVGLS AWLKPKGT
Sequence 27 (SEQ ID NO: 27)
Amino acid sequence of chimeric DNA polymerase Pod-II.
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 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 ETILKHGDVE EAVRIVKEVI QKLANYEIPP
661	EKLAIYEQIT RPLHEYKAIG PHVAVAKKLA AKGVKIKPGM VIGYIVLRGD GPISNRAILA
721	EEYDPKKHKY DAEYYIENQV LPAVLRILEG FGYRKEDLRY QKTRQVGLTS WLNIKKS
Sequence 28 (SEQ ID NO: 28)
Amino acid sequence of chimeric DNA polymerase Kofu-III.
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 YVKEPEKGLW ENIVYLDFRA LYPSIIITHN
421	VSPDTLNLEG CKNYDIAPQV GHKFCKDIPG FIPSLLGHLL EERQKIKTKM KETQDPIEKI
481	LLDYRQKAIK LLANSFYGYY GYAKARWYCK ECAESVTAWG RKYIELVWKE LEEKFGFKVL
541	YIDTDGLYAT IPGGESEEIK KKALEFLKYI NAKLPGALEL EYEGFYKRGF FVTKKKYAVI
601	DEEGKITTRG LEIVRRDWSE IAKETQARVL EALLKDGDVE KAVRIVKEVT EKLSKYEVPP
661	EKLVIHEQIT RDLKDYKATG PHVAVAKRLA ARGVKIRPGT VISYIVLKGS GRIGDRAIPF
721	DEFDPTKHKY DAEYYIENQV LPAVERILRA FGYRKEDLRY QKTRQVGLSA WLKPKGT
Sequence 29 (SEQ ID NO: 29)
Amino acid sequence of chimeric DNA polymerase Pod-III.
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 PMEIQLSRLV GQPLWDVSRS STGNLVEWFL
361	LRKAYERNEV APNKPSEEEY QRRLRESYTG GFVKEPERGL WENIVYLDFR SLYPSIIITH
421	NVSPDTLNRE GCKEYDVAPQ VGHRFCKDFP GFIPSLLGDL LEERQKIKKK MKATIDPIER
481	KLLDYRQRAI KILANSYYGY YGYARARWYC KECAESVTAW GREYITMTIK EIEEKYGFKV
541	IYSDTDGFFA TIPGADAETV KKKAMEFVKY INSKLPGLLE LEYEGFYKRG FFVTKKRYAV
601	IDEEGKVITR GLEIVRRDWS EIAKETQARV LETILKHGDV EEAVRIVKEV IQKLANYEIP
661	PEKLAIYEQI TRPLHEYKAI GPHVAVAKKL AAKGVKIKPG MVIGYIVLRG DGPISNRAIL
721	AEEYDPKKHK YDAEYYIENQ VLPAVLRILE GFGYRKEDLR YQKTRQVGLT SWLNIKKS

[0144] 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.

[0145] 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. Where the claims recite a composition, it should be understood that the invention encompasses methods of using the composition and methods of making the composition.

[0146] 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.