WO2025029707A2

WO2025029707A2 - Mutant dna polymerases and methods of use

Info

Publication number: WO2025029707A2
Application number: PCT/US2024/039979
Authority: WO
Inventors: Achim Karger; Remigijus Skirgaila; Dominyka ERLECKIENĖ; Gintarė TAULAVIČIŪTĖ; Karolina MUKAUSKAITĖ; Anushka JIRAPURE; Ramandeep Kaur; Jolanta VITKUTĖ; Anadelia VALLADARES POPOCA; Linlin Huang; Carole Bornarth
Original assignee: Life Technologies Corp
Current assignee: Life Technologies Corp
Priority date: 2023-07-28
Filing date: 2024-07-29
Publication date: 2025-02-06
Anticipated expiration: 2026-01-28
Also published as: WO2025029707A3

Abstract

The present invention provides mutant DNA polymerases, polynucleotides encoding the polymerases, cassettes and vectors including such polynucleotides, and cells containing the polymerases, polynucleotides, cassettes, and/or vectors of the invention. The present invention also provides methods for synthesizing polynucleotides and kits including a DNA polymerase of the invention.

Description

MUTANT DNA POLYMERASES AND METHODS OF USE CROSS-REFERENCE APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Number 63/516,396 filed on July 28, 2023, and U.S. Provisional Application Number 63/516,412 filed on July 28, 2023, and U.S. Provisional Application Number 63/516,435 filed on July 28, 2024 FIELD OF THE INVENTION [0002] The present invention is generally related to mutant DNA polymerases. BACKGROUND OF THE INVENTION [0003] DNA polymerases are enzymes that synthesize DNA molecules using a template DNA strand and a complementary synthesis primer annealed to a portion of the template. A detailed description of DNA polymerases and their enzymological characterization can be found in Kornberg (1989). [0004] The amino acid sequences of many DNA polymerases have been determined, and sequence comparisons between different DNA polymerases have identified many regions of homology between the different enzymes. Studies of the tertiary structures of DNA polymerases and amino acid sequence comparisons have revealed numerous structural similarities between diverse DNA polymerases. In general, DNA polymerases have a large cleft that is thought to accommodate the binding of duplex DNA. This cleft is formed by two sets of helices, the first set is referred to as the "fingers" region and the second set of helices is referred to as the "thumb" region. The bottom of the cleft is formed by anti-parallel beta sheets and is referred to as the "palm" region. Reviews of DNA polymerase structure can be found in Joyce and Steitz (1994). Computer readable data files describing the three-dimensional structure of some DNA polymerases have been publicly disseminated. [0005] DNA polymerases have a variety of uses in molecular biology techniques suitable for both research and clinical applications. Foremost among these techniques are DNA sequencing and polynucleotide amplification techniques such as the polymerase chain reaction (PCR). [0006] However, while widely used, available DNA polymerases can display any number of attributes that can decrease the enzyme's efficiency for synthesizing DNA, including: the polymerase may not efficiently read through all regions of the template; the polymerase may have decreased efficiency at higher salt concentrations; the polymerase may display 5'-3' nuclease activity; and/or the polymerase may discriminate against the efficient incorporation of fluorescently labeled nucleotides into the resulting DNA strand. [0007] Accordingly, there is a need for DNA polymerases having increased efficiency for synthesizing DNA molecules from, e.g., fluorescently labeled nucleotides. SUMMARY OF THE INVENTION [0008] Provided herein are mutant polymerases useful, e.g., for sequencing DNA. In some embodiments, the mutations of a mutant polymerase (1) increase polymerase speed relative to wild-type DNA polymerase; (2) increase affinity to DNA substrate; (3) increase resistance to a DNA polymerase inhibitor; (3) decrease 5'-3' nuclease activity; (4) allow for more efficient incorporation of fluorescently labeled nucleotides into the resulting DNA strand; (5) improve the ability of the polymerase to read through templates, e.g., with secondary structure; (6) increase resistance to higher salt concentrations; and/or (7) increase sequencing read lengths of DNA templates. [0009] Accordingly, certain embodiments of the present invention provide a mutant DNA polymerase including F667Y and G46D mutations and one or more substitutions selected from the group consisting of E681I, D732N, A743H, E507N, E742H, and M747K. In further embodiments, the mutant DNA polymerase includes the E189K substitution. In further embodiments, the mutant DNA polymerase includes the S543N substitution. [0010] Other embodiments of the present invention provide a mutant DNA polymerase including E189K, F667Y and G46D substitutions and one or more substitutions selected from the group consisting of D732N, E507N, S543N, E742H, and M747K. [0011] The invention also provides in certain embodiments polynucleotides encoding the polymerases of the invention, expression cassettes and vectors including such polynucleotides, and cells containing such polymerases and polynucleotides. [0012] Also provided are methods for synthesizing polynucleotides in a reaction, including contacting at least one polymerase of the invention with a primed template and nucleotides, e.g., fluorescently labeled nucleotides, under conditions effective to synthesize polynucleotides. The present invention in certain embodiments also provides kits containing packaging material and at least one polymerase of the invention. [0013] Also provided are methods for sequencing polynucleotides, e.g., sequencing a DNA sequence, using a polymerase of the invention. BRIEF DESCRIPTION OF THE FIGURES [0014] FIG.1 illustrates the protein yields obtains for various mutant DNA polymerases of the invention. Protein expression of all 20 mutant DNA polymerases and the control enzyme was successful with an average yield of 2.6 mg of active DNA polymerase activity (specific activity of 86,000 U/mg) per 1 L of bacterial culture. [0015] FIG.2 illustrates the ability of the mutant DNA polymerase of the invention to shorten the time of a thermocycling protocol. [0016] FIG.3 depicts the results obtained when assessing the effect of extension time on the success rate of sequencing reactions using the mutant DNA polymerases of the invention according to the BigDye Terminator (“BDT”) v3.1 thermocycling protocol. The extension time in the cycle sequencing was varied (5-60 seconds) using pGEM as the control plasmid in DNA replicate BDT v3.1 sequencing reactions, with both strands (forward (F) and reverse (R)). Performance was measured as Contiguous Read Length QV20 (recited as QV20 in the figure). Success rate was determined as compared to the specification limit (LSL=850 nt) of the run module (50 cm, POP-7 StdSeq). The data demonstrated that increasing extension time results in increasing average CRL, less variability in the read length, and increased success rate (vs. specification limit). [0017] FIG.4 depicts the results obtained when the polymerase speed of the mutant DNA polymerases of the invention was assessed in multiplex PCR in GeneAmp^TM PCR Buffer II. The maximum fragment length of the multiplex PCR agarose gel electrophoresis was recorded for each enzyme and each extension time. Three mutations (E507K, E742H, and E189K) conferred an approximately 3-fold increase in polymerase speed in the GeneAmp^TM PCR Buffer II. Two additional mutations (E681I and A743H) conferred a more modest approximately 1.5-fold increase in polymerase speed (dashed arrows). [0018] FIG.5 depicts the results obtained when identifying mutant DNA polymerase of the invention with high-speed polymerase activity. All mutant enzymes were used for sequencing plasmid DNA (pGEM) in both directions in ReadyReaction Sequencing (RR) buffer using very short (5 and 10 seconds) cycle sequencing extension times. Performance was measured as Contiguous Read Length QV20 for all reactions. The E189K mutation alone and in combination with additional mutations (E507K, E742H, S543N) confer significant increase in polymerase speed in dye-terminator sequencing as compared to a control DNA polymerase having the G46D and F667Y mutations. Polymerase speed assessment by sequencing confirms the results of the polymerase speed assessment by PCR. [0019] FIG.6 illustrates the sequencing read length of various mutant DNA polymerases of the invention using a 5 second extension time. The five fastest mutant DNA polymerases producing the longest read lengths with 5 seconds of thermocycler extension time all carried the E189K mutation. [0020] FIG. 7 depicts the results obtained when assessing the effect of the E189K, G46D, and F667Y mutations in the DNA polymerase on sequencing speed. For 5 of the 6 mutant DNA polymerases that were tested, the presence of the E189K mutation resulted in unexpectedly longer sequencing read lengths with short 5 and 10 seconds of thermocycler extension time. [0021] FIG.8 illustrates the effect of the E189K, G46D, and F667Y mutations in the DNA polymerase on peak quality. In a side-by-side comparison, in the case of five of six mutant DNA polymerases of the invention tested, the addition of the E189K mutation produced better peak quality (as measured by Trace Score) with short 5 and 10 seconds of thermocycler extension time. [0022] FIG.9 illustrates that mutant DNA polymerases of the invention can generate high quality sequencing data with control plasmid template DNA. Sequencing quality of the mutant DNA polymerases of the invention were compared with BDT^TM v3.1 (control) using pGEM as the control plasmid DNA and 5x ReadyReaction Buffer 3.1. All 21 mutant DNA polymerases tested generated high quality sequencing data. Depicted in this figure are the sequencing results of the E507K, G46D, F667Y mutant DNA polymerase. [0023] FIG.10 illustrates the results observed when mutant DNA polymerases of the invention were used in connection with difficult target templates as compared to the BDT v3.1 DNA polymerase. Plasmids pGEM (control) and p4009-1 (difficult template) were sequenced in replicate reactions, both strands (F and R) using BDT v3.1. Performance was measured as Contiguous Read Length QV20. Success rate was determined as percent reactions meeting the specification (LSL=850 nt) for the run module (50 cm POP-5 StdSeq). As compared to a regular DNA template, difficult-to-sequence DNA template results reduced average CRL, increased variability of read length, and a decrease in success rate of sequencing reactions (vs. specification limit). [0024] FIG. 11 illustrates the read length (CRL QV-20) analysis of BDT v3.1 DNA polymerase versus the mutant DNA polymerases of the invention. Two regular (pGEM, AAV400-polyA-v01) and 7 difficult DNA templates were sequenced in replicate reactions, both strands (F and R) using BDT v3.1 and six mutant DNA polymerases of the invention carrying the E189K mutation. Performance was measured as the average base quality TraceScore. DNA polymerases carrying the E189K mutation generally provided higher or equal read lengths than the BDT v3.1 DNA polymerase for all 9 DNA templates tested. [0025] FIG. 12 illustrates the base quality (TraceScore) analysis of BDT v3.1 DNA polymerase versus the mutant DNA polymerases of the invention. Two regular (pGEM, AAV400-polyA-v01) and 7 difficult DNA templates were sequenced in replicate reactions, both strands (F and R) using BDT v3.1 and six mutant DNA polymerases of the invention carrying the E189K mutation. Performance was measured as the average base quality TraceScore. DNA polymerases carrying the E189K mutation provided higher or equal average base quality score than BDT v3.1 for all 9 DNA templates tested. [0026] FIG.13 illustrates the read length (CRL QV-20) observed with 2 mutant DNA polymerases of the invention as compared to BDT v3.1 using 2 regular and 7 difficult DNA templates. The mutant DNA polymerases of the invention perform equal to or better than BDT v3.1 for all 9 DNA templates tested. [0027] FIG.14 illustrates the effects of additional mutations (D732N, E507K, E742H, M747K, and S543N) in the E189K, G46D, F667Y mutant DNA polymerase on sequencing read length for 2 different plasmid DNA templates. For all 6 mutant DNA polymerases tested, the presence of the E189K mutation resulted in longer sequencing read lengths with regular (240 seconds) thermocycler extension time on both plasmid DNA templates. [0028] FIG. 15 illustrates the effect of additional mutations (D732N, E507K, E742H, M747K, and S543N) in the E189K, G46D, F667Y mutant DNA polymerase on sequencing read length for pGEM-3Zf(+) sequencing standard plasma DNA template. For 5 of the 6 mutant DNA polymerases of the invention that were tested, the presence of the E189K mutation resulted in longer sequencing read lengths with regular (240 seconds) thermocycler extension time. [0029] FIG. 16 illustrates the effect of additional mutations (D732N, E507K, E742H, M747K, and S543N) in the E189K, G46D, F667Y mutant DNA polymerase on sequencing read length for the p4009-1 plasmid DNA template (“difficult-to-sequence” template). For 5 of the 6 mutant DNA polymerases of the invention that were tested, the presence of the E189K mutation resulted in longer sequencing read lengths with regular (240 seconds) thermocycler extension time. [0030] FIG.17 illustrates the multiplex amplification of 15 DNA targets of varying length by DNA mutant polymerases of the invention. [0031] FIG.18 illustrates 1.3 KB amplification from λDNA by DNA mutant polymerases of the invention. DETAILED DESCRIPTION [0032] Described herein are polymerases that combine mutations to produce an enhanced polymerase useful, e.g., for sequencing DNA. In some embodiments, these mutations (1) increase polymerase speed relative to wild-type DNA polymerase; (2) increase affinity to DNA substrate; (3) increase resistance to a DNA polymerase inhibitor; (3) decrease 5'-3' nuclease activity; (4) allow for more efficient incorporation of fluorescently labeled nucleotides into the resulting DNA strand; (5) improve the ability of the polymerase to read through templates, e.g., with secondary structure; (6) increase resistance to higher salt concentrations; and/or (7) increase sequencing read lengths of DNA templates. [0033] Accordingly, certain embodiments of the present invention provide a mutant DNA polymerase including an amino acid substitution at amino acid position 189 and one or more substitutions selected from the group consisting of G46D, S543N, F667Y, D732N, E742H, and M747K. In some embodiments, the amino acid substitution at amino acid position is a Lys residue. In some embodiments, the mutant DNA polymerase includes E189K, S543N, F667Y, and E742H substitutions. In further embodiments, the mutant DNA polymerase includes an E507K substitution. [0034] Certain embodiments of the present invention provide a mutant DNA polymerase including F667Y and G46D substitutions and one or more substitutions selected from the group consisting of E681I, D732N, A743H, E507N, E742H, and M747K. [0035] Other embodiments of the present invention provide a mutant DNA polymerase including E189K, F667Y and G46D substitutions and one or more substitutions selected from the group consisting of D732N, E507N, S543N, E742H, and M747K. [0036] The DNA polymerase may be a thermostable Taq DNA polymerase. In certain embodiments, the DNA polymerase may include SEQ ID NO: 3-8. [0037] The present invention also provides polynucleotides encoding the polymerases of the invention, and cassettes and vectors including such polynucleotides. The polynucleotide may be operably linked to a promoter. Also provided are cells containing the polymerases, polynucleotides, cassettes, and/ or vectors of the invention. [0038] A wild type polymerase from Thermus aquaticus is SEQ ID NO: 1. A nucleotide sequence encoding such a wild type polymerase is SEQ ID NO: 2. (see accession number 104636) (SEQ ID NO: 1) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0039] In the sequence below, the start codon (atg) at position 121 is underlined. Also underlined are codons that may be mutated in some embodiments of the invention to produce a polymerase of the invention. (SEQ ID NO: 2) 1 aagctcagat ctacctgcct gagggcgtcc ggttccagct ggcccttccc gagggggaga 61 gggaggcgtt tctaaaagcc cttcaggacg ctacccgggg gcgggtggtg gaagggtaac 121 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 181 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 241 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 301 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 361 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 421 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 481 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 541 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 601 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 661 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 721 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 781 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 841 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 901 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 961 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 1021 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 1081 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1141 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1201 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1261 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1321 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1381 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1441 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1501 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1561 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1621 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1681 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1741 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1801 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1861 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1921 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1981 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 2041 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 2101 gcggccaaga ccatcaac ttcggggtcctc tacggcatgt cggcccaccg cctctcccag 2161 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2221 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2281 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2341 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2401 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2461 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2521 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2581 gggatag gggaggactg gctctccgcc aaggagtgat accacc [0040] A mutant DNA polymerase of the invention (E189K, G46D, F667Y; SEQ ID NO: 3) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 3) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0041] A mutant DNA polymerase of the invention (E189K, G46D, E507K, F667Y; SEQ ID NO: 4) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 4) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTKKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0042] A mutant DNA polymerase of the invention (E189K, G46D, S543N, F667Y; SEQ ID NO: 5) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 5) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKNTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0043] A mutant DNA polymerase of the invention (E189K, G46D, F667Y, D732N; SEQ ID NO: 6) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 6) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPNLEARVKSVREAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0044] A mutant DNA polymerase of the invention (E189K, G46D, F667Y, E742H; SEQ ID NO: 7) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 7) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRHAAERMAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0045] A mutant DNA polymerase of the invention (E189K, G46D, F667Y, M747K; SEQ ID NO: 8) is provided below. Mutated amino acids are underlined below. (SEQ ID NO: 8) MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYDFAKSLLKALKEDGDAVIVVFDAK APSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRI LTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDKSDNLPGVKGIGEKTARKL LEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERL EFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDL KEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFA NLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFN LNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDL IHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAH LSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINYGVLYGMSAHRLSQELAIPYEEAQ AFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERKAFNMPVQGTAAD LMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWL SAKE [0046] Certain embodiments of the present invention also provide methods for synthesizing a polynucleotide in a reaction, including contacting at least one DNA polymerase of the invention with a primed template and nucleotides. The reaction may be, e.g., a chain termination sequencing reaction or a polymerase chain reaction. The nucleotides may include labeled nucleotides, e.g., fluorescently labeled nucleotides. [0047] Such polymerase chain reactions may be useful in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses, and diagnosis of genetic disorders. [0048] Certain embodiments of the present invention also provide kits including packaging material and a DNA polymerase of the invention. The kit may contain nucleotides, e.g., labeled nucleotides, e.g., fluorescently labeled nucleotides. The kits may also include unlabeled nucleotides. The kits may also include at least one primer. [0049] Thus, a new polymerase has been developed that combines mutations to produce an enhanced polymerase useful, e.g., in DNA sequencing. These mutations may include: G46D, which reduces, e.g., eliminates, the 5'-3' nuclease activity; F667Y, which allows more efficient incorporation of dideoxy nucleotides; S543N, which enhances the processivity of the polymerase. S543N also improves the ability of the polymerase to read through regions in templates with secondary structure that would normally disrupt the sequencing ability of the polymerase. In addition, the S543N mutation enhances the salt tolerance of the polymerase. [0050] Thus, methods utilizing certain polymerases of the invention will demonstrate a reduction in failures in sequencing due to template secondary structure. Certain polymerases also have increased salt tolerance, which reduces sensitivity of the polymerase to salts, e.g., carried over from template preparations or from PCR reactions. Use of certain polymerases also reduces the number of false stops in dye primer reactions. The mutations in certain polymerases also improve the ability of polymerases of the invention to tolerate dITP and dUTP in the extending strand. [0051] The polymerases of the invention could be used to make, e.g., dye terminator sequencing kits or dye-labeled primer kits. The polymerases of the invention could also be used in, e.g., direct PCR sequencing chemistry, e.g., in combination with a polymerase without the F667Y mutation. In some embodiments of the invention, the polymerases of the invention may be used, e.g., with dye-labeled primers and/or dye- labeled terminators, e.g., to perform simultaneous amplification and sequencing. [0052] Thus, embodiments of the invention include the mutant polymerases and polynucleotide sequences encoding the mutant polymerases. Polynucleotide sequences encoding the mutant polymerases of the invention may be used for the recombinant production of the mutant polymerases. Polynucleotide sequences encoding mutant polymerases may be produced by a variety of methods. One method of producing polynucleotide sequences encoding mutant polymerases is by using site- directed mutagenesis to introduce desired mutations into polynucleotides encoding the parent, wildtype polymerase. [0053] Polynucleotides encoding the mutant polymerases of the invention may be used for the recombinant expression of the mutant polymerases. Generally, the recombinant expression of the mutant polymerase is effected by introducing a polynucleotide encoding a mutant polymerase into an expression vector adapted for use in particular type of host cell. Thus, another aspect of the invention is to provide vectors including a polynucleotide encoding a mutant polymerase of the invention, such that the polymerase encoding polynucleotide is functionally inserted into the vector. The invention also provide host cells that include the vectors of the invention. Host cells for recombinant expression may be prokaryotic or eukaryotic. Example of host cells include bacterial cells, yeast cells, cultured insect cell lines, and cultured mammalian cells lines. A wide range of vectors, e.g., expression vectors, are well known in the art, and the expression of polymerases in recombinant cell systems is a well-established technique. [0054] The invention also provides kits for synthesizing polynucleotides, e.g., fluorescently labeled polynucleotides. The kits may be adapted for performing specific polynucleotide synthesis procedures such as DNA sequencing or PCR. Kits of certain embodiments of the invention include a mutant DNA polymerase of the invention. Kits preferably contain instructions on how to perform the procedures for which the kits are adapted. Optionally, the kits may further include at least one other reagent for performing the method the kit is adapted to perform. Examples of such additional reagents include labeled nucleotides, unlabeled nucleotides, buffers, cloning vectors, restriction endonucleases, sequencing primers, and amplification primers. The reagents include in the kits of the invention may be supplied in premeasured units so as to provide for greater precision and accuracy. [0055] The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity." [0056] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a segment of or the entirety of a specified sequence. [0057] As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the sequences. Generally, the comparison window is at least 5, 10 or 20 contiguous nucleotides or polypeptide in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide or polypeptide sequence, a gap penalty can be introduced and is subtracted from the number of matches. [0058] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CABIOS, 4: 11 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, JMB, 48:443 (1970); the search-for-similarity method of Pearson and Lipman, PNAS, 85:2444 (1988); the algorithm of Karlin and Altschul, PNAS, 87:2264 (1990), modified as in. Karlin and Altschul, PNAS, 90:5873 (1993). [0059] Computer implementation of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program and GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package. Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al., Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151 (1989); Carpet et al., Nucl. Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155 (1992); and Pearson et al., Meth. Mal. Biol., 24:307 (1994). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., JMB, 215:403 (1990); Nucl. Acids Res., 25:3389 (1990), are based on the algorithm of Karlin and Altschul supra. [0060] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm generally involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. [0061] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test polynucleotide sequence to the reference polynucleotide sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. [0062] To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25:3389 (1997). Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See http://www.ncbi.nlm.nih.gov. Alignments may also be performed manually by inspection. [0063] For purposes of the present invention, comparison of sequences for determination of percent sequence identity to the sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters, or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. [0064] As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). [0065] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical polynucleotide base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. [0066] The term "substantial identity" of sequences means that a sequence includes a sequence that has at least about 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91 %, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. [0067] Another indication that sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1 ° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. [0068] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. [0069] As noted above, another indication that two sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe polynucleotide and a target polynucleotide and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence. [0070] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of polynucleotide hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of posthybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267 (1984); Tm 81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1 ° C. for each 1 % of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of polynucleotides is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes, part I chapter 2 "Overview of principles of hybridization and the strategy of polynucleotide probe assays" Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. [0071] An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2xSSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lxSSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6xSSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. and at least about 60° C. for long probes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Polynucleotides that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a polynucleotide is created using the maximum codon degeneracy permitted by the genetic code. [0072] Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1 % SDS at 37° C., and a wash in 0.lxSSC at 60 to 65° C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, IM NaCl, 1 % SDS (sodium dodecyl sulphate) at 37° C., and a wash in lx to 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1 % SDS at 37° C., and a wash in 0.5x to lxSSC at 55 to 60° C. [0073] Thus, certain embodiments of the present invention are directed to polynucleotide and polypeptide sequences that specifically hybridize to, or are substantially identical to the polypeptide sequences of the polymerases of the invention and the polynucleotide sequences that encode such polypeptide sequences. The activity of such polymerases may be determined using assays known to the art worker. [0074] The polymerases of certain embodiments of the invention include polymerases with substitutions of at least one amino acid residue in the polypeptide. In some embodiments of the invention, amino acid substitutions falling within the scope of the invention include those that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: [0075] (1) hydrophobic: norleucine, met, ala, val, leu, ile; [0076] (2) neutral hydrophilic: cys, ser, thr; [0077] (3) acidic: asp, glu; [0078] (4) basic: asn, gin, his, lys, arg; [0079] (5) residues that influence chain orientation: gly, pro; and [0080] (6) aromatic; trp, tyr, phe. [0081] Substitution of like amino acids can also be made on the basis of hydrophilicity. As detailed in U.S. Pat. No.4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine ( +3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine ( +0.3); asparagine ( +0.2); glutamine ( +0.2); glycine (0); praline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In such changes, the substitution of amino acids whose hydrophilicity values can be within ±2, within ±1, or within ±0.5. [0082] In one embodiment of the invention, the polymerase has a conservative amino acid substitution, for example, aspartic-glutamic as acidic amino acids; lysine/ arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid substitutions also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. [0083] Exemplary substitutions include those in Table 1. TABLE 1 Original Residue Exemplary Substitutions Ala Gly; Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg Met Met; Leu; Tyr Ser Thr; Ala; Leu Thr Ser; Ala Trp Tyr Tyr Trp; Phe Val Ile; Leu [0084] After the substitutions are introduced, the resulting polymerase can be screened for activity by the art worker using assays known to the art worker. [0085] Positions of amino acid residues within a DNA polymerase are indicated by either numbers or number/letter combinations. The numbering starts at the amino terminus residue. The letter is the single letter amino acid code for the amino acid residue at the indicated position in the naturally occurring polymerase from which the mutant is derived. Unless specifically indicated otherwise, an amino acid residue position designation should be construed as referring to the analogous position in all DNA polymerases, even though the single letter amino acid code specifically relates to the amino acid residue at the indicated position in Taq DNA polymerase. [0086] Individual substitution mutations are indicated by the form of a letter/number/letter combination. The letters are the single letter code for amino acid residues. The numbers indicate the amino acid residue position of the mutation site. The numbering system starts at the amino terminus residue. The numbering of the residues in Taq DNA polymerase is as described in U.S. Pat. No.5,079,352. Amino acid sequence homology between different DNA polymerases permits corresponding positions to be assigned to amino acid residues for DNA polymerases other than Taq. Unless indicated otherwise, a given number refers to position in Taq DNA polymerase. The first letter, i.e., the letter to the left of the number, represents the amino acid residue at the indicated position in the non-mutant polymerase. The second letter represents the amino acid residue at the same position in the mutant polymerase. For example, the term "R660D" indicates that the arginine at position 660 has been replaced by an aspartic acid residue. [0087] Genes encoding DNA polymerases have been isolated and sequenced. This sequence information is available on publicly accessible DNA sequence databases such as GENBANK. A compilation of the amino acid sequences of DNA polymerases from a range of organism can be found in Braithwaite and Ito (1993). This information may be used in designing various embodiments of polymerases of the invention and polynucleotides encoding these polymerases. The publicly available sequence information may also be used to clone genes encoding DNA polymerases through techniques such as genetic library screening with hybridization probes. EXAMPLE 1 MULTIPLEX AMPLIFICATION [0088] Amplification of 15 targets (99, 131, 160, 199, 251, 300, 345, 400, 516, 613, 735, 908, 1,005, 1,190, and 1,606 bp) was performed from 200 ng of human genomic DNA, in 50 μL reaction containing 100 nM of each primer, 1x Platinum™ II PCR buffer, 4 U of polymerase. The cycling protocol was: 1 cycle at 94°C for 2 min; 35 cycles at 94°C for 15 sec, 60°C for 30 sec, and 68°C for 96 sec. Different time for elongation step was tested to determine faster variants of the polymerase: 1.68°C for 96 sec.2.68°C for 60 sec.3.68°C for 30 sec.4.68°C for 10 sec. Primer sequences are provided in Table 2. The “Mut 4” DNA polymerase carries the G46D, F667Y, and E507K mutations. The “Mut 9” DNA polymerase carries the G46D, F667Y, and D732N mutations. The “Mut 10” DNA polymerase carries the G46D, Y667Y, and E742H mutations. The “Mut 12” DNA polymerase carries G46D, F667Y, and M747K mutations. The “Mut 15” DNA polymerase carries the G46D, F667Y and E189K mutations. The “Mut 16” DNA polymerase carries the G46D, F667Y, E189K, and E507K mutations. The “Mut 17” DNA polymerase carries the G46D, F667Y, E189K, and S542N mutations. The “Mut 18” DNA polymerase carries the G46D, F667Y, E189K, and D732N mutations. The “Mut19” DNA polymerase carries the G46D, F667Y, E189K, and E742H mutations. The “Mut 21” DNA polymerase carries the G46D and F667Y mutations. TABLE 2 S^{EQ ID NO.} _{Primer name Primer sequence} ‘

17 85-Dir 5‘-TCTGGACGGGCATCTCAAGT 18 86-Rev 5‘-TTCACAGGAAGCACTCACCA

EXAMPLE 2 1.3 KB AMPLIFICATION FROM λDNA [0089] Amplification of a 1.3 kb fragment was performed from 10 ng of λ DNA, in 50 μL reaction containing 400 nM of each primer, 1x Platinum™ II PCR buffer, 4 U of polymerase. The cycling protocol was: 1 cycle at 94°C for 2 min; 25 cycles at 94°C for 15 sec, 60°C for 15 sec, and 68°C for 60 sec. Different time for elongation step was tested to determine faster variants of the polymerase: 1.68°C for 60 sec.2.68°C for 30 sec.3.68°C for 15 sec.4.68°C for 0 sec. Primers sequences provided in the Table 3. The “Mut 4” DNA polymerase carries the G46D, F667Y, and E507K mutations. The “Mut 9” DNA polymerase carries the G46D, F667Y, and D732N mutations. The “Mut 10” DNA polymerase carries the G46D, Y667Y, and E742H mutations. The “Mut 12” DNA polymerase carries G46D, F667Y, and M747K mutations. The “Mut 15” DNA polymerase carries the G46D, F667Y and E189K mutations. The “Mut 16” DNA polymerase carries the G46D, F667Y, E189K, and E507K mutations. The “Mut 17” DNA polymerase carries the G46D, F667Y, E189K, and S542N mutations. The “Mut 18” DNA polymerase carries the G46D, F667Y, E189K, and D732N mutations. The “Mut19” DNA polymerase carries the G46D, F667Y, E189K, and E742H mutations. The “Mut 21” DNA polymerase carries the G46D and F667Y mutations. TABLE 3 S^{EQ ID NO.} _{Primer name Primer sequence} 39 Lambda_1.3_F 5‘-GTCACCAGTGCAGTGCTTGATAACAGG

Claims

WHAT IS CLAIMED IS: 1. A Thermus (Taq) DNA polymerase, wherein the Taq DNA polymerase comprises F667Y and G46D mutations and at least one substitution selected from the group consisting of E681I, D732N, A743H, E507N, E742H, and M747K, and wherein the Taq DNA polymerase does not retain 5’ to 3’ exonuclease activity.

2. A Thermus (Taq) DNA polymerase, wherein the Taq DNA polymerase comprises E189K, F667Y, and G46D mutations and at least one substitution selected from the group consisting of D732N, E507N, S543N, E742H, and M747K, and wherein the Taq DNA polymerase does not retain 5' to 3' exonuclease activity.

3. The DNA polymerase according to any of claims 1-2, having 5 or fewer of said substitutions.

4. The DNA polymerase according to any of claims 1-3, having 4 or fewer of said substitutions.

5. The DNA polymerase according to any of claims 1-4, having 3 or fewer of said substitutions.

6. The DNA polymerase according to any of claims 1-5, having 2 of said substitutions.

7. The DNA polymerase according to any of claims 1-6, wherein the DNA polymerase further comprises the E189K substitution.

8. The DNA polymerase according to any of claims 1-7, wherein the DNA polymerase further comprises the S543 substitution.

9. A polynucleotide comprising a sequence encoding the DNA polymerase of any of claims 1-7.

10. A vector comprising the polynucleotide of claim 9.

11. The vector of claim 9, further comprising a promoter operably linked to the polynucleotide.

12. A cell comprising the DNA polymerase of any of claims 1-8.

13. A cell comprising the polynucleotide of claim 9.

14. A cell comprising the vector of claim 10.

15. A method for synthesizing a polynucleotide in a reaction, comprising contacting the DNA polymerase of any of claims 1-8 with a primed template and nucleotides.

16. The method of claim 15, wherein the reaction is a chain termination sequencing reaction.

17. The method of claim 15, wherein the reaction is a polymerase chain reaction.

18. The method of claim 15, wherein the nucleotides comprise labeled nucleotides.

19. The method of claim 18, wherein the labeled nucleotides are fluorescently labeled nucleotides.

20. A kit for nucleic acid amplification, which comprises a DNA polymerase according to any of claims 1-8 and one or more reagents for a DNA synthesis reaction under conditions to synthesize a new DNA complementary to the target DNA.

21. The kit of claim 20, further comprising labeled nucleotides.

22. The kit of claim 21, wherein the labeled nucleotides are fluorescently labeled nucleotides.

23. The kit of claim 22, further comprising unlabeled nucleotides.

24. The kit of claim 20, further comprising at least one primer.

25. A method for determining a nucleic acid sequence of a nucleic acid molecule comprising: (a) contacting a nucleic acid molecule with a primer capable of hybridizing to the nucleic acid molecule, a ddNTP, and a Taq DNA polymerase of any of claims 1-8, (b) hybridizing the primer to the nucleic acid molecule; (c) incorporating a ddNTP at the 3’ end of the primer to form an extended primer product; and (d) determining the nucleic acid sequence of the nucleic acid molecule based on the ddNTP incorporated at the 3’ end of the extended primer product.

26. The method of claim 25, wherein the ddNTP is a ddATP, ddTTP, ddCTP, ddGTP, ddUTP, derivatives thereof, or combinations thereof.

27. The method of claim 25, wherein the ddNTP is fluorescently labeled.

28. The method of claim 25, wherein the method further comprises a combination of dNTPs, wherein the combination of dNTPs is selected from one or more of dATP, dGTP, dCTP, dTTP, dUTP, dTTP, or derivatives thereof.

29. The method of claim 25, wherein the determining includes separating the extended primer product based on molecular weight and/or capillary electrophoresis.

30. The method of claim 25, wherein the nucleic acid sequence of the nucleic acid molecule is determined by Sanger sequencing.

31. The method of claim 25, wherein the nucleic acid sequence of the nucleic acid molecule is determined by PCR.