Thermus brockianus nucleic acid polymerases

Abstract

The invention provides nucleic acids and polypeptides for nucleic acid polymerases from a thermophilic organism, Thermus brockianus . The invention also provides methods for using these nucleic acids and polypeptides.

What is claimed is: 
     
         1 . An isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         2 . An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that decreases 5-3′ exonuclease activity. 
     
     
         3 . The isolated nucleic acid of  claim 2 , wherein the derivative nucleic acid polymerase has decreased 5-3′ exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         4 . An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 9-10 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         5 . The isolated nucleic acid of  claim 4 , wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         6 . An isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-8. 
     
     
         7 . An isolated nucleic acid comprising a nucleotide sequence complementary to any one of SEQ ID NO:1-8. 
     
     
         8 . An isolated nucleic acid encoding a nucleic acid polymerase from  Thermus brockianus  comprising amino acid sequence with at least 96% identity to any one of SEQ ID NO:9-16. 
     
     
         9 . A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         10 . A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that decreases 5-3′ exonuclease activity. 
     
     
         11 . The vector of  claim 10 , wherein the derivative nucleic acid polymerase has decreased 5-3′ exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         12 . A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 9-10 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         13 . The vector of  claim 12 , wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         14 . A vector comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-8. 
     
     
         15 . A vector comprising an isolated nucleic acid comprising a nucleotide sequence complementary to any one of SEQ ID NO:1-8. 
     
     
         16 . A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase from  Thermus brockianus  comprising amino acid sequence with at least 96% identity to any one of SEQ ID NO:9-16. 
     
     
         17 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         18 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that decreases 5-3′ exonuclease activity. 
     
     
         19 . The expression vector of  claim 18 , wherein the derivative nucleic acid polymerase has decreased 5-3′ exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         20 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 9-10 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         21 . The expression vector of  claim 20 , wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         22 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-8. 
     
     
         23 . An expression vector comprising a promoter operably linked to an isolated nucleic acid comprising a nucleotide sequence complementary to any one of SEQ ID NO:1-8. 
     
     
         24 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase from Thermos brockianus comprising amino acid sequence with at least 96% identity to any one of SEQ ID NO:9-16. 
     
     
         25 . A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         26 . A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that decreases 5-3′ exonuclease activity. 
     
     
         27 . The host cell of  claim 26 , wherein the derivative nucleic acid polymerase has decreased 5-3′ exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         28 . A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO: 9-10 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         29 . The host cell of  claim 28 , wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         30 . A host cell comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-8. 
     
     
         31 . A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase from  Thermus brockianus  comprising amino acid sequence with at least 96% identity to any one of SEQ ID NO:9-16. 
     
     
         32 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         33 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-49. 
     
     
         34 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that decreases 5-3′ exonuclease activity. 
     
     
         35 . The isolated nucleic acid polymerase of  claim 34 , wherein the derivative nucleic acid polymerase has decreased 5-3′ exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         36 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         37 . The isolated nucleic acid polymerase of  claim 36 , wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:9-10. 
     
     
         38 . A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises any one of amino acid sequences SEQ ID NO:9-49. 
     
     
         39 . A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises any one of amino acid sequences SEQ ID NO:9-16. 
     
     
         40 . The kit of  claim 38  or  39  further comprising a container containing an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer. 
     
     
         41 . The kit of  claim 38  or  39 , adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension. 
     
     
         42 . A method of making a nucleic acid polymerase comprising any one of SEQ ID NO:9-16 that comprises, incubating under conditions sufficient for RNA transcription and translation a host cell comprising a nucleic acid encoding a polypeptide comprising any one of SEQ ID NO:9-16 operably linked to a promoter. 
     
     
         43 . The method of  claim 42  wherein the nucleic acid comprises any one SEQ ID NO:1-8. 
     
     
         44 . A nucleic acid polymerase made by the method of  claim 42 . 
     
     
         45 . A method of synthesizing DNA comprising contacting a polypeptide comprising any one of SEQ ID NO:9-16 with a DNA under conditions sufficient to permit polymerization of DNA. 
     
     
         46 . A method of synthesizing DNA comprising contacting a polypeptide comprising any one of SEQ ID NO:9-49 with a DNA under conditions sufficient to permit polymerization of DNA. 
     
     
         47 . A method for thermocyclic amplification of nucleic acid comprising:
 (a) contacting a nucleic acid with a thermostable polypeptide having any one of SEQ ID NO:9-49 under conditions suitable for amplification of said nucleic acid; and   (b) amplifying the nucleic acid.   
     
     
         48 . The method of  claim 47  that further comprises cycling the temperature to denature nucleic acids, to renature a primer onto a template nucleic acid and to polymerize a nucleic acid strand complementary to the template nucleic acid. 
     
     
         49 . The method of  claim 47  wherein the thermocyclic amplification of DNA is performed by Strand Displacement Amplification. 
     
     
         50 . The method of  claim 47  wherein thermocyclic amplification of DNA is performed by Polymerase Chain Reaction. 
     
     
         51 . A method of primer extension comprising contacting a polypeptide comprising any one of SEQ ID NO:9-16 with a primer and a nucleic acid that is complementary to the primer under conditions sufficient to permit polymerization of DNA. 
     
     
         52 . The method of  claim 51  wherein the nucleic acid is DNA. 
     
     
         53 . The method of  claim 51  wherein the primer extension is done to sequence the nucleic acid. 
     
     
         54 . The method of  claim 51  wherein the primer extension is done to amplify the nucleic acid.