Thermus thermophilus nucleic acid polymerases

Abstract

The invention provides novel nucleic acid polymerases from strains GK24 and RQ-1 of Thermus thermophilus , and nucleic acids encoding those polymerases, as well as methods for using the polymerases and nucleic acids.

What is claimed: 
     
         1 . An isolated nucleic acid encoding a  Thermus thermophilus  strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase. 
     
     
         2 . The isolated nucleic acid of  claim 1  wherein the nucleic acid polymerase is a DNA polymerase. 
     
     
         3 . An isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-24. 
     
     
         4 . An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that decreases 5′-3′ exonuclease activity. 
     
     
         5 . The isolated nucleic acid of  claim 4 , 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:13-15. 
     
     
         6 . An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         7 . The isolated nucleic acid of  claim 6 , 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:13-15. 
     
     
         8 . An isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-12. 
     
     
         9 . An isolated nucleic acid comprising a nucleotide sequence complementary to any one of SEQ ID NO:1-12. 
     
     
         10 . A vector comprising an isolated nucleic acid encoding a  Thermus thermophilus  strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase. 
     
     
         11 . The vector of  claim 10  wherein the nucleic acid polymerase is a DNA polymerase. 
     
     
         12 . A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-24. 
     
     
         13 . A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that decreases 5′-3′ exonuclease activity. 
     
     
         14 . The vector of  claim 13 , 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:13-15. 
     
     
         15 . A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         16 . The vector of  claim 15 , 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:13-15. 
     
     
         17 . A vector comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-12. 
     
     
         18 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a  Thermus thermophilus  strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase. 
     
     
         19 . The expression vector of  claim 18  wherein the nucleic acid polymerase is a DNA polymerase. 
     
     
         20 . 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:13-24. 
     
     
         21 . 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:13-15 having a mutation that decreases 5′-3′ exonuclease activity. 
     
     
         22 . The expression vector of  claim 21 , 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:13-15. 
     
     
         23 . 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:13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         24 . The expression vector of  claim 23 , 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:13-15. 
     
     
         25 . An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-12. 
     
     
         26 . A host cell comprising an isolated nucleic acid encoding a  Thermus thermophilus  strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase. 
     
     
         27 . The host cell of  claim 26  wherein the nucleic acid polymerase is a DNA polymerase. 
     
     
         28 . A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-24. 
     
     
         29 . A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that decreases 5′-3′ exonuclease activity. 
     
     
         30 . The host cell of  claim 29 , 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:13-15. 
     
     
         31 . A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         32 . The host cell of  claim 31 , 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:13-15. 
     
     
         33 . A host cell comprising an isolated nucleic acid encoding a nucleic polymerase comprising any one of SEQ ID NO:1-12. 
     
     
         34 . An isolated nucleic acid polymerase from  Thermus thermophilus  strain RQ-1 (DSM catalog number 9247). 
     
     
         35 . The isolated nucleic acid polymerase of  claim 34  wherein the nucleic acid polymerase is a DNA polymerase. 
     
     
         36 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-24. 
     
     
         37 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that decreases 5′-3′ exonuclease activity. 
     
     
         38 . The isolated nucleic acid polymerase of  claim 37 , 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:13-15. 
     
     
         39 . An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates. 
     
     
         40 . The isolated nucleic acid polymerase of  claim 31 , 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:13-15. 
     
     
         41 . 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:13-24. 
     
     
         42 . The kit of  claim 41  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. 
     
     
         43 . The kit of  claim 41 , adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension. 
     
     
         44 . A method of making a nucleic acid polymerase comprising any one of SEQ ID NO:13-24 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:13-24 operably linked to a promoter. 
     
     
         45 . The method of  claim 44  wherein the nucleic acid comprises any one SEQ ID NO:1-12. 
     
     
         46 . A nucleic acid polymerase made by the method of  claim 44 . 
     
     
         47 . A method of synthesizing DNA comprising contacting a polypeptide comprising any one of SEQ ID NO:13-24 with a DNA under conditions sufficient to permit polymerization of DNA. 
     
     
         48 . 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: 13-24 under conditions suitable for amplification of said nucleic acid; and   (b) amplifying the nucleic acid.   
     
     
         49 . The method of  claim 48  wherein the thermocyclic amplification of the nucleic acid includes cycles of denaturation, primer annealing and primer extension. 
     
     
         50 . The method of  claim 48  wherein the thermocyclic amplification of the nucleic acid is performed by Strand Displacement Amplification. 
     
     
         51 . The method of  claim 48  wherein thermocyclic amplification of the nucleic acid is performed by Polymerase Chain Reaction. 
     
     
         52 . A method of primer extension comprising contacting a polypeptide comprising any one of SEQ ID NO:13-24 with a primer and a nucleic acid that is complementary to the primer under conditions sufficient to permit polymerization of DNA. 
     
     
         53 . The method of  claim 52  wherein the nucleic acid is DNA. 
     
     
         54 . The method of  claim 52  wherein the primer extension is done to sequence the nucleic acid. 
     
     
         55 . The method of  claim 52  wherein the primer extension is done to amplify the nucleic acid.