Thermus oshimai nucleic acid polymerases
AssigneeApplera Corporation
InventorsElena V. Bolchakova, James E. Rozzelle
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Abstract
The invention provides nucleic acids and polypeptides for a nucleic acid polymerase enzyme from a thermophilic organism, Thermus oshimai. The invention also provides methods for using these nucleic acids and polypeptides.
Claims46
1. An isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that decreases 5-3′ exonuclease activity.
An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
The isolated nucleic acid of claim 5, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5.
An isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
An isolated nucleic acid comprising a sequence complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that decreases 5-3′ exonuclease activity.
A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
A vector comprising an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that decreases 5-3′ exonuclease activity.
An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that decreases 5-3′ exonuclease activity.
A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
A host cell comprising an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
An isolated nucleic acid polymerase comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
An isolated derivative nucleic acid polymerase comprising SEQ ID NO:5 having a mutation that decreases 5-3′ exonuclease activity.
An isolated derivative nucleic acid polymerase comprising SEQ ID NO:5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.
A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises an amino acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
A method of making a nucleic acid polymerase comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 that comprises, incubating under conditions sufficient for RNA transcription and translation a host cell comprising a nucleic acid encoding a polypeptide comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 operably linked to a promoter.
A method of synthesizing DNA comprising contacting a polypeptide comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 with a DNA under conditions sufficient to permit polymerization of DNA.
A method for thermocyclic amplification of nucleic acid comprising:
- (a) contacting a nucleic acid with a thermostable polypeptide having SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 under conditions suitable for amplification of said nucleic acid;
- and
- (b) amplifying the nucleic acid.
A method of primer extending DNA comprising contacting a polypeptide comprising SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 with a nucleic acid under conditions sufficient to permit polymerization of DNA.
Dependencies are inferred from the claim text.