DNA Polymerases Having Improved Labeled Nucleotide Incorporation Properties

• US 20100311959A1
• Filed: 07/01/2010
• Published: 12/09/2010
• Est. Priority Date: 03/12/1997
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Brandis, John, Richards, John H., Bloom, Curtis
• Application Number:
  US12/829,335
• Publication Number:
  US 20100311959A1
• Time in Patent Office: Days
• US Class Current: 536/23.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates to mutant DNA polymerases that exhibit reduced discrimination against labeled nucleotides into polynucleotides. The DNA polymerases of the invention have at least one mutation in the nucleotide label interaction region of the enzyme such the mutation results in reduced discrimination against labeled nucleotides. The nucleotide label interaction regions is located at portions of the O-helix, (ii) the K helix, and (iii) the inter O—P helical loop of Taq DNA polymerase or analogous positions in other DNA polymerases. In addition to providing novel mutant DNA polymerases, the invention also provides polynucleotides encoding the subject mutant DNA polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerases. The invention also provides host cells containing the subject polynucleotides. The invention also includes numerous methods of using the subject DNA polymerases, including uses for chain termination sequencing and PCR. Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerase of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerase in the kit.

Claims

1. An isolated thermophilic Taq DNA polymerase having reduced discrimination against incorporating a nucleotide labeled with a fluorescein-type dye, the polymerase comprising an amino acid substitution of a glutamic acid at position 615 of SEQ ID NO:

4.

2. The isolated thermophilic DNA polymerase of claim 1, wherein the fluorescein-type dye is a 6-FAM, 5-FAM, TET, HEX, HOE, or ZOE dye.

3. The isolated thermophilic DNA polymerase of claim 1, wherein the nucleotide is a deoxynucleotide or a dideoxynucleotide.

4. The isolated thermophilic DNA polymerase of claim 1, wherein the dideoxynucleotide is a 2′

3′

-dideoxynucleotide.

5. The isolated thermophilic DNA polymerase of claim 1, wherein the amino acid substitution is in a region in the DNA polymerase that interacts with the labeled nucleotide.

6. The isolated thermophilic DNA polymerase of claim 5, wherein the region comprises a helical structure.

7. The isolated thermophilic DNA polymerase of claim 1, wherein the substitute amino acid is a non-polar amino acid.

8. The isolated thermophilic DNA polymerase of claim 7, wherein the non-polar amino acid is alanine, glycine, leucine, isoleucine, methionine, phenylalanine, proline, or valine.

The isolated thermophilic DNA polymerase of claim 1, wherein the substitute amino acid comprises a smaller side chain compared to the side chain of an amino acid in a corresponding non-mutated thermophilic DNA polymerase.

10. The isolated thermophilic DNA polymerase of claim 1, further comprising a glycine at position 46 substituted with aspartic acid to confer 3′

-5′

exonuclease activity.

11. A polynucleotide encoding the thermophilic DNA polymerase of claim 1.

14. A method of synthesizing a fluorescently labeled polynucleotide, comprising the steps of mixing the thermophilic DNA polymerase of claim 1 with a primed nucleic acid template and a labeled nucleotide.

18. A kit comprising the thermophilic DNA polymerase of claim 1.

9. The isolated thermophilic DNA polymerase of claim 9, wherein the substitute amino is alanine, aspartic acid, isoleucine, or lysine.

12. An expression vector comprising the polynucleotide of claim 12.

13. A host cell comprising the expression vector of claim 13.

15. The method of claim 15 which is a chain terminating sequencing reaction.

16. The method of claim 15 which is a polymerase chain reaction (PCR).

17. A fluorescently labeled polynucleotide produced by the method of claim 15.