DNA polymerases having improved labeled nucleotide incorporation properties

• US 6,265,193 B1
• Filed: 03/12/1998
• Issued: 07/24/2001
• Est. Priority Date: 03/12/1997
• Status: Expired due to Term

• Current Assignee: California Institute of Technology, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: California Institute of Technology, PQ Corporation (Ecovyst, Inc.)
• Inventors: Brandis, John, Bloom, Curtis, Richards, John H.
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): HUTSON, RICHARD G
• Application Number:
  US09/041,878
• Time in Patent Office: 1,230 Days
• Field of Search: 435/6, 435/194, 536/350
• US Class Current: 435/194
• 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 provide 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. A purified DNA polymerase having at least one mutation as defined with respect to a naturally occurring DNA polymerase, wherein the mutation is at an amino acid residue position selected from the group consisting of E520, A531, L522, R523, E524, A525, H526, P527, I528, V529, E530, K531, I532, R536, E537, R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612, Q613, R636, D637, T640, F647, V654, D655, P656, L657, R659, R660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832, wherein the position of said amino acid residue within the DNA polymerase is defined with respect to Taq DNA polymerase and wherein the DNA polymerase has at least 2 fold reduced discrimination for a fluorescein-type dye labeled nucleotide as compared with the naturally occurring DNA polymerase.

2. A DNA polymerase according to claim 1, wherein the mutation is at a position selected from the group consisting of, R595, D655, R660, T664 and E681.

3. A DNA polymerase according to claim 2, wherein the mutation is selected from the group consisting of R660D, D655L, E681G, and R595E.

4. A DNA polymerase according to claim 3, comprising a mutation set belonging to the gorup consisting of (G46D, R660D, F667Y), (G46D, R595D, R660D, F667Y), and (G46D, R660D, F667Y, E681G), and (G46D,F667Y, E681G).

5. A DNA polymerase according to claim 1, wherein the DNA polymerase is a thermostable DNA polymerase.

6. A kit for fluorescently labeling a polynucleotide, the kit comprising a DNA polymerase according to claim 1 and a fluorescent dye labeled nucleotide.

7. A DNA polymerase according to claim 5, wherein the naturally occurring DNA polymerase is Taq DNA polymerase.

8. A kit according to claim 6, wherein the DNA polymerase is a thermostable polymerase.

9. A kit according to claim 8, wherein the naturally occurring DNA polymerase is Taq DNA polymerase.

10. A DNA polymerase according to claim 5, wherein the mutation is at a position selected from the group consisting of, R595, D655, R660, T664 and E681.

11. A DNA polymerase according to claim 10, wherein the DNA polymerase is Taq DNA polymerase.

12. A DNA polymerase according to claim 11, wherein the mutation is selected from the group consisting of R660D, D655L, E681G, and R595E.

13. A DNA polymerase according to claim 12, wherein said DNA comprises a mutation set belonging to the group consisting of (G46D, R660D, F667Y), (G46D, R595D, R660D, F667Y), and (G46D, R660D, F667Y, E681G), and (G46D, F667Y, E681G).