DNA polymerases having improved labeled nucleotide incorporation properties

• US 20060088879A1
• Filed: 12/09/2005
• Published: 04/27/2006
• Est. Priority Date: 03/12/1997
• Status: Active Grant

• Current Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Brandis, John, Richards, John H., Bloom, Curtis
• Granted Patent: US 7,897,738 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-15. -15. (canceled)

16. A method of performing a chain terminating sequencing reaction, said method comprising,

forming a mixture comprising a first DNA polymerase, a second DNA polymerase, that is different in structure from the first DNA polymerase, a first fluorescent dye labeled terminator, and a second fluorescent dye labeled terminator, wherein the first DNA polymerase is receptive with respect to a first dye-labeled nucleotide and the second DNA polymerase is receptive with respect to a different dye-labeled nucleotide, and performing a chain terminating sequencing reaction.

17. The method of claim 16, wherein the first and second DNA polymerase are receptive with respect to nucleotides that differ with respect to one another by a nucleotide base.

18. The method of claim 17 wherein the first and second DNA polymerase are receptive with respect to nucleotides that differ with respect to one another by a fluorescent dye.

19. The method of claim 18 wherein the first and second DNA polymerase are receptive with respect to nucleotides that differ with respect to one another by a fluorescent dye and a nucleotide base.

20. The method according to claim 16, wherein the chain terminator sequencing reaction is a cycle sequencing reaction.

21. The method according to claim 20, wherein the sequencing reaction does not take place concurrently with a PCR amplification reaction.

22. The method of claim 16, wherein the first DNA polymerase and the second DNA polymerase are derived from the same naturally occurring DNA polymerase.

23. A method of performing a chain terminating sequencing reaction, said method comprising,

forming a mixture comprising a first DNA polymerase, a second DNA polymerase, first fluorescent dye labeled terminator, and a second fluorescent dye labeled terminator, wherein the first and second DNA polymerase are receptive with respect to nucleotides, and performing a chain terminating sequencing reaction, wherein the first and second DNA polymerase are receptive with respect to nucleotides that differ with respect to one another by a fluorescent dye.

24. The method of claim 23 wherein the first and second DNA polymerase are receptive with respect to nucleotides that differ with respect to one another by a fluorescent dye and a nucleotide base.