Polymerases with charge-switch activity and methods of generating such polymers

• US 20040259082A1
• Filed: 04/24/2002
• Published: 12/23/2004
• Est. Priority Date: 04/24/2001
• Status: Abandoned Application

• Current Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G. K.
• Application Number:
  US10/131,998
• Publication Number:
  US 20040259082A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

This invention provides DNA polymerases with mutations in the charge-switch nucleotide interaction region that increase activity for charge-switch nucleotides. Such polymerases can be generated by introducing mutations in specific residues which are identified as being in the appropriate region through structural models, by homology to polymerases with known structures, or experimental analysis. In some embodiments, the mutant DNA polymerases have additional mutations that decrease activity for non-charge-switch nucleotides and mutations that decrease exonuclease activity. In another aspect, the invention provides methods of sequencing a target nucleic acid with the above described mutated DNA polymerases. In yet another aspect, the invention provides methods of generating polypeptides having charge-switch nucleotide polymerase activity by introducing “random” mutations and selecting those mutated polypeptides that encode polypeptides having charge-switch nucleotide activity

Claims

1-36. -36. Cancel

37. A purified mutant DNA polymerase having at least one mutation in a nucleotide γ

-phosphate region, wherein said mutant DNA polymerase has increased activity for a γ

-phosphate labeled nucleotide compared to a naturally occurring DNA polymerase, and wherein said mutant DNA polymerase is capable of synthesizing DNA at a rate of at least 1 nucleotide per second.

38. The purified mutant DNA polymerase of claim 37, wherein said increased activity for said γ

-phosphate labeled nucleotide is increased by at least 2-fold.

39. The purified mutant DNA polymerase of claim 37, wherein said polymerase has decreased activity for a non-γ

-phosphate labeled nucleotide.

40. The purified mutant DNA polymerase of claim 39, wherein said decreased activity for said non-γ

-phosphate labeled nucleotide is about 2-fold to about 20-fold.

41. The purified mutant DNA polymerase of claim 37, wherein said mutant DNA polymerase has decreased exonuclease activity but retains strand displacement activity.

42. The purified mutant DNA polymerase of claim 37, wherein said mutant DNA polymerase has at least two mutations.

43. The purified mutant DNA polymerase of claim 37, wherein said mutant DNA polymerase has at least three mutations.

44. A purified mutant DNA polymerase having at least one mutation in a nucleotide γ

-phosphate region, wherein said mutant DNA polymerase has increased activity for a γ

-phosphate labeled nucleotide compared to a naturally occurring DNA polymerase, and wherein said mutant DNA polymerase is capable of synthesizing DNA at a rate of at least 10 nucleotides per second.

45. The purified mutant DNA polymerase of claim 44, wherein said increased activity for said γ

-phosphate labeled nucleotide is increased by at least 2-fold.

46. The purified mutant DNA polymerase of claim 44, wherein said polymerase has decreased activity for a non-γ

-phosphate labeled nucleotide.

47. The purified mutant DNA polymerase of claim 46, wherein said decreased activity for said non-γ

-phosphate labeled nucleotide is about 2-fold to about 20-fold.

48. The purified mutant DNA polymerase of claim 44, wherein said mutant DNA polymerase has decreased exonuclease activity but retains strand displacement activity.

49. The purified mutant DNA polymerase of claim 44, wherein said mutant DNA polymerase has at least two mutations.

50. The purified mutant DNA polymerase of claim 44, wherein said mutant DNA polymerase has at least three mutations.

51. A purified mutant DNA polymerase having at least one mutation in a nucleotide γ

-phosphate region, wherein said mutant DNA polymerase has increased activity for a nucleotide coupled to a detectable moiety at a γ

-phosphate compared to a naturally occurring DNA polymerase, and wherein said mutation is found in regions of a nucleotide binding pocket of said DNA polymerase, wherein said pocket interacts with said detectable moiety of the nucleotide.

52. The purified mutant DNA polymerase of claim 51, wherein said detectable moiety is selected from the group consisting of PPi-Dye, PP—

F, P-Dye and P—

F.

53. The purified mutant DNA polymerase of claim 51, wherein said detectable moiety is a phosphate detectable moiety that is cleaved from γ

-labeled dNTPs.

54. The purified mutant DNA polymerase of claim 51, wherein said increased activity for a nucleotide coupled to a detectable moiety at a γ

-phosphate is increased by at least 2-fold.

55. The purified mutant DNA polymerase of claim 51, wherein said polymerase has decreased activity for a non-γ

-phosphate labeled nucleotide.

56. The purified mutant DNA polymerase of claim 55, wherein said decreased activity for said non-γ

-phosphate labeled nucleotide is about 2-fold to about 20-fold.