Compositions and methods utilizing DNA polymerases
First Claim
Patent Images
1. A composition for identifying a nucleotide at a given position of a template DNA molecule, said composition comprising:
- a Family B DNA polymerase deficient in 3′
to 5′
exonuclease activity comprising the amino acid sequence of one of SEQ ID NOs;
2 and 62-78, excepta first mutation at amino acid D or E or both in the exo I (DXE) motif within said SEQ ID NO, anda second mutation at an amino acid selected from the group consisting of;
amino acid L, P, and both L and P in Region II (DXXSLYPSII, SEQ ID NO;
7) within said SEQ ID NO, wherein said second mutation confers a reduction in discrimination.
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Abstract
The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.
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Citations
95 Claims
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1. A composition for identifying a nucleotide at a given position of a template DNA molecule, said composition comprising:
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a Family B DNA polymerase deficient in 3′
to 5′
exonuclease activity comprising the amino acid sequence of one of SEQ ID NOs;
2 and 62-78, excepta first mutation at amino acid D or E or both in the exo I (DXE) motif within said SEQ ID NO, and a second mutation at an amino acid selected from the group consisting of;
amino acid L, P, and both L and P in Region II (DXXSLYPSII, SEQ ID NO;
7) within said SEQ ID NO, wherein said second mutation confers a reduction in discrimination. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 95)
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19. A kit for identifying a nucleotide at a given position of a template DNA molecule, said kit comprising:
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a Family B DNA polymerase deficient in 3′
to 5′
exonuclease activity comprising the amino acid sequence of one of SEQ ID NOs;
2 and 62-78, excepta first mutation at amino acid D or E or both in the exo I (DXE) motif within said SEQ ID NO, and a second mutation at an amino acid selected from the group consisting of;
amino acid L, P, and both L and P in Region II (DXXSLYPSII, SEQ ID NO;
7) within said SEQ ID NO wherein said second mutation confers a reduction in discrimination. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method for identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising:
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contacting a first primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′
of said nucleotide at the given position of said template DNA molecule, so as to form a duplex between said first primer and said template DNA molecule;incubating said duplex in the presence of at least one chain-terminating nucleotide analog and a Family B DNA polymerase deficient in 3′
to 5′
exonuclease activity, said Family B DNA polymerase comprising the amino acid sequence of one of SEQ ID NOs;
2 and 62-78, excepta first mutation at amino acid D or E or both in the exo I (DXE) motif within said SEQ ID NO, and a second mutation at an amino acid selected from the group consisting of;
amino acid L, P, and both L and P in Region II (DXXSLYPSII, SEQ ID NO;
7) within said SEQ ID NO, wherein said second mutation confers a reduction in discrimination,wherein said at least one chain-terminating nucleotide analog is labeled with a first detectable label, wherein said incubating allows the incorporation of a labeled chain-terminating nucleotide analog into said first primer by said DNA polymerase in a template-dependent manner; and determining the presence or identity of said duplex by a signal generated from said first detectable label as a result of said incubating. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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52. A DNA polymerase comprising the sequence of SEQ ID NO:
- 2 except an amino acid mutation at P410 and A485.
- View Dependent Claims (53, 54, 55)
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56. A DNA polymerase comprising the sequence of SEQ ID NO:
- 2 except amino acid mutations at
1) P410 and A485; and
2) D141, E143, or both. - View Dependent Claims (57)
- 2 except amino acid mutations at
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58. A composition comprising a DNA polymerase comprising the sequence of SEQ ID NO:
- 2 except mutations at P410 and A485.
- View Dependent Claims (59, 60, 61, 63, 64, 65, 66, 67, 68)
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62. A DNA polymerase comprising the sequence of SEQ ID NO:
- 2 except a P410L mutation, an A485T mutation, a D141A mutation, and a E143 mutation.
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69. A kit comprising
a DNA polymerase comprising the sequence of SEQ ID NO: - 2 except mutations at positions P410 and A485 and optionally at D141 and E143, and
packaging material therefor. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81)
- 2 except mutations at positions P410 and A485 and optionally at D141 and E143, and
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82. A DNA polymerase produced by expressing a polynucleotide encoding a DNA polymerase, wherein said DNA polymerase comprises the sequence of SEQ ID NO:
- 2 except a mutation at P410 and A485.
- View Dependent Claims (84, 85, 86)
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83. A DNA polymerase produced by the steps:
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(a) introducing a mutation into a polynucleotide encoding a wild-type DNA polymerase comprising the sequence of SEQ ID NO;
2 to produce a mutant polynucleotide encoding a mutant DNA polymerase comprising the sequence of SEQ ID NO;
2 except mutations at positions P410 and A485; and(b) expressing said mutant polynucleotide to produce said mutant DNA polymerase.
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87. A DNA polymerase produced by expressing a polynucleotide encoding a mutant DNA polymerase, wherein said mutant DNA polymerase comprises the sequence of SEQ ID NO:
- 2 except one or more mutations at D141, E143, P410, and A485.
- View Dependent Claims (88)
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89. A composition comprising a DNA polymerase produced by expressing in said composition a polynucleotide encoding a DNA polymerase comprising the sequence of SEQ ID NO:
- 2 except mutations at P410 and A485 and optionally D141 and E143.
- View Dependent Claims (91, 92, 93, 94)
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90. A composition comprising a DNA polymerase produced by the steps:
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(a) introducing a mutation into a polynucleotide encoding a wild-type DNA polymerase to produce a mutant polynucleotide encoding a mutant DNA polymerase comprising the sequence of SEQ ID NO;
2 except mutations at P410 and A485 and optionally D141 and E143; and(b) expressing said mutant polynucleotide to produce said composition comprising said mutant DNA polymerase.
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Specification