Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
First Claim
Patent Images
1. A composition comprising a polymerase and at least one compound selected from the group consisting of compounds of the following general formulas:
-
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(e)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(f)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(g)
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(h)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent, E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.
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Abstract
Nucleotide triphosphate probes containing a molecular and/or atomic tag on a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.
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Citations
138 Claims
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1. A composition comprising a polymerase and at least one compound selected from the group consisting of compounds of the following general formulas:
-
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(e)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(f)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(g)
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(h)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent, E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.- View Dependent Claims (2, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 72, 73, 74, 75, 76, 77, 78)
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3. A composition comprising a polymerase and at least one compound of the general formula:
-
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(e)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent and E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.- View Dependent Claims (4, 40, 41, 42, 43, 44, 45, 46, 47)
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5. A composition comprising a polymerase and at least one compound of the general formula:
-
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(f)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent and E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.- View Dependent Claims (6, 48, 49, 50, 51, 52, 53, 54, 55)
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7. A composition comprising a polymerase and at least one compound of the general formula:
-
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(g)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent and E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.- View Dependent Claims (8, 56, 57, 58, 59, 60, 61, 62, 63)
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9. A composition comprising a polymerase and at least one compound of the general formula:
-
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(h)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent and E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2.- View Dependent Claims (10, 64, 65, 66, 67, 68, 69, 70, 71)
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24. A method for performing a nucleotide incorporation, comprising:
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contacting a polymerase with a template nucleic acid molecule and a tagged nucleotide or analog thereof, the tagged nucleotide or analog including an optically detectable nucleotide tag, wherein the contacting is performed in the presence of a compound selected from the group consisting of compounds of the following general formulas;
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(e)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(f)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(g)and
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(h)where Z and Z′
are each independently a hydrogen atom or a thermally stable substituent, E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2, andincorporating the tagged nucleotide or analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase. - View Dependent Claims (25, 26, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)
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27. A method for obtaining sequence information from a template nucleic acid molecule, comprising:
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contacting a template nucleic acid molecule with a polymerase and at least one tagged nucleotide or analog thereof, wherein the at least one tagged nucleotide or analog includes an optically detectable nucleotide tag, and where the contacting is performed in the presence of at least one compound selected from the group consisting of compounds of the following general formulas;
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(e)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(f)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(g)and
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(h)where Z or Z′
are each independently a hydrogen atom or a thermally stable substituent, E and E′
are each independently an oxygen atom or a thermally stable substituent and n is an integer having a value of at least 2;incorporating the tagged nucleotide or analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase; detecting a signal that indicates the incorporation of the at least one tagged nucleotide or analog into the nascent nucleic acid molecule by the polymerase; and identifying the at least one tagged nucleotide or analog, thereby obtaining sequence information from the template nucleic acid molecule. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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97. A composition comprising:
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a polymerase; and a tagged nucleotide or analog thereof, where the tagged nucleotide or analog includes; (a) a polyphosphate chain including at least four phosphate groups, and (b) an optically detectable nucleotide label linked to a phosphate group of the polyphosphate chain. - View Dependent Claims (98, 99, 100, 101, 102, 103, 104, 105, 106)
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107. A composition comprising:
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a polymerase; a tagged nucleotide or analog thereof, where the tagged nucleotide or analog includes an optically detectable nucleotide tag, and a polyphosphate compound including at least four phosphate groups linked to each other to form a polyphosphate chain. - View Dependent Claims (108, 109, 110, 111, 112, 113)
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114. A method for performing a nucleotide incorporation, comprising:
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contacting a polymerase with a template nucleic acid molecule and a tagged nucleotide or analog thereof, where the tagged nucleotide or analog includes; (i) a polyphosphate chain including at least four phosphate groups, and (ii) an optically detectable nucleotide tag linked to a phosphate group of the polyphosphate chain; and incorporating the tagged nucleotide or analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase. - View Dependent Claims (115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125)
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126. A method for performing a nucleotide incorporation, comprising:
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contacting a polymerase with a template nucleic acid molecule and a tagged nucleotide or analog thereof and a polyphosphate compound including at least four phosphate groups linked to form a polyphosphate chain, where the tagged nucleotide or analog includes an optically detectable nucleotide tag; and incorporating the tagged nucleotide or analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase. - View Dependent Claims (127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138)
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Specification