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 of the 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
98 Claims
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1. A composition comprising a polymerase and at least one compound of the 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 (21, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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2-20. -20. (canceled)
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22-29. -29. (canceled)
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30. 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 (31, 67, 68, 69, 70, 71, 72, 73, 74)
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-
32. 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 (33, 75, 76, 77, 78, 79, 80, 81, 82)
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-
34. 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 (35, 83, 84, 85, 86, 87, 88, 89, 90)
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-
36. 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 (37, 91, 92, 93, 94, 95, 96, 97, 98)
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51. A method for performing a nucleotide incorporation reaction, comprising:
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contacting a polymerase with a tagged nucleotide or nucleotide analog including an optically detectable tag, and a compound selected from the group consisting of;
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, wherein the contacting is performed under conditions where the tagged nucleotide or nucleotide analog is incorporated into a nucleic acid molecule by the polymerase and the optically detectable tag is released from the tagged nucleotide or nucleotide analog.- View Dependent Claims (52, 53)
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54. A method for sequencing a nucleic acid molecule, comprising:
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conducting a nucleic acid polymerization reaction including a target nucleic acid template, a polymerase, at least one tagged nucleotide or nucleotide analog including a fluorescent tag that is covalently bound to a β
-phosphate, γ
-phosphate or other terminal phosphate of the tagged nucleotide or nucleotide analog, and a compound selected from the group consisting of;
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;detecting, a signal emitted by the tag of the at least one tagged nucleotide or nucleotide analog that indicates the incorporation of the at least one tagged nucleotide or nucleotide analog by the polymerase; and identifying the at least one tagged nucleotide or nucleotide analog, thereby determining a partial or complete nucleic acid sequence of the target nucleic acid molecule. - View Dependent Claims (55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66)
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Specification