Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
First Claim
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1. A method for sequencing nucleic acids, comprising:
- a) flowing labeled deoxynucleotide triphosphates and modified pyrophosphates onto template nucleic acids which are hybridized to a primer nucleic acid, where the template nucleic acids or primers are attached to a support, and wherein the labeled nucleotide triphosphates include a molecular tag bonded to the beta- or gamma-phosphate, wherein the molecular tag emits a detectable signal, and wherein the modified pyrophosphates include at least one compound selected from the group consisting of compound having the following general formulas
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(i)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(ii)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(iii)
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(iv)
where Z and Z′
are each independently a hydrogen atom, E and E′
are each independently an oxygen atom and n is an integer having a value of 2, 3, 4 or 5;
b) incorporating one of the labeled nucleotide triphosphates into at least one of the primers with a polymerase;
c) releasing a tagged pyrophosphate which includes the molecular tag bonded to the beta- or gamma-phosphate; and
d) detecting the released tagged pyrophosphates.
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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.
178 Citations
8 Claims
-
1. A method for sequencing nucleic acids, comprising:
-
a) flowing labeled deoxynucleotide triphosphates and modified pyrophosphates onto template nucleic acids which are hybridized to a primer nucleic acid, where the template nucleic acids or primers are attached to a support, and wherein the labeled nucleotide triphosphates include a molecular tag bonded to the beta- or gamma-phosphate, wherein the molecular tag emits a detectable signal, and wherein the modified pyrophosphates include at least one compound selected from the group consisting of compound having the following general formulas
Z—
OPO2—
(OP(EE′
)O)n—
PO2O—
Z′
(i)
Z—
OPO2—
(OP(EE′
)O)nPO2—
Z′
(ii)
Z—
PO2—
(OP(EE′
)O)nPO2O—
Z′
(iii)
Z—
PO2—
(OP(EE′
)O)nPO2—
Z′
(iv)
where Z and Z′
are each independently a hydrogen atom, E and E′
are each independently an oxygen atom and n is an integer having a value of 2, 3, 4 or 5;b) incorporating one of the labeled nucleotide triphosphates into at least one of the primers with a polymerase; c) releasing a tagged pyrophosphate which includes the molecular tag bonded to the beta- or gamma-phosphate; and d) detecting the released tagged pyrophosphates. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
-
Specification