Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
AssigneeVisigen Biotechnologies, Inc.
InventorsSusan H. Hardin, Xiaolian Gao, James Briggs, Richard Willson, Shiao-Chun Tu
Life of this patent
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Abstract
Nucleotide triphosphate probes containing a molecular and/or atomic tag on a 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.
Claims31
A method for using modified nucleotides to alter base incorporation fidelity comprising the step of adding a modified nucleotide including a molecular tag to a nucleotide polymerization medium comprising a nucleotide polymerizing agent, a primer, a template, and a pyrophosphorolysis inhibitor selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:
- Z-OPO2O-Z′ (a)
- Z-PO2O-Z′ (b)
- Z-OPO2-Z′ (c)
- Z-PO2-Z′ (d)
- 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 or Z′ is a hydrogen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements, E and E′ are an oxygen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements and n is an integer having a value between 0 and about 5, where the modified nucleotide increases base incorporation fidelity of the nucleotide polymerizing agent relative to a base incorporation fidelity of the nucleotide polymerizing agent in the absence of the modified nucleotide to produce an extended primer having reduced incorrect base incorporations and where the pyrophosphorolysis inhibits pryosphosphorolysis reduces the amount of pyrophosphorolysis occurring in the reaction.
A method for using modified nucleotides to alter base incorporation fidelity comprising the step of adding a modified nucleotide including a molecular tag to an assay for extending a nucleotide sequence, and a pyrophosphorolysis inhibitor selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:
- Z-OPO2O-Z′ (a)
- Z-PO2O-Z′ (b)
- Z-OPO2-Z′ (c)
- Z-PO2-Z′ (d)
- 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 or Z′ is a hydrogen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements, E and E′ are an oxygen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements and n is an integer having a value between 0 and about 5 and where the modified nucleotide alters base incorporation fidelity of a nucleotide polymerizing agent relative to abase incorporation fidelity of the polymerizing agent in the absence of the modified nucleotide, and the assay is selected from the group consisting of genotyping for in vitro reproductive methods (human and other organisms);
- single nucleotide polymorphism (SNP) detection;
- DNA sequencing;
- RNA sequencing;
- single nucleotide extension assays;
- amplified DNA product assays;
- rolling circle product assays;
- PCR product assays;
- allele-specific primer extension assays;
- single-molecule arrays (DNA, RNA, protein) assays;
- and drug toxicity evaluation assays.
A kit for performing a nucleotide polymerizing reaction comprising using at least one modified nucleotide including a molecular tag in the presence of a polymerizing agent, a primer, a template and a pyrophosphorolysis inhibitor selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:
- Z-OPO2O-Z′ (a)
- Z-PO2O-Z′ (b)
- Z-OPO2-Z′ (c)
- Z-PO2-Z′ (d)
- 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 or Z′ is a hydrogen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements, E and E′ are an oxygen atom or a thermally stable substituent comprising primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements and n is an integer having a value between 0 and about 5 and where the modified nucleotide alters polymerizing agent extension fidelity for the at least one modified nucleotide compared to the polymerizing agent extension fidelity in the presence of the unmodified nucleotide corresponding to the at least one modified nucleotide.
Dependencies are inferred from the claim text.