Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
First Claim
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1. A composition comprising a modified nucleotide including a molecular and/or atomic tag, where the nucleotide alters base incorporation fidelity in a nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide.
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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.
81 Citations
20 Claims
- 1. A composition comprising a modified nucleotide including a molecular and/or atomic tag, where the nucleotide alters base incorporation fidelity in a nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide.
- 7. A method comprising the step of adding a modified nucleotide including a molecular and/or atomic tag, where the nucleotide alters base incorporation fidelity of a nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide, to a nucleotide polymerization medium comprising a nucleotide polymerizing agent.
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13. A method comprising the step of adding a modified nucleotide including a molecular and/or atomic tag, where the nucleotide alters base incorporation fidelity of a nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide, to an assay for extending a nucleotide sequence, 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.
- single nucleotide polymorphism (SNP) detection;
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14. A method for making blunt-ended fragments comprising the steps of amplifying a DNA fragment in the presence of a nucleotide including a molecular and/or atomic tag on a γ
- phosphate group and/or a base moiety, where the tag alters fidelity of base incorporation and decreases or eliminates non-templated addition of a base to the 3′
end of the DNA fragment being amplified.
- phosphate group and/or a base moiety, where the tag alters fidelity of base incorporation and decreases or eliminates non-templated addition of a base to the 3′
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15. A kit for performing a nucleotide polymerizing reaction comprising polymerizing reagents and at least one modified nucleotide including an atomic and/or molecular tag, where the modified nucleotide alters extension fidelity.
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16. A method of inhibiting or preventing pyrophosphorolysis during synthesis of a nucleic acid molecule, said method comprising
(a) combining a primer with a nucleic acid template under conditions sufficient to form a hybridized product; - and
(b) incubating the hybridized product with a polymerase in the presence or absence of an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase, under conditions sufficient to form a second nucleic acid molecule complementary to all or a portion of the nucleic acid template, where a tagged nucleotide comprises an atomic and/or molecular tag or moiety attached to and/or associated with a β and
/or γ
-phosphate and/or a base moiety of the nucleotide is added at either or both steps to inhibit or prevent pyrophosphorolysis during synthesis of a nucleic acid molecule.
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- 17. A composition comprising a nucleotide including a molecular and/or atomic tag on a phosphate group adapted to alter the fidelity of viral replication.
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19. A method for increasing the fidelity of replication comprising administering an therapeutically effective amount of a nucleotide including a molecular and/or atomic tag on a γ
- phosphate group to an animal including a human, where the nucleotide is designed to increase base incorporation fidelity during replication.
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Current AssigneeLife Technologies Corporation (Thermo Fisher Scientific Incorporated)
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Original AssigneeVisiGen Biotechnologies, Inc. (Thermo Fisher Scientific Incorporated)
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InventorsWillson, Richard, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian, Hardin, Susan H.
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current514/44
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CPC Class CodesC07H 19/06 Pyrimidine radicalsC07H 19/10 with the saccharide radical...C07H 19/20 with the saccharide radical...C12N 9/1241 Nucleotidyltransferases (2....C12N 9/1252 DNA-directed DNA polymerase...C12Q 1/6806 Preparing nucleic acids for...C12Q 1/6853 using modified primers or t...C12Q 1/6869 Methods for sequencingC12Q 2525/101 incorporating non-naturally...C12Q 2525/113 incorporating modified back...C12Q 2533/101 Primer extensionC12Q 2565/301 Pyrophosphate (PPi)
