NUCLEIC ACID SYNTHESIS COMPOSITIONS AND METHODS AND SYSTEMS FOR USING SAME
First Claim
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1. A method of sequencing a nucleic acid, comprising:
- providing a complex comprising a polymerase enzyme, a template nucleic acid, and a primer sequence complementary to at least a portion of the template nucleic acid;
contacting the complex with a reaction mixture that comprises a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion, and one or more types of nucleotides or nucleotide analogs; and
detecting incorporation of one or more of the nucleotide into the complex.
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Abstract
The Application relates to compositions, kits, methods, and systems for nucleotide sequencing comprising producing polymerase reactions that comprise both catalytic and non-catalytic divalent metal ions. Effective ratios and amounts of catalytic and non-catalytic divalent metal ions are described.
75 Citations
15 Claims
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1. A method of sequencing a nucleic acid, comprising:
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providing a complex comprising a polymerase enzyme, a template nucleic acid, and a primer sequence complementary to at least a portion of the template nucleic acid; contacting the complex with a reaction mixture that comprises a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion, and one or more types of nucleotides or nucleotide analogs; and detecting incorporation of one or more of the nucleotide into the complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of sequencing a nucleic acid, comprising:
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providing a complex comprising a polymerase enzyme, a template nucleic acid, and a primer sequence complementary to at least a portion of the template nucleic acid; contacting the complex with a reaction mixture that comprises a plurality of types of nucleotides, and a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion at first and second concentrations, respectively, wherein the mixture of divalent metal ions is selected to provide improved sequencing accuracy over a complex exposed to the first concentration of the catalytic metal in the absence of the non-catalytic metal; and detecting incorporation of a nucleotide into the complex.
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13. A composition, comprising:
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a complex comprising a template nucleic acid, a primer sequence and a polymerase enzyme; a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion; and at least a first incorporatable nucleotide or nucleotide analog.
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14. A method of modulating a polymerase activity, comprising:
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sequestering a bound nucleotide in a non-exchangeable state with a polymerase enzyme by contacting the polymerase enzyme with a first non-catalytic exchangeable co-factor; and contacting the polymerase enzyme with a catalytic exchangeable co-factor to exchange the non-catalytic co-factor, rendering the bound nucleotide into an exchangeable state with the polymerase enzyme.
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15. A kit, comprising:
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one or more components of a nucleic acid synthesis complex, selected from a DNA polymerase enzyme and a primer sequence; a reaction buffer comprising a mixture of catalytic metal ions and non-catalytic metal ions; and a plurality of types of fluorescently labeled nucleotide analogs.
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Specification