Dye-labeled ribonucleotide triphosphates
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Abstract
The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.
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Citations
123 Claims
- 1. A compound having the formula:
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14. A compound of the formula I:
- 22. A compound of the formula II:
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26. A compound of the formula III:
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34. A compound of the formula IV:
- 42. A compound of the formula V:
- 50. A compound of the formula VI:
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58. A compound of the formula VII:
- 63. A compound of the formula VIII:
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68. A compound of the formula IX:
- 73. A compound of the formula X:
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78. A compound having the formula:
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101. A method for determining the sequence of a DNA template, comprising
(i) annealing at least one oligonucleotide primer to a template; -
(ii) incubating said at least one oligonucleotide primer with a DNA polymerase that can incorporate both dNTPs and rNTPs in a reaction comprising a mixture of unlabeled dNTPs and at least one dye-labeled ribonucleotide of the invention so that primer extension products are formed;
(iii) treating the primer extension products with a means for hydrolyzing the extension products at each occurrence of a ribonucleotide;
(iv) separating the resulting fragments that contain said at least one primer from other fragments, (v) resolving the primer-containing extension products by size; and
(vi) detecting the fragments. - View Dependent Claims (102, 103, 104, 105, 106, 107)
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108. A method for detecting mutations in DNA, comprising
annealing two oligonucleotide primers to a template; -
incubating the two oligonucleotide primers with a DNA polymerase that can incorporate both dNTPs and rNTPs in a reaction comprising a mixture of unlabeled dNTPs and at least one dye-labeled ribonucleotide of the invention so that primer extension products are formed;
treating the primer extension products with a means for hydrolyzing the extension products at each occurrence of a ribonucleotide to produce fragments;
resolving the fragments by size; and
detecting the fragments. - View Dependent Claims (109, 110, 111, 112, 113, 114, 116, 119, 120, 121, 122, 123)
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115. A method for preparing polynucleotide fragments, comprising
incubating a DNA template with a DNA polymerase, dATP, dGTP, dCTP, dTTP, at least two oligonucleotide primers complementary to the DNA template, and at least one dye-labeled ribonucleotide so that the primers are extended and said at least one dye-labeled ribonucleotide is incorporated in the primer extension products; - and
hydrolyzing 3′
-5′
phosphodiester linkages between adjacent ribo- and deoxyribonucleotides.
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117. A method for preparing dye-labeled RNA complementary to a sequence of interest comprising:
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preparing a mixture of a template, an RNA polymerase, rATP, rGTP, rCTP, rUTP, and at least one dye-labeled ribonucleotide, wherein said sequence of interest is operably linked to a site for the initiation of RNA synthesis by the RNA polymerase; and
incubating the mixture so that the RNA polymerase catalyzes the synthesis of RNA.
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118. A method for detecting 5-methylcytosine in a DNA template comprising:
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treating the DNA template with a bisulfite salt under conditions whereby 5-methylcytosine remains non-reactive;
incubating the DNA template with a DNA polymerase, dATP, dGTP, dCTP, dTTP, at least two oligonucleotide primers complementary to the DNA template, and a dye-labeled rCTP compound so that the primers are extended and the dye-labeled rCTP compound is incorporated in the primer extension products;
hydrolyzing 3′
-5′
phosphodiester linkages between adjacent ribo- and deoxyribonucleotides to produce fragments;
resolving the fragments by size; and
detecting the fragments.
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Specification