METHODS AND COMPOSITIONS FOR LABELING NUCLEIC ACIDS
First Claim
1. A method of labeling a nucleic acid polymer, comprising steps of:
- providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises a first reactive unsaturated group and a base, wherein the first reactive unsaturated group is directly covalently attached to the base; and
contacting the nucleic acid polymer with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second unsaturated groups;
wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile, orwherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.
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Accused Products
Abstract
The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a [3+2] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixation and denaturation and therefore can be applied to the labeling of nucleic acid polymers in living cells and in organisms. Also provided are methods for measuring cellular proliferation. In these methods, the amount of label incorporated into the DNA is measured as an indication of cellular proliferation. The methods of the invention can be used in a wide variety of applications including clinical diagnosis of diseases and disorders in which cellular proliferation is involved, toxicity assays, and as a tool for the study of chromosomes'"'"' ultrastructures.
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Citations
119 Claims
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1. A method of labeling a nucleic acid polymer, comprising steps of:
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providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises a first reactive unsaturated group and a base, wherein the first reactive unsaturated group is directly covalently attached to the base; and contacting the nucleic acid polymer with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second unsaturated groups; wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile, or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole. - View Dependent Claims (3, 4, 5, 6, 7, 10, 41, 42, 43, 44, 45, 46, 105, 108, 109)
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2. (canceled)
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8-9. -9. (canceled)
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11-25. -25. (canceled)
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26. A method of differentially labeling nucleic acid polymers, comprising steps of:
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providing a first nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and a base, wherein the first reactive unsaturated group is directly covalently attached to the base; providing a second nucleic acid polymer comprising at least one second nucleotide analogue that comprises a second reactive unsaturated group and a base, wherein the second reactive unsaturated group is directly covalently attached to the base; contacting the first nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; and contacting the second nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups; wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole, or wherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile. - View Dependent Claims (29, 30, 31, 32, 33, 37, 38, 39, 106, 107, 112, 113)
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27-28. -28. (canceled)
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34-36. -36. (canceled)
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40. (canceled)
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53. (canceled)
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54. A kit for labeling a nucleic acid polymer comprising:
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at least one nucleoside analogue that comprises a first reactive unsaturated group; and a reagent comprising a second reactive unsaturated group attached to a label; wherein the first reactive unsaturated group comprises a 1,3-dipole, the second reactive unsaturated group comprises a dipolarophile and the first and second reactive unsaturated groups can react via [3+2] cycloaddition, or wherein the first reactive unsaturated group comprises a dipolarophile, the second reactive unsaturated group comprises a 1,3-dipole, and the first and second reactive unsaturated groups can react via [3+2] cycloaddition. - View Dependent Claims (57, 58, 59, 60, 61, 62, 114, 115)
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55-56. -56. (canceled)
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63. A kit for dually labeling a nucleic acid polymer, comprising
at least one first nucleoside analogue that comprises a first reactive unsaturated group; -
at least one second nucleoside analogue that comprises a second reactive unsaturated group; a first reagent comprising a third reactive unsaturated group attached to a first label; and a second reagent comprising a fourth reactive unsaturated group attached to a second label; wherein the first reactive unsaturated group comprises a first 1,3-dipole, the third reactive unsaturated group comprises a first dipolarophile, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition, or wherein the first reactive unsaturated group comprises a first dipolarophile, the third reactive unsaturated group comprises a first 1,3-dipole, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition; and
wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition. - View Dependent Claims (66, 67, 68, 69, 70, 71, 116, 117, 118, 119)
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64-65. -65. (canceled)
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72-104. -104. (canceled)
Specification