Methods and compositions for labeling nucleic acids
First Claim
1. A method of differentially labeling DNA, comprising steps of:
- contacting a first cell with an effective amount of a first nucleoside analogue that comprises a first azide group such that the nucleoside analogue is incorporated into DNA of the first cell;
contacting a second cell with an effective amount of a second nucleoside analogue that comprises a second azide group such that the nucleoside analogue is incorporated into DNA of the second cell;
contacting the first cell with a first reagent comprising a first label attached to a first substituted triarylphoshine, such that a Staudinger ligation occurs between the first azide and first substituted triarylphosphine; and
contacting the second cell with a second reagent comprising a second label attached to a second substituted triarylphosphine, such that a Staudinger ligation occurs between the second azide and second substituted triarylphosphine.
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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. Certain methods are provided that include a [3+2] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Other methods are provided that include a Staudinger ligation between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent comprising a substituted triarylphosphine 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.
33 Citations
14 Claims
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1. A method of differentially labeling DNA, comprising steps of:
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contacting a first cell with an effective amount of a first nucleoside analogue that comprises a first azide group such that the nucleoside analogue is incorporated into DNA of the first cell; contacting a second cell with an effective amount of a second nucleoside analogue that comprises a second azide group such that the nucleoside analogue is incorporated into DNA of the second cell; contacting the first cell with a first reagent comprising a first label attached to a first substituted triarylphoshine, such that a Staudinger ligation occurs between the first azide and first substituted triarylphosphine; and contacting the second cell with a second reagent comprising a second label attached to a second substituted triarylphosphine, such that a Staudinger ligation occurs between the second azide and second substituted triarylphosphine. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of differentially labeling DNA, comprising steps of:
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administering to a first organism an effective amount of a first nucleoside analogue that comprises a first azide group such that the nucleoside analogue is incorporated into DNA of cells of the first organism; administering to a second organism an effective amount of a second nucleoside analogue that comprises a second azide group such that the nucleoside analogue is incorporated into DNA of cells of the second organism; contacting at least one cell of the first organism with a first reagent comprising a first label attached to a first substituted triarylphoshine, such that a Staudinger ligation occurs between the first azide and first substituted triarylphosphine; and contacting at least one cell of the second organism with a second reagent comprising a second label attached to a second substituted triarylphosphine, such that a Staudinger ligation occurs between the second azide and second substituted triarylphosphine. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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Specification