DUAL LABELING METHODS FOR MEASURING CELLULAR PROLIFERATION
First Claim
1. A method for measuring a change in cellular nucleic acid synthesis:
- a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample;
b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample;
c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample;
d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured,wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular nucleic acid synthesis,with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety.
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Abstract
The present invention provides a method for measuring cellular nascent nucleic acid synthesis by dual pulse labeling of nucleic acid. The first pulse labeling of nucleic acid with a nucleoside analog allows establishment of a baseline nucleic acid synthesis rate. Pulse labeling of the nucleic acid with a second nucleoside analog then allows measurement of any changes to nucleic acid synthesis. The nucleic acid synthesis can be measured as cell proliferation, DNA, or gene expression, RNA. This method does not require a potentially artifact-inducing intermediary wash step between pulse labels. Additionally, this method may be used to screen compounds for their affect on cellular proliferation by treating cells or an organism with the test compound simultaneous to or before treatment with a competitive nucleoside analog.
68 Citations
33 Claims
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1. A method for measuring a change in cellular nucleic acid synthesis:
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a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample; b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample; c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample; d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured, wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular nucleic acid synthesis, with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32)
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2. A method for measuring a change in cellular DNA synthesis:
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a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample; b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample; c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample; d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured, wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular DNA synthesis, with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety.
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3. A method for measuring a change in cellular RNA synthesis:
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a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample; b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample; c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample; d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured, wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular RNA synthesis, with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety.
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31. A method for screening compounds for effects on cellular proliferation or gene expression comprising:
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a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample; b) treating the primary incubated sample with a test compound to form a treated sample; c) incubating the treated sample with at least one second nucleoside or nucleotide analog simultaneous to or after treating the primary incubated sample with the test compound to form a secondary incubated sample; d) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample; e) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured, wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as an effect of the screening compounds on cellular proliferation or gene expression, with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety.
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33. A kit for measuring a change in cellular nucleic acid synthesis, wherein the kit comprises:
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a) a first nucleoside or nucleotide analog; b) at least one second nucleoside or nucleotide analog, wherein in at least the first analog or the at least one second nucleoside or nucleotide analog contains a bioorthogonal functional moiety; c) a first labeling reagent; and d) a second labeling reagent.
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Specification