Dual labeling methods for measuring cellular proliferation

• US 10,138,510 B2
• Filed: 06/10/2014
• Issued: 11/27/2018
• Est. Priority Date: 05/16/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Clarke, Scott, Bradford, Jolene
• Primary Examiner(s): Ault, Addison D
• Application Number:
  US14/300,728
• Publication Number:
  US 20160130637A1
• Time in Patent Office: 1,631 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity

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.

Claims

1. A method for measuring a change in cellular DNA synthesis:

a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog comprising an ethynyl group to form a primary incubated sample,

wherein the first nucleoside or nucleotide analog is ethynyl-deoxyuracil (EdU);

b) incubating the primary incubated sample with a second nucleoside or nucleotide analog comprising a halogen moiety to form a secondary incubated sample,

wherein the second nucleoside or nucleotide analog is BrdU, and wherein the first nucleoside or nucleotide analog is not incorporated into a DNA polymer when the second nucleoside or nucleotide analog is present;

c) incubating the secondary incubated sample with a first labeling reagent comprising an azide group that can undergo a [3+2] cycloaddition reaction with the ethynyl group of the first nucleoside or nucleotide analog and a second labeling reagent that is an antibody that binds to the second nucleoside or nucleotide analog to form a labeled sample; and

d) detecting the labeled sample wherein a level of incorporation of the first nucleoside or nucleotide analog is measured and allows establishment of a baseline rate of the cellular DNA synthesis and a level of incorporation of the second nucleoside or nucleotide analog is measured,

wherein a difference between the level of incorporation of the second nucleoside or nucleotide analog relative to the baseline rate indicates a change in cellular DNA synthesis,

with the proviso that there is no wash step prior to adding the second nucleoside or nucleotide analog.

2. 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 comprising an ethynyl group to form a primary incubated sample,

wherein the first nucleoside or nucleotide analog is ethynyl-deoxyuracil (EdU);

b) incubating the primary incubated sample with a second nucleoside or nucleotide analog comprising a halogen moiety to form a secondary incubated sample,

wherein the second nucleoside or nucleotide analog is BrdU, and wherein the first nucleoside or nucleotide analog is not incorporated into a nucleic acid polymer when the second nucleoside or nucleotide analog is present;

c) incubating the secondary incubated sample with a first labeling reagent comprising an azide group that can undergo a [3+2] cycloaddition reaction with the ethynyl group of the first nucleoside or nucleotide analog and a second labeling reagent that is an antibody that binds to the second nucleoside or nucleotide analog to form a labeled sample; and

d) detecting the labeled sample wherein a level of incorporation of the first nucleoside or nucleotide analog is measured and allows establishment of a baseline rate of the cellular nucleic acid synthesis and a level of incorporation of the second nucleoside or nucleotide analog is measured,

wherein a difference between the level of incorporation of the second nucleoside or nucleotide analog relative to the baseline rate indicates a change in cellular nucleic acid synthesis,

with the proviso that there is no wash step prior to adding the second nucleoside or nucleotide analog.

3. The method according to claim 2 wherein the sample is treated with a test compound simultaneous to or before treatment with the second nucleoside or nucleotide analog.

4. The method according to claim 2, wherein the first labeling reagent or the second labeling reagent comprises a fluorescent dye.

5. The method according to claim 2, wherein the antibody is an anti-BrdU antibody.

6. The method according to claim 2, wherein the first labeling reagent is a dye-labeled azide.

7. The method according to claim 2, wherein incorporation of said first nucleoside analog and said second nucleoside analog is detected by flow cytometry.

8. The method according to claim 2, wherein incorporation of said first nucleoside analog and said second nucleoside analog is detected by fluorescence microscopy.

9. The method according to claim 2, wherein the sample is an organism or cells in cell culture.