Methods and compositions for labeling nucleic acids

• US 8,541,570 B2
• Filed: 05/24/2010
• Issued: 09/24/2013
• Est. Priority Date: 10/27/2005
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gee, Kyle R., Agnew, Brian, Salic, Adrian, Mitchison, Timothy J.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/785,999
• Publication Number:
  US 20100311063A1
• Time in Patent Office: 1,219 Days
• Field of Search: None
• US Class Current: 536/28.4
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/582 with fluorescent label

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.

Claims

1. A kit for labeling a nucleic acid polymer comprising:

at least one nucleoside analogue that comprises an azide group; and

a first reagent comprising a first label attached to a first substituted triarylphosphine;

wherein the azide group is attached to the nucleoside analogue at a 5-position of a pyrimidine base.

2. The kit of claim 1, wherein one of the aryl groups of the first triarylphosphine is substituted with an alkyl ester.

3. The kit of claim 2, wherein the alkyl ester is in ortho to the phosphorus atom of the first triarylphosphine.

4. The kit of claim 1, wherein the first label is directly detectable.

5. The kit of claim 4, wherein the first label is a fluorescent agent.

6. The kit of claim 1, wherein the first label is indirectly detectable.

7. The kit of claim 6, wherein the first label comprises a hapten.

8. The kit of claim 1 further comprising a second reagent comprising a second label attached to a second substituted triarylphosphine.

9. The kit of claim 8, wherein one of the aryl groups of the second triarylphosphine is substituted with an alkyl ester.

10. The kit of claim 9, wherein the alkyl ester is in ortho to the phosphorus atom of the second triarylphosphine.

11. The kit of claim 8, wherein the first and second labels are directly detectable.

12. The kit of claim 11, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agents produce a dual-color fluorescence upon excitation.

13. The kit of claim 8 wherein the first and second labels are indirectly detectable.

14. The kit of claim 13, wherein the first label comprises a first hapten and the second label comprises a second hapten.

15. The kit of claim 1, wherein the nucleoside analogue is 5-azido-2′

-deoxyuridine.