# US 8,541,570 B2 · Methods and compositions for labeling nucleic acids

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- Number: US 8,541,570 B2
- Document ID: US8541570B2
- Status: Granted
- Kind: B2
- Application number: 12/785,999
- Filed: 2010-05-24
- Published: 2010-12-09
- Granted: 2013-09-24
- Earliest claimed: 2005-10-27
- Assignees: President and Fellows of Harvard College; Life Technologies Corporation
- Applicants: Kyle R. Gee; Brian Agnew; Adrian Salic; Timothy J. Mitchison
- Inventors: Kyle R. Gee; Brian Agnew; Adrian Salic; Timothy J. Mitchison
- Web page: https://patentgrep.com/patent/US8541570B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/68: involving nucleic acids
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6841: In situ hybridisation
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/582: {with fluorescent label}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 8)

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

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 8)

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

### Claim 14 (depends on 13)

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

### Claim 15 (depends on 1)

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