# US 9,005,893 B2 · Methods to fix and detect nucleic acids

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- Number: US 9,005,893 B2
- Document ID: US9005893B2
- Status: Granted
- Kind: B2
- Application number: 13/759,363
- Filed: 2013-02-05
- Published: 2013-05-23
- Granted: 2015-04-14
- Earliest claimed: 2009-03-11
- Assignees: The Rockefeller University
- Applicants: The Rockefeller University
- Inventors: Thomas Tuschl; John Pena; Pavol Cekan; Cherin Sohn; Sara H. Rouhanifard; Janos Ludwig
- Web page: https://patentgrep.com/patent/US9005893B2

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6841: In situ hybridisation
- 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)
- 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

## Abstract

In one aspect, the invention relates to a method for fixing a short nucleic acid in a biological sample. In another aspect, the invention relates to a method for detecting a target short nucleic acid in a biological sample. The method includes contacting the biological sample with an aldehyde-containing fixative, and subsequently contacting the sample with a water-soluble carbodiimide. In a further aspect, the invention relates to a kit for fixing a short nucleic acid in a biological sample. The kit includes a support substrate for holding the sample; an aldehyde-containing fixative; and a water-soluble carbodiimide.

## Claims

### Claim 1

A method for fixing a nucleic acid in a biological sample, said method comprising
a) contacting the biological sample with an aldehyde-containing fixative; and
b) subsequently contacting the sample with a water-soluble carbodiimide, wherein said carbodiimide is in solution with a buffer.

### Claim 2 (depends on 1)

The method of claim 1, wherein the aldehyde-containing fixative is formaldehyde.

### Claim 3 (depends on 1)

The method of claim 1, wherein the carbodiimide is selected from the group consisting of: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); 1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIC).

### Claim 4 (depends on 3)

The method of claim 3, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

### Claim 5 (depends on 4)

The method of claim 4, wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a concentration of about 50 mM to about 250 mM.

### Claim 6 (depends on 4)

The method of claim 4, wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide contacts the sample at a temperature of about 20° C. to about 70° C.

### Claim 7 (depends on 4)

The method of claim 4, wherein the carbodiimide has a pH of 8.0 in solution.

### Claim 8 (depends on 1)

The method according to claim 1, wherein said nucleic acid is between 5-100 nucleotides.

### Claim 9

A method for detecting a target nucleic acid in a biological sample, said method comprising
a) contacting the biological sample with an aldehyde-containing fixative;
b) subsequently contacting the sample with a water-soluble carbodiimide to produce a crosslinked nucleic acid;
c) contacting the cross-linked nucleic acid with a probe, said probe being complementary to all or a part of a region of interest of the nucleic acid, thereby producing a hybridized nucleic acid; and
d) detecting the hybridized nucleic acid as the target nucleic acid.

### Claim 10 (depends on 9)

The method of claim 9, wherein the carbodiimide is selected from the group consisting of: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); 1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIC).

### Claim 11 (depends on 10)

The method according to claim 10, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a concentration of about 50 mM to about 250 mM.

### Claim 13 (depends on 11)

The method according to claim 11, wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a temperature of about 20° C. to about 70° C.

### Claim 14 (depends on 9)

The method according to claim 9, wherein the probe comprises a locked nucleic acid.

### Claim 15 (depends on 9)

The method according to claim 9 wherein said nucleic acid is between 5-100 nucleotides.