# US 7,745,119 B2 · System for detecting polynucleotides

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- Number: US 7,745,119 B2
- Document ID: US7745119B2
- Status: Granted
- Kind: B2
- Application number: 11/285,025
- Filed: 2005-11-21
- Published: 2006-07-06
- Granted: 2010-06-29
- Earliest claimed: 2003-05-20
- Assignees: Investigen, Inc.
- Inventors: Heather Koshinsky; Michael S. Zwick; K. Yeon Choi
- Web page: https://patentgrep.com/patent/US7745119B2

## CPC

- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present invention relates to methods for detecting the presence or amount of a target polynucleotide. A polynucleotide, target nucleic acid analog, and dye are combined to form a mixture. The optical property of the dye is observed after the mixture is exposed to a stimulating means. Optionally, after the stimulating means is employed, the mixture is compared to a reference value characteristic of the rate of change in the optical property of the dye in a similar mixture containing a known amount of a target polynucleotide/nucleic acid analog hybrid to determine a relative rate of change in the optical property. The change in a property of the mixture after exposure thereof to a stimulating means or the relative rate of change in the optical property of dye in the mixture is correlated with the presence or amount of the specified target polynucleotide in the sample.

## Claims

### Claim 1

A method comprising the steps of:
(a) producing a mixture comprising
(i) a sample that may or may not contain a target polynucleotide having a target nucleic acid sequence,
(ii) a nucleic acid analog that is complementary to the target nucleic acid sequence and
(iii) a cyanine dye, wherein some of the cyanine dye forms a complex with the nucleic acid analog and a target polynucleotide, if present in the sample;
(b) exposing the mixture to a light stimulus, wherein the light stimulus catalyzes a reaction that causes a decrease in the amount of the cyanine dye in the mixture; and
(c) correlating the rate of change in an optical property of the mixture to the light stimulus during the period of time subsequent to formation of the complex, wherein the rate of change in the optical property of the mixture caused by the light stimulus is indicative of the presence or amount of the target polynucleotide in the sample.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the step of
correlating an alteration in the rate of change in the optical property of the mixture with the presence or amount of the target polynucleotide in the sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the optical property is determined by observing one or more of color, absorbance, reflectance or fluorescence.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nucleic acid analog is a peptide nucleic acid (PNA) or a locked nucleic acid (LNA).

### Claim 5 (depends on 1)

The method of claim 1, wherein the target polynucleotide is DNA or RNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the nucleic acid analog includes a complementary region that is greater than about 4 nucleic acid bases in length and less than about 24 nucleic acid bases in length.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleic acid analog or the target polynucleotide is immobilized on a solid substrate.

### Claim 8 (depends on 2)

The method of claim 2, wherein the light stimulus-causes a higher rate of change in the optical property of the mixture when the nucleic acid analog and the target polynucleotide are present in the sample.

### Claim 9 (depends on 2)

The method of claim 2, wherein the light stimulus causes a lower rate of change in the optical property of the mixture when the nucleic acid analog and the target polynucleotide are present in the sample.

### Claim 10 (depends on 1)

A method for detecting a single nucleotide polymorphism (SNP), comprising detecting the target polynucleotide in the sample according to the method of claim 1, wherein the target nucleic acid sequence is specific to the SNP, and wherein the detecting of the target polynucleotide identifies the SNP.

### Claim 11 (depends on 1)

A method of detecting an organism in a sample, comprising detecting the target polynucleotide in the sample according to the method of claim 1, wherein the target nucleic acid sequence is specific to the organism, and wherein the detecting of the target polynucleotide identifies the presence or amount of the organism.

### Claim 12 (depends on 3)

The method of claim 3, wherein the light stimulus, in the presence of the target nucleic acid and complementary nucleic acid analog, results in a reduction in the color of the mixture.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cyanine dye is thiacyanine dye.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cyanine dye is a carbocyanine dye.

### Claim 15 (depends on 14)

The method of claim 14, wherein the cyanine dye is selected from the group consisting of 3,3′-diethylthiacarbocyanine iodide, 3,3′diethylthiadicarbocyanine iodide, 3,3′-diethylthiatricarbocyanine iodide, and 3,3′diethylthiacyanine iodide.

### Claim 16 (depends on 1)

The method of claim 1, wherein the cyanine dye is 3,3′diethylthiacarbocyanine iodide.

### Claim 17 (depends on 1)

The method of claim 1, wherein the temperature following step (a) is substantially constant.

### Claim 18 (depends on 1)

The method of claim 1, wherein the method is performed at a substantially constant temperature.

### Claim 19 (depends on 1)

The method of claim 1, wherein the mixture is exposed to the light stimulus for a period of less than 30 seconds.

### Claim 20 (depends on 1)

The method of claim 1, wherein the mixture is exposed to the light stimulus for a period of time less than 1 minute.

### Claim 21 (depends on 1)

The method of claim 1, wherein the mixture is exposed to the light stimulus for a period of time less than 20 minutes.

### Claim 22 (depends on 1)

The method of claim 1, wherein the light stimulus is a filtered light.

### Claim 23 (depends on 1)

The method of claim 1, wherein the light stimulus comprises a specific range of wavelengths.

### Claim 24 (depends on 23)

The method of claim 23, wherein the range of wavelengths is from a blue light stimulus.

### Claim 25 (depends on 23)

The method of claim 23, wherein the range of wavelengths is from a green light stimulus.

### Claim 26 (depends on 1)

The method of claim 1, further comprising the step of comparing the rate of change in the optical property of the mixture to a reference mixture either containing a known amount of a target polynucleotide or not containing the target polynucleotide.

### Claim 27 (depends on 1)

The method of claim 1, wherein the rate of change in the optical property of the mixture is observed over constant temperature conditions.

### Claim 28 (depends on 1)

The method of claim 1, wherein the light stimulus causes a breakdown of the cyanine dye in the mixture.

### Claim 29 (depends on 28)

The method of claim 28, further comprising detecting the breakdown of the cyanine dye in the mixture.

### Claim 30 (depends on 29)

The method of claim 29, further comprising correlating the breakdown of the cyanine dye with the presence or amount of the target polynucleotide.

### Claim 31 (depends on 30)

The method of claim 30, further comprising determining whether exposure to the light stimulus alters the rate of the breakdown of the cyanine dye in the mixture.

### Claim 32 (depends on 1)

The method of claim 1, wherein the nucleic acid analog is a peptide nucleic acid (PNA).

### Claim 33 (depends on 1)

The method of claim 1, further comprising the step of correlating the rate of change in the optical property of the mixture to the amount of the target polynucleotide in the sample.

### Claim 34 (depends on 1)

The method of claim 1, wherein the correlating step is at a point of time that allows differentiation between the presence or absence or differentiation of the amount of the target polynucleotide in the sample.