# US 10,246,739 B2 · Exonuclease cycling assay

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- Number: US 10,246,739 B2
- Document ID: US10246739B2
- Status: Granted
- Kind: B2
- Application number: 14/212,151
- Filed: 2014-03-14
- Published: 2014-11-27
- Granted: 2019-04-02
- Earliest claimed: 2013-03-15
- Assignees: Georgetown University
- Applicants: Georgetown University
- Inventors: Mark Danielsen; Berenice Alfonso; Bolor Tumurpurev
- Web page: https://patentgrep.com/patent/US10246739B2

## CPC

- C12Q 1/6823: Release of bound markers
- 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

The present invention relates to a novel method for detecting a target polynucleotide having a target sequence, comprising hybridizing the target polynucleotide with a probe to form a hybrid; exposing the hybrid to a 5′ exonuclease so that the probe in the hybrid is digested and the target polynucleotide is dissociated from the digested probe; repeating the hybridization step and the digestion step; and detecting the digested probes. The presence of the digested probes indicates the presence of the target polynucleotide.

## Claims

### Claim 1

A method for detecting a target polynucleotide in a test sample, wherein the target polynucleotide has a target sequence and is protected against digestion by a 5′ exonuclease, wherein the 5′ exonuclease is lambda exonuclease, comprising
(a) incubating the test sample with a probe having a sequence complementary with the target sequence and lacking a phosphorylated 5′ end, wherein the probe has a quencher at its 5′ end and a fluor, whereby a hybrid of the probe and the target polynucleotide is formed, wherein the hybrid comprises a hybridization region formed between the target sequence and the probe, wherein the fluor of the probe is quenched by the quencher before forming the hybrid, and wherein the fluor of the probe is quenched by the quencher in the hybrid;
(b) exposing the hybrid to the 5′ exonuclease to digest the probe in the hybrid and forming a reaction mixture, whereby a digested probe comprising the fluor is generated, wherein the fluorescence of the fluor of the digested probe is not quenched;
(c) dissociating the target polynucleotide from the digested probe in the reaction mixture;
(d) repeating steps (a)-(c) by adding the probe and the 5′ exonuclease to the reaction mixture after step (c), wherein step (b) of step (d) is performed for a first time period until no further increase in the fluorescence in the reaction mixture;
(e) determining a first fluorescence intensity of the fluor in the reaction mixture after step (d);
(f) repeating steps (a)-(d) with a control sample in place of the test sample, wherein step (b) in step (f) is performed for a second time period, wherein the second time period equals to the first time period, wherein the control sample is identical to the test sample except without the target polynucleotide; and
(g) determining a second fluorescence intensity of the fluor in the control sample after step (f), wherein the first fluorescence intensity greater than the second fluorescence intensity indicates the presence of the target polynucleotide in the test sample.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

The method of claim 1, wherein the probe has the fluor internally.

### Claim 5 (depends on 1)

The method of claim 1, wherein the probe has the fluor at its 3′ end.

### Claim 6 (depends on 1)

The method of claim 1, wherein the target polynucleotide has one or more nuclease resistant nucleotides upstream of the hybridization region.

### Claim 7 (depends on 1)

The method of claim 1, wherein the target polynucleotide has one or more uncleavable linkages upstream of the hybridization region.

### Claim 8 (depends on 1)

The method of claim 1, wherein the target polynucleotide is prepared by a polymerase synthesis reaction using a 5′ exonuclease protected primer.

### Claim 9 (depends on 8)

The method of claim 8, wherein the 5′ exonuclease protected primer lacks a 5′ phosphate group.

### Claim 10 (depends on 8)

The method of claim 8, wherein the 5′ exonuclease protected primer contains one or more uncleavable nucleotide linkages.

### Claim 11 (depends on 8)

The method of claim 8, wherein the 5′ exonuclease protected primer is chemically modified.

### Claim 12 (depends on 8)

The method of claim 8, wherein the polymerase synthesis reaction is selected from the group consisting of multiple displacement amplification (MDA), polymerase chain reaction (PCR), rolling circle amplification, nondisplacement amplification and reverse transcription.

### Claim 13 (depends on 1)

The method of claim 1, wherein the target DNA polynucleotide is synthesized from one or more short polynucleotides, wherein the one or more short oligonucleotides are resistant to the digestion by the 5′ exonuclease.

### Claim 14 (depends on 1)

The method of claim 1, further comprising determining the amount of the digested probe.

### Claim 15 (depends on 1)

The method of claim 1, wherein the test sample is a homogenous mixture.

### Claim 16 (depends on 1)

The method of claim 1, wherein the amount of the target polynucleotide in the test sample is less than 1 pmole.

### Claim 17 (depends on 1)

The method of claim 1, wherein the amount of the target polynucleotide in the test sample is less than 10 fmole.