# US 11,499,179 B2 · Real time cleavage assay

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- Number: US 11,499,179 B2
- Document ID: US11499179B2
- Status: Granted
- Kind: B2
- Application number: 16/789,276
- Filed: 2020-02-12
- Published: 2020-08-06
- Granted: 2022-11-15
- Earliest claimed: 2010-11-15
- Assignees: Exact Sciences Development Company, LLC
- Applicants: Exact Sciences Development Company, LLC
- Inventors: Rebecca Oldham-Haltom; Hongzhi Zou; Graham P. Lidgard; Michael J. Domanico; Hatim Allawi
- Web page: https://patentgrep.com/patent/US11499179B2

## CPC

- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression 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

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

## Claims

### Claim 1

A method for detecting a target nucleic acid, comprising:
(a) subjecting a PCR reaction mixture comprising a flap endonuclease activity to thermocycling conditions that comprise:
i. a first set of 5 to 15 cycles each comprising a denaturation step followed by one or more steps in which all temperatures are in the range of 60° C. to 75° C.; followed by:
ii. a second set of 20 to 50 cycles each comprising a denaturation step followed by one or more steps comprising a temperature that is in the range of 50° C. to 57° C.;
wherein the PCR reaction mixture comprises at least a first set of PCR primers, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites that flank the target nucleic acid and wherein the target nucleic acid is amplified by the first set of PCR primers and not any other primers that may be present in the PCR reaction mixture; and
(b) detecting a fluorescent signal in at least some cycles of the second set, wherein the fluorescent signal is generated by cleavage of a probe by the flap endonuclease activity and indicates the presence of a target nucleic acid
wherein no additional reagents are added to the PCR reaction mixture between said first and second sets.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cycles of the second set of 20 to 50 cycles each comprises a temperature that is in the range of 50° C. to 55° C.

### Claim 3 (depends on 1)

The method of claim 1, wherein the measuring step of (b) comprises measuring cleavage of a fluorophore from an oligonucleotide in each of the second set of 20 to 50 cycles.

### Claim 4 (depends on 1)

The method of claim 1, wherein the fluorescent signal of (b) indicates a mutation.

### Claim 5 (depends on 1)

The method of claim 1, wherein step (a) comprises an initial pre-incubation step that activates a thermostable polymerase.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises graphing the amount of fluorescent signal that is detected in each of the 20-50 cycles, thereby providing an estimate of the abundance of the target nucleic acid in the reaction mix.

### Claim 7 (depends on 1)

The method of claim 1, wherein step (b) comprises measuring two fluorescent signals in at least some of cycles of the second set, wherein the fluorescent signals respectively indicate the presence of two different mutations.

### Claim 8

A method for detecting a target nucleic acid comprising:
(a) subjecting a PCR reaction mixture comprising a flap endonuclease activity to thermocycling conditions that comprise:
i. a first set of 5 to 15 cycles each comprising a denaturation step followed by one or more steps in which all temperatures are in the range of 65° C. to 75° C.; followed by
ii. a second set of 20 to 50 cycles each comprising a denaturation step followed by one or more steps comprising a temperature that is in the range of 50° C. to 65° C.,
wherein the PCR reaction mixture comprises at least a first set of PCR primers, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites that flank the target nucleic acid and wherein the target nucleic acid is amplified by the first set of PCR primers and not any other primers that may be present in the PCR reaction mixture; and
(b) detecting a fluorescent signal in at least some cycles of the second set, wherein the fluorescent signal is generated by cleavage of a probe by the flap endonuclease activity and indicates the presence of a target nucleic acid, wherein no additional reagents are added to the PCR reaction mixture between said first and second sets.

### Claim 9 (depends on 8)

The method of claim 8, wherein the cycles of the second set of 20 to 50 cycles each comprises a temperature that is in the range of 50° C. to 55° C.

### Claim 10 (depends on 8)

The method of claim 8, wherein the measuring step of (b) comprises measuring cleavage of a fluorophore from an oligonucleotide in each of the second set of 20 to 50 cycles.

### Claim 11 (depends on 8)

The method of claim 8, wherein the fluorescent signal of (b) indicates a mutation.

### Claim 12 (depends on 8)

The method of claim 8, wherein step (a) comprises an initial pre-incubation step that activates a thermostable polymerase.

### Claim 13 (depends on 8)

The method of claim 8, wherein the method further comprises graphing the amount of fluorescent signal that is detected in each of the 20-50 cycles, thereby providing an estimate of the abundance of the target nucleic acid in the reaction mix.

### Claim 14 (depends on 8)

The method of claim 8, wherein step (b) comprises measuring two fluorescent signals in at least some of the cycles of the second set, wherein the fluorescent signals respectively indicate the presence of two different mutations.