Granted Patent B2

US 12,655,466 · App. 17/760,709 · Granted Jun 16, 2026

Inventors: Zubin Gagrat (Madison, WI); Michael B. Matter (Madison, WI); Nicholas J. Domanico (Madison, WI); Michael J. Domanico (Madison, WI)

Assignee: Exact Sciences Corporation

C12Q1/6818C12Q1/686C12Q1/6876

Abstract

Claims (20)

1 . A composition comprising a 5′ hairpin invasive cleavage structure having a target cleavage site and a FRET labeling system, wherein a first member of the FRET labeling system is attached at a first position on the 5′ hairpin invasive cleavage structure and a second member of the FRET labeling system is attached at a second position on the 5′ hairpin invasive cleavage structure, wherein the target cleavage site is between the first position and the second position;

wherein the 5′ hairpin invasive cleavage structure comprises a 5′ flap, a downstream duplex stem, and an upstream duplex stem that define an invasive cleavage structure, wherein the 5′ flap comprises a hairpin-forming region configured to form a hairpin at a temperature below a 5′ hairpin melting temperature, and wherein formation of a hairpin in the hairpin-forming region suppresses FEN-1 endonuclease cleavage of the 5′ hairpin invasive cleavage structure at the target cleavage site.

2 . The composition of claim 1 , wherein the 5′ hairpin-forming region of the 5′ hairpin invasive cleavage structure is configured to form a hairpin structure at room temperature.

3 . The composition of claim 1 , further comprising a buffer solution comprising Mg ++ .

4 . The composition of claim 1 , further comprising a thermostable flap endonuclease.

5 . The composition of claim 4 , wherein the flap endonuclease comprises a FEN-1 endonuclease from an Archaeal organism.

6 . The composition of claim 1 , further comprising one or more components selected from:

a DNA polymerase;

deoxynucleoside triphosphates; and

primers.

7 . A method, comprising:

i) providing a 5′ hairpin invasive cleavage structure having a target cleavage site and a FRET labeling system, wherein a first member of the FRET labeling system is attached at a first position on the 5′ hairpin invasive cleavage structure and a second member of the FRET labeling system is attached at a second position on the 5′ hairpin invasive cleavage structure, wherein the target cleavage site is between the first position and the second position;

wherein the 5′ hairpin invasive cleavage structure comprises a 5′ flap, a downstream duplex stem, and an upstream duplex stem that define an invasive cleavage structure, wherein the 5′ flap comprises a hairpin-forming region, and wherein formation of a hairpin in the hairpin-forming region suppresses FEN-1 endonuclease cleavage of the 5′ hairpin invasive cleavage structure at the target cleavage site;

ii exposing the 5′ hairpin invasive cleavage structure to a flap endonuclease in a reaction mixture; and

iii) detecting the presence or absence of cleavage of the 5′ hairpin invasive cleavage structure at the target cleavage site.

8 . The method of claim 7 , wherein the 5′ hairpin-forming region of the 5′ hairpin invasive cleavage structure is configured to form a hairpin structure at room temperature.

9 . The method of claim 7 , wherein the 5′ hairpin invasive cleavage structure is exposed to the flap endonuclease at a temperature at which the 5′ hairpin-forming region is not in the form of a hairpin.

10 . The method of claim 7 , wherein the reaction mixture comprises a buffer solution comprising Mg ++ .

11 . The method of claim 7 , wherein the flap endonuclease comprises a thermostable flap endonuclease.

12 . The method of claim 11 , wherein the thermostable flap endonuclease comprises a FEN-1 endonuclease from an Archaeal organism.

Assignments (4)

Continuity (2)

References Cited (138)