Granted Patent B2

US 12,612,664 · App. 17/314,099 · Granted Apr 28, 2026

Inventors: Hatim Allawi (Middleton, WI); Graham P. Lidgard (Middleton, WI); Maria Giakoumopoulos (Middleton, WI); David A. Ahlquist (Rochester, MN); William R. Taylor (Lake City, MN); Douglas Mahoney (Rochester, MN)

Assignees: Exact Sciences Corporation; Mayo Foundation for Medical Education and Research

C12Q1/6886C12Q1/6806C12Q2600/154C12Q2600/16

Abstract

Claims (32)

1 . A composition comprising a reaction mixture comprising:

i) nucleic acid from a human sample;

ii) for each of at least three methylation marker genes selected from the group consisting of SHOX2, HOXA9, ZNF781, BARX1, and EMX1, a pair of primers that specifically hybridize to and are extendible by a DNA polymerase from a region within a methylation marker gene in a target DNA when the target DNA is present in the nucleic acid from the human sample, wherein the target DNA comprises a differentially methylated region of the methylation marker gene that is differently methylated between DNA from a human subject having a lung neoplasm and DNA from a human subject who does not have a lung neoplasm;

iii) amplicon DNA amplified from at least one target DNA by extension of a pair of primers of ii) and comprising a differentially methylated region of the methylation marker gene;

iv) a pair of B3GALT6 primers that specifically hybridize to and are extendible by a DNA polymerase from a region within a B3GALT6 DNA;

v) B3GALT6 amplicon DNA amplified from the B3GALT6 DNA by extension of a pair of primers of iv); and

vi) one or more of a FRET cassette, a FEN-1 endonuclease, and a thermostable DNA polymerase.

2 . The composition of claim 1 , wherein the nucleic acid from a human sample comprises DNA treated with a methylation-sensitive restriction enzyme or with a reagent that selectively modifies unmethylated cytosine nucleotides in DNA.

3 . The composition of claim 1 , wherein the at least three methylation marker genes comprises the group consisting of ZNF781, BARX1, and EMX1.

4 . The composition of claim 1 , wherein the at least three methylation marker genes comprises the group consisting of ZNF781, BARX1, EMX1, and HOXA9.

5 . The composition of claim 1 , further comprising one or more oligonucleotides that each specifically hybridize to a differentially methylated region of methylation marker gene in one or more amplicon DNAs amplified from the at least one target DNA.

6 . The composition of claim 5 , wherein the one or more oligonucleotides comprise reporter molecules.

7 . The composition of claim 6 , where the reporter molecules comprise fluorophores.

8 . The composition of claim 5 , wherein at least one of the one or more oligonucleotides comprises a flap sequence.

9 . The composition of claim 1 , wherein the at least three methylation marker genes comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 21, 106, 146, 267, and 371.

10 . The composition of claim 2 , wherein the at least three methylation marker genes treated with a reagent that selectively modifies unmethylated cytosine residues in DNA comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS:22, 107, 147, 268, and 372.

11 . The composition of claim 1 , wherein the reaction mixture comprises:

i) thermostable DNA polymerase;

ii) dNTPs;

iii) a buffer comprising Mg ++ ;

iv) a flap endonuclease;

v) a flap oligonucleotide; and

vi) a hairpin oligonucleotide comprising a region that is complementary to a portion of said flap oligonucleotide.

12 . The composition of claim 1 , wherein nucleic acid from a human sample comprises DNA prepared from a plasma sample.

13 . A composition comprising a reaction mixture comprising:

i) nucleic acid from a human sample comprising DNA treated with a methylation-sensitive restriction enzyme or with a reagent that selectively modifies unmethylated cytosine nucleotides in DNA;

ii) for each of at least three methylation marker genes selected from the group consisting of SHOX2, HOXA9, ZNF781, BARX1, and EMX1, a pair of primers that specifically hybridize to and are extendible by a DNA polymerase from a region within the methylation marker gene in a target DNA when the target DNA is present in the nucleic acid from the human sample, wherein the target DNA comprises a differentially methylated region of the methylation marker gene that is differently methylated between DNA from a human subject having a lung neoplasm and DNA from a human subject who does not have a lung neoplasm;

iii) amplicon DNA amplified from at least one target DNA by extension of a pair of primers of ii) and comprising a differentially methylated region of the methylation marker gene;

iv) a pair of B3GALT6 primers that specifically hybridize to and are extendible by a DNA polymerase from a region within a B3GALT6 DNA; and

v) B3GALT6 amplicon DNA amplified from the B3GALT6 DNA by extension of a pair of primers of iv).

14 . The composition of claim 13 , wherein the at least three methylation marker genes comprises the group consisting of ZNF781, BARX1, and EMX1.

15 . The composition of claim 13 , wherein the at least three methylation marker genes comprises the group consisting of ZNF781, BARX1, EMX1, and HOXA9.

Assignments (6)

Continuity (5)

References Cited (400)