Detection and analysis of methylated DNA
Provided herein is technology for lung neoplasia screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of lung cancer.
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.