Methylation assay
Inventors
Lidgard, Graham P. • Domanico, Michael J. • Allawi, Hatim • Zou, Hongzhi
Assignees
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Publication Number
US-12297510-B2
Publication Date
2025-05-13
Expiration Date
Abstract
A method for detecting a methylated genomic locus is provided. In certain embodiments, the method comprises: a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies cytosine to uracil to produce a treated nucleic acid; b) amplifying a product from the treated nucleic acid using a first primer and a second primer, wherein the first primer hybridizes to a site in the locus that contain methylcytosines and the amplifying preferentially amplifies the methylated copies of the genomic locus, to produce an amplified sample; and c) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.
Core Innovation
The patent provides a method for detecting methylation status of a target genomic locus by combining amplification of methylation-preferentially amplified methylated copies with flap assay detection. The amplification is performed on a treated nucleic acid sample in a reaction mixture that includes a thermostable polymerase, nucleotides, and primers for amplifying the target genomic locus, and the first primer hybridizes to a methylated sequence and contains a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in the genomic locus.
The amplified methylated copies are then detected using a flap assay employing flap assay reagents comprising a flap endonuclease, a FRET cassette, and a flap oligonucleotide. The flap oligonucleotide comprises a G or C nucleotide at a position corresponding to the methylated cytosine, and the method is configured so that an invasive oligonucleotide distinct from the first primer is not included, with the first primer serving as an invasive oligonucleotide.
The disclosed approach is positioned as an integrated workflow that supports detection across multiple targets and sample types, including FFPE and stool. The patent also describes internal or control concepts and quantitative sensitivity observations for methylated targets in mixtures with abundant unmethylated copies, with experimental results documented for methylated C6ORF150, ZNF804B, and vimentin (VIM).
Claims Coverage
The independent claim coverage centers on a single integrated detection method with three inventive features: methylation-preferential amplification using a methylation-specific primer whose 3′ terminal G/C corresponds to a methylated cytosine, flap assay detection using a FRET cassette and a flap oligonucleotide aligned to the methylated cytosine, and use of the first primer as an invasive oligonucleotide without including a separate invasive oligonucleotide.
Methylation-preferential amplification from treated nucleic acid sample
Amplifying a target genomic locus in a reaction mixture comprising a treated nucleic acid sample, thermostable polymerase, nucleotides, a first primer and a second primer, wherein the first primer hybridizes to a methylated sequence in the locus and contains a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in the genomic locus, and wherein the reagents preferentially amplify methylated copies of the genomic locus to produce an amplified sample.
Flap assay detection with FRET cassette and flap oligonucleotide aligned to methylated cytosine
Detecting the presence of amplified methylated copies of the genomic locus using a flap assay employing flap assay reagents comprising a flap endonuclease, a FRET cassette, and a flap oligonucleotide that comprises a G or C nucleotide at a position that corresponds to the methylated cytosine.
No distinct invasive oligonucleotide; first primer serves as invasive oligonucleotide
Configuring the flap assay such that an invasive oligonucleotide distinct from the first primer is not included, and wherein the first primer serves as an invasive oligonucleotide.
Overall, the claim coverage is anchored in an integrated methylation detection workflow where a methylation-specific primer with a 3′ terminal G/C directs preferential amplification of methylated copies, and the same primer architecture is linked to flap assay detection using flap endonuclease and a FRET cassette. The flap oligonucleotide is positioned around the methylated cytosine, and no separate invasive oligonucleotide is included because the first primer serves as the invasive oligonucleotide.
Stated Advantages
Integrated detection workflow combining methylation-preferential amplification with flap assay detection using a FRET cassette.
Enables detection of amplified methylated copies by employing flap assay reagents aligned to a methylated cytosine position.
Supports detection in mixtures where unmethylated copies are present in abundance relative to methylated copies.
Documented Applications
Detecting methylation status of methylated genomic loci including methylated C6ORF150, ZNF804B, and vimentin (VIM).
Use of sample types including FFPE and stool/fecal DNA for methylation detection.
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