US 12,297,510 B2
Methylation assay
Graham P. Lidgard, Middleton, WI (US); Michael J. Domanico, Middleton, WI (US); Hatim Allawi, Middleton, WI (US); and Hongzhi Zou, Middleton, WI (US)
Assigned to Exact Sciences Corporation, Madison, WI (US)
Filed by Exact Sciences Corporation, Madison, WI (US)
Filed on May 12, 2023, as Appl. No. 18/316,883.
Application 18/316,883 is a continuation of application No. 17/170,541, filed on Feb. 8, 2021, granted, now 11,685,956.
Application 17/170,541 is a continuation of application No. 16/522,500, filed on Jul. 25, 2019, abandoned.
Application 16/522,500 is a continuation of application No. 14/539,841, filed on Nov. 12, 2014, abandoned.
Application 14/539,841 is a continuation of application No. 12/946,745, filed on Nov. 15, 2010, granted, now 8,916,344, issued on Dec. 23, 2014.
Prior Publication US 2023/0407407 A1, Dec. 21, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/686 (2013.01); C12Q 2600/154 (2013.01)] 20 Claims
OG exemplary drawing
 
1. A method for detecting methylation status of a target genomic locus, the method comprising:
1) amplifying a target genomic locus in a reaction mixture comprising:
a) a treated nucleic acid sample;
b) amplification reagents comprising a thermostable polymerase, nucleotides, a first primer and a second primer for amplifying the target genomic locus, wherein:
i. 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
ii. the reagents preferentially amplify methylated copies of the genomic locus, to produce an amplified sample;
2) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs 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 and wherein an invasive oligonucleotide distinct from the first primer is not included and wherein the first primer serves as an invasive oligonucleotide.