US 11,952,633 B2
Digital sequence analysis of DNA methylation
David A. Ahlquist, Rochester, MN (US); William R. Taylor, Lake City, MN (US); Hongzhi Zou, Middleton, WI (US); and Graham P. Lidgard, Madison, WI (US)
Assigned to Exact Sciences Corporation, Madison, WI (US); and Mayo Foundation for Medical Research and Education, Rochester, MN (US)
Filed by Exact Sciences Corporation, Madison, WI (US); and Mayo Foundation for Medical Education and Research, Rochester, MN (US)
Filed on Nov. 23, 2020, as Appl. No. 17/101,904.
Application 15/278,697 is a division of application No. 13/364,978, filed on Feb. 2, 2012, granted, now 9,637,792, issued on May 2, 2017.
Application 17/101,904 is a continuation of application No. 16/665,738, filed on Oct. 28, 2019, granted, now 10,870,893.
Application 16/665,738 is a continuation of application No. 15/278,697, filed on Sep. 28, 2016, granted, now 10,519,510, issued on Dec. 31, 2019.
Claims priority of provisional application 61/438,649, filed on Feb. 2, 2011.
Prior Publication US 2021/0095350 A1, Apr. 1, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6858 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/6858 (2013.01); C12Q 2600/154 (2013.01)] 19 Claims
 
1. A method of making an assay for detecting DNA amplified from a methylated marker DNA in the presence of DNA amplified from the unmethylated marker DNA that exhibits background methylation, the method comprising:
a) isolating marker DNA from a first group of samples in which the marker DNA is unmethylated DNA exhibiting background methylation and from a second group of samples in which the marker DNA is methylated;
b) processing the isolated marker DNA using digital sequencing and/or digital polymerase chain reaction (PCR) to determine the methylation status for three or more CpG loci in each of at least 1000 individual copies of the isolated marker DNA from each of the first and second groups of samples;
c) for each of the three or more CpG loci, determining a ratio between a mean methylation at that CpG locus in the first group of samples to a mean methylation at the corresponding CpG locus in the second group of samples;
d) selecting a subset of at least three CpG loci for which the percentage of individual copies of the marker DNA from the first group of samples that are methylated at all of the at least three CpG loci in the subset is less than the percentage of individual copies of the marker DNA from the second group of samples that are methylated at all of the at least three CpG loci in the subset; and
e) creating a detection assay that detects the methylation status of all of the at least three CpG loci in the subset of CpG loci in a strand of the marker DNA, wherein the absence of methylation at any one of the CpG loci in the subset of CpG loci in a strand of the marker DNA produces an assay result classifying that strand of the marker DNA as not methylated,
wherein creating the detection assay comprises synthesizing at least one oligonucleotide complementary to a segment of nucleic acid comprising the subset of CpG loci.