US 12,404,546 B2
Detection of methylation status of a DNA sample
Clément Cassart, Vaux-et-Borset (BE); and Renaud Schoemans, Seraing (BE)
Assigned to Diagenode S.A., Ougree (BE)
Filed by DIAGENODE S.A., Seraing (BE)
Filed on Sep. 13, 2024, as Appl. No. 18/885,331.
Application 18/885,331 is a continuation of application No. PCT/IB2023/052012, filed on Mar. 3, 2023.
Claims priority of provisional application 63/320,053, filed on Mar. 15, 2022.
Prior Publication US 2025/0002987 A1, Jan. 2, 2025
Int. Cl. C12Q 1/6851 (2018.01)
CPC C12Q 1/6851 (2013.01) 14 Claims
 
1. A method for determining methylation of a test DNA sample comprising:
(a) digesting a test DNA sample with one or more methylation-sensitive restriction endonuclease(s) (MSRE), resulting in a digested test DNA sample,
(b) inactivating the endonuclease(s);
(c) subjecting the digested test sample DNA to a quantitative amplification reaction with at least two primer pairs configured to amplify first and second loci of the test sample DNA, wherein the second locus includes one or more recognition sites for the enzyme(s) used in step (a) and the first locus lack recognition sites for the enzyme(s) used in step (a);
(d) determining from the quantitative amplification reaction values representing amounts of intact first and second locus in the digested test sample DNA;
(e) obtaining values representing amounts of intact first and second locus in a digested reference sample DNA prepared by an in vitro methylation reaction on reference DNA to provide the reference DNA sample, which values were determined by:
(a′) a reference DNA sample being digested with the one or more methylation-sensitive restriction endonuclease(s) (MSRE), resulting in a digested reference DNA sample, the reference DNA sample being a methylated reference DNA sample;
(b′) the endonuclease(s) being inactivated; and
(c′) the digested reference DNA sample being subjected to separate quantitative amplification reactions with the at least two primer pairs to amplify first and second loci of the reference sample DNA, wherein the second locus includes one or more recognition sites for the enzymes used in step (a) and the first locus lack recognition sites for the enzymes used in step (a); and
(f) determining a value of methylation of the test DNA sample from the relative values of intact first and second locus in the digested test sample DNA normalized by the relative values of intact first and second locus in the digested reference DNA, wherein the relative values are determined by calculating ΔCtts as Cttsl2−Cttsl1 and ΔCtrs as Ctrsl2−Ctrsl1, wherein ts is the test sample, rs is the reference sample, l1 is the first locus and l2 is the second locus and the normalization is performed by calculating ΔΔCt as ΔCtts−ΔCtrs, wherein ΔΔCt is the value representing methylation of the test sample DNA.