	US 12,152,273 B2
	Methods and systems for detecting methylation changes in DNA samples
Danny Frumkin, Rehovot (IL); Adam Wasserstrom, Ness Ziona (IL); Revital Knirsh, Rosh Haayin (IL); and Orna Savin, Herzliya (IL)
Assigned to NUCLEIX LTD., Rehovot (IL)
Filed by NUCLEIX LTD., Rehovot (IL)
Filed on Sep. 5, 2022, as Appl. No. 17/902,979.
Application 17/902,979 is a division of application No. 17/439,906, granted, now 11,434,528, previously published as PCT/IL2020/050314, filed on Mar. 17, 2020.
Claims priority of provisional application 62/820,866, filed on Mar. 20, 2019.
Claims priority of application No. 265451 (IL), filed on Mar. 18, 2019.
Prior Publication US 2023/0220458 A1, Jul. 13, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6858 (2018.01); G01N 33/49 (2006.01)

CPC C12Q 1/6858 (2013.01) [G01N 33/491 (2013.01)]

28 Claims

1. A method for sensitive detection of methylation changes in a DNA sample, the method comprising:

(a) providing a DNA sample, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA); and

(b) subjecting the DNA sample to methylation ratio analysis by digesting the DNA sample with at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease, co-amplifying from the digested DNA at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus in a single reaction mixture, and comparing a ratio between signal intensities of the amplification products of each of said at least one restriction locus and the control locus to at least one reference ratio,

thereby detecting methylation changes in the DNA sample at a detection sensitivity of at least 1:100, wherein the DNA is cell-free DNA extracted from a biological fluid sample.