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US 2025/0051839 A1Published application
Methods and systems for detecting methylation changes in dna samples
AssigneeNucleix Ltd.
InventorsDanny FRUMKIN, Adam WASSERSTROM, Revital KNIRSH, Orna SAVIN
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Abstract
Methods and systems for highly sensitive detection of methylation changes in DNA samples are provided, particularly in DNA samples obtained from biological fluids such as plasma and urine.
Claims28
A method for sensitive detection of methylation changes in a DNA sample from a subject, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA) from the subject, the method comprising:
- 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.
1 more element
A method for measuring a methylation ratio of DNA obtained from a subject, the method comprising:
- subjecting a DNA sample from the subject that contains less than 5% ssDNA from the subject 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
- calculating, using software stored in non-transitory memory and implemented on a computer processor, a ratio between the signal intensities of the amplification products of each of said at least one restriction locus and the control locus,
- thereby measuring the methylation ratio of the DNA from the subject.
2 more elements
A system for detecting methylation changes in a DNA sample, the system comprising:
- (a) a DNA sample comprising less than 5% ssDNA;
- and
- (b) components for carrying out a methylation ratio analysis, comprising:
- (i) at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease for digesting the DNA sample;
- (ii) a plurality of primer pairs for co-amplification of a plurality of genomic loci in a single reaction mixture from the DNA sample following digestion, wherein the plurality of genomic loci comprises at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus;
- and (iii) computer software stored on non-transitory computer readable medium, the computer software directs a computer processor to determine methylation changes in the DNA sample based on a comparison of a ratio of signal intensities of the restriction locus and the control locus following amplification to a reference ratio.
3 more elements
Dependencies are inferred from the claim text.