Dual-probe digital droplet pcr strategy for specific detection of tissue-specific circulating dna molecules
AssigneesYissum Research Development Company of the Hebrew University of Jerusalem, Hadasit Medical Research Services and Development Ltd.
InventorsYuval DOR, Ruth SHEMER, Benjamin GLASER, Judith MAGENHEIM
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Abstract
A digital droplet PCR method of analyzing the methylation status of methylation sites of a double-stranded DNA molecule which comprises at least two methylation sites per single strand of the double-stranded DNA molecule is disclosed.
Claims20
A method of analyzing the methylation status of methylation sites of a double-stranded DNA molecule which comprises at least two methylation sites per single strand of said double-stranded DNA molecule, the double-stranded DNA molecule being comprised in a specimen, the method comprising:
- (a) contacting the double-stranded DNA with bisulfite to generate single-stranded DNA molecules of which demethylated cytosines of said single-stranded DNA molecules are converted to uracils;
- (b) fractionating the specimen into a plurality of specimen fractions wherein more than 50% of the fractions contain no more than one single-stranded DNA molecule per specimen fraction;
- and
- (c) determining the methylation status of said at least two methylation sites of said single-stranded DNA molecule in at least one of said specimen fractions, wherein a methylation status of each of said at least two methylation sites on said single-stranded DNA molecule is indicative of the methylation status of methylation sites of a double-stranded DNA molecule.
A kit for analyzing methylation sites of a double-stranded DNA molecule, the kit comprising:
- (i) a first oligonucleotide probe, wherein the 3′ end of said first probe comprises a quenching moiety and the 5′ end is labeled with a first detectable moiety;
- (ii) a second oligonucleotide probe, wherein the 3′ end of said second probe comprises a quenching moiety and the 5′ end is labeled with a second detectable moiety;
- wherein the sequence of said first probe is selected so as to determine a methylation status at a first methylation site of the double-stranded DNA molecule and the sequence of said second probe is selected so as determine a methylation status at a second methylation site of the same double-stranded DNA molecule, wherein said first methylation site and said second methylation site are no more than 300 base pairs apart;
- and
- (iii) a Taqman™ polymerase.
A kit for analyzing methylation sites of a double-stranded DNA molecule, the kit comprising:
- (i) at least two oligonucleotides, wherein the sequence of said first oligonucleotide of said at least two oligonucleotides is selected so as to determine a methylation status at a first methylation site of the double-stranded DNA molecule and the sequence of said second oligonucleotide of said at least two oligonucleotides is selected so as determine a methylation status at a second methylation site of the same double-stranded DNA molecule, wherein said first methylation site and said second methylation site are no more than 300 base pairs apart;
- and
- (ii) a droplet forming oil.
Dependencies are inferred from the claim text.