Dual-probe digital droplet pcr strategy for specific detection of tissue-specific circulating dna molecules

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.

What is claimed is: 
     
         1 . 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.   
     
     
         2 . The method of  claim 1 , wherein said determining comprises contacting said amplified DNA with:
 (i) a first probe that hybridizes to said amplified DNA at a site which comprises the first of said at least two methylation sites; and   (ii) a second probe that hybridizes to said amplified DNA at a site which comprises a second of said at least two methylation sites.   
     
     
         3 . The method of  claim 2 , wherein said first probe and said second probe are labeled with non-identical detectable moieties. 
     
     
         4 . The method of  claim 1 , wherein
 a. said double-stranded DNA molecule is no longer than 150 bp;   b. said at least two methylation sites are not more than 150 bp apart;   c. wherein each strand of said double-stranded DNA comprises at least four methylation sites; or   d. wherein each strand of said double-stranded DNA comprises at least four methylation sites which are not more than 150 bp apart.   
     
     
         5 . The method of  claim 1 , wherein said determining is affected using at least two non-identical labels or using a single label. 
     
     
         6 . The method of  claim 5 , wherein said first probe and said second probe comprise a quenching moiety;
 wherein said contacting is affected under conditions that separate said quenching moiety from said first probe and said second probe to generate a non-quenched first probe and a non-quenched second probe; and   wherein the method further comprises analyzing the amount of said non-quenched first probe and said non-quenched second probe in at least one specimen fraction of said plurality of specimen fractions; or   wherein said first probe hybridizes to the forward strand of said amplified DNA and said second probe hybridizes to the reverse strand of said amplified DNA.   
     
     
         7 . The method of  claim 1 , wherein said method further comprises contacting said single-stranded DNA with amplification primers under conditions that generate amplified DNA from said single-stranded DNA following step (b) and prior to step (c) and wherein said determining comprises contacting said amplified DNA with:
 (i) a first probe that hybridizes to said amplified DNA at a site which comprises the first of said at least two methylation sites; and   (ii) a second probe that hybridizes to said amplified DNA at a site which comprises a second of said at least two methylation sites, wherein said first probe and said second probe are labeled with non-identical detectable moieties.   
     
     
         8 . The method of  claim 1 , wherein said double-stranded DNA molecule is differentially methylated in a cell or tissue of interest. 
     
     
         9 . The method of  claim 8 , wherein said cell of interest is selected from the group consisting of a pancreatic beta cell, a pancreatic exocrine cell, a hepatocyte, a brain cell, a lung cell, a uterus cell, a kidney cell, a breast cell, an adipocyte, a colon cell, a rectum cell, a cardiomyocyte, a skeletal muscle cell, a prostate cell and a thyroid cell or wherein said tissue is selected from the group consisting of pancreatic tissue, liver tissue, lung tissue, brain tissue, uterus tissue, renal tissue, breast tissue, fat, colon tissue, rectum tissue, cardiac tissue, skeletal muscle tissue, prostate tissue and thyroid tissue. 
     
     
         10 . The method of  claim 9 , wherein said tissue is cardiac tissue and wherein said double-stranded DNA molecule is non-methylated in cells of cardiac tissue and methylated in leukocytes. 
     
     
         11 . The method of  claim 10 , wherein said double-stranded DNA molecule comprises at least a part of the sequence of human chromosome 12, between coordinates 124692462-124692551 or comprises a sequence which is comprised in SEQ ID NOs: 56 or 57. 
     
     
         12 . The method of  claim 9 , wherein said tissue is liver tissue and wherein said double-stranded DNA molecule comprises a sequence which is comprised in SEQ ID NOs: 65 or 66. 
     
     
         13 . The method of  claim 6 , wherein said first probe comprises a sequence as set forth in SEQ ID NO: 118 and said second probe comprises a sequence as set forth in SEQ ID NO: 119, said first probe comprises a sequence as set forth in SEQ ID NO: 128 and said second probe comprises a sequence as set forth in SEQ ID NO: 129 or said first probe comprises a sequence as set forth in SEQ ID NO: 125 and said second probe comprises a sequence as set forth in SEQ ID NO: 126. 
     
     
         14 . The method of  claim 5 , wherein said single label is comprised on a probe and wherein said probe comprises a sequence as set forth in SEQ ID NO: 130. 
     
     
         15 . The method of  claim 6 , wherein the sequence of said first probe is selected such that said first probe binds to said amplified DNA when said methylation site of said double-stranded DNA molecule is non-methylated or wherein the sequence of said second probe is selected such that said second probe binds to said amplified DNA when said methylation site of said double-stranded DNA molecule is non-methylated. 
     
     
         16 . The method of  claim 6 , wherein said detectable moiety is FAM or HEX. 
     
     
         17 . The method of  claim 1 , wherein
 a. said double-stranded DNA is cell-free DNA;   b. said double-stranded DNA is cellular DNA and wherein said method further comprises lysing the cells of said cellular DNA prior to said determining;   c. said specimen is a fluid specimen;   d. said specimen is a body fluid specimen;   e. said specimen is a body fluid specimen selected from the group consisting of blood, plasma, sperm, milk, urine, saliva and cerebral spinal fluid;   f. said specimen comprises DNA from a plurality of cell-types;   said specimen is blood; or   g. said method further comprises quantitating the amount of DNA of said cell or tissue origin.   
     
     
         18 . 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.   
     
     
         19 . The kit of  claim 16 , further comprising a droplet forming oil. 
     
     
         20 . 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.