Method and kit for determining the tissue or cell origin of dna

Abstract

A method of detecting death of a cell type or tissue in a subject is disclosed. The method comprises determining whether cell-free DNA comprised in a fluid sample of the subject is derived from the cell type or tissue, wherein the determining is effected by ascertaining the methylation status of at least four methylation sites on a continuous sequence of the cell-free DNA, the sequence comprising no more than 300 nucleotides, wherein a methylation status of each of the at least four methylation sites on the continuous sequence of the DNA characteristic of the cell type or tissue is indicative of death of the cell type or tissue. Kits for detecting cell death are also disclosed.

What is claimed is: 
     
         1 . A method for quantifying molecules of cell-free DNA having a methylation pattern of a cell- or tissue-type of interest in a sample derived from a subject, the method comprising:
 (a) identifying a DNA sequence of 50-200 bp that has at least four methylation sites, wherein a methylation pattern of said at least four methylation sites in the cell- or tissue-type of interest is different as compared to a methylation pattern of said at least four methylation sites in a same DNA sequence in a second different cell- or tissue-type, whereby the methylation pattern of said at least four methylation sites in the cell- or tissue-type of interest is characteristic of said cell- or tissue-type of interest, wherein a methylation pattern comprises a methylation status of each of said at least four methylation sites on a given DNA molecule;   (b) amplifying said identified DNA sequence in the cell-free DNA of said sample to obtain amplified DNA molecules;   (c) sequencing each of a plurality of said amplified DNA molecules to obtain a sequence that includes consecutive nucleotides encompassing the at least four methylation sites;   (d) ascertaining from each of said sequences the methylation status of each of said at least four methylation sites, thereby obtaining a methylation pattern for each of said sequenced DNA molecules; and   (e) quantifying the amount of sequenced DNA molecules having a specific methylation pattern corresponding to the cell- or tissue-type of interest that is present in the total of said sequenced DNA molecules.   
     
     
         2 . The method of  claim 1 , wherein said sample is derived from a subject having a disease. 
     
     
         3 . The method of  claim 1 , wherein said sample is a body fluid selected from the group consisting of blood, plasma, serum, sperm, milk, urine, saliva and cerebral spinal fluid. 
     
     
         4 . The method of  claim 1 , wherein said amplifying step (b) is conducted using primers that are methylation-independent. 
     
     
         5 . The method of  claim 1 , further comprising contacting the DNA of the sample with bisulfite to convert unmethylated cytosines of the DNA to uracils prior to said amplifying step (b). 
     
     
         6 . The method of  claim 1 , wherein the sample comprises cell-free DNA which is derived from said second different cell- or tissue-type. 
     
     
         7 . The method of  claim 6 , comprising repeating said step (e) using the second different cell- or tissue-type as the cell- or tissue-type of interest so as to quantify both the amount of sequenced DNA molecules having the specific methylation pattern of DNA molecules of the cell- or tissue-type of interest and the amount of sequenced DNA molecules having the specific methylation pattern of DNA molecules of the second different cell- or tissue-type in the total sequenced DNA molecules. 
     
     
         8 . The method of  claim 7 , further comprising determining a ratio of the amount of DNA molecules having the specific methylation pattern of said cell- or tissue-type of interest to the amount of DNA molecules having the specific methylation pattern of said second cell- or tissue-type. 
     
     
         9 . The method of  claim 6 , wherein said step (e) quantifies the amount of sequenced DNA molecules having the specific methylation pattern of DNA molecules of both the cell or tissue-type of interest and the second different cell- or tissue-type that are present in the total of said sequenced DNA molecules. 
     
     
         10 . The method of  claim 1 , further comprising, after said quantifying step (e), determining a ratio of the amount of cell-free DNA derived from said cell- or tissue-type of interest to the total amount of cell-free DNA in the sequenced DNA molecules. 
     
     
         11 . The method of  claim 1 , wherein said cell- or tissue-type 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. 
     
     
         12 . The method of  claim 1 , wherein said cell- or tissue-type of interest 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, heart tissue, skeletal muscle tissue, prostate tissue and thyroid tissue. 
     
     
         13 . The method of  claim 1 , wherein the sample is a blood sample. 
     
     
         14 . The method of  claim 1 , wherein said ascertaining step (d) is effected using a multiplex reaction. 
     
     
         15 . The method of  claim 1 , wherein said cell- or tissue-type of interest is a non-diseased cell-type. 
     
     
         16 . The method of  claim 1 , wherein said second different cell- or tissue-type is a hematopoietic cell. 
     
     
         17 . The method of  claim 1 , wherein said DNA sequence of 50-200 bp has at least five methylation sites and said methylation pattern comprises the methylation status of each of the at least five methylation sites on a given DNA molecule. 
     
     
         18 . The method of  claim 1 , wherein said DNA sequence is no longer than 170 bp. 
     
     
         19 . The method of  claim 1 , wherein the amplified sequences in said amplifying step (b) are no larger than the size of said DNA sequence identified in step (a). 
     
     
         20 . A method for quantifying molecules of cell-free DNA having a methylation pattern of a cell- or tissue-type of interest in a sample derived from a subject, the method comprising:
 (a) identifying a DNA sequence of 50-200 bp that has at least four methylation sites, wherein a methylation pattern of said at least four methylation sites in the cell- or tissue-type of interest is different as compared to a methylation pattern of said at least four methylation sites in a same DNA sequence in a second different cell- or tissue-type, whereby the methylation pattern of said at least four methylation sites in the cell- or tissue-type of interest is characteristic of said cell- or tissue-type of interest, wherein a methylation pattern comprises a methylation status of each of said at least four methylation sites on a given DNA molecule;   (b) chemically converting unmethylated cytosines of said identified DNA sequence in the cell-free DNA of said sample to uracils, to obtain converted DNA molecules;   (c) amplifying, with at least 15 cycles of polymerase chain reaction (PCR), said converted DNA molecules to obtain amplified DNA molecules;   (d) sequencing each of a plurality of said amplified DNA molecules to obtain a sequence that includes consecutive nucleotides encompassing the at least four methylation sites;   (e) ascertaining from each of said sequences the methylation status of each of said at least four methylation sites, thereby obtaining a methylation pattern for each of said sequenced DNA molecules; and   (e) quantifying the amount of sequenced DNA molecules having a specific methylation pattern corresponding to the cell- or tissue-type of interest that is present in the total of said sequenced DNA molecules.   
     
     
         21 . The method of  claim 20 , wherein, in said step (d), at least 10,000 amplified DNA molecules are sequenced.