COMPOSITIONS AND METHODS FOR CHARACTERIZING MARKER DNA FROM A SAMPLE
Provided herein is technology relating compositions and methods for detecting tissue cell-specific DNA, such as epithelial cell-specific DNA, in blood or blood products from a subject. The technology also relates to use of tissue cell-specific DNAs as internal controls for methylation assays.
1 . A method of characterizing blood or blood product, comprising:
a) providing a blood or blood product sample from a subject;
b) assaying said sample to detect the presence of epithelial cell-specific DNA;
c) creating a record reporting the presence or absence of epithelial cell-specific DNA in the blood or blood product from said subject,
wherein the presence of epithelial cell-specific DNA is indicative of the presence of epithelial cells or epithelial cell-specific DNA in said blood or blood product sample.
2 . The method of claim 1 , wherein said epithelial cell-specific DNA comprises a DNA that is methylated in epithelial cells and is not methylated in blood cells, and wherein the method comprises treating DNA from said sample with a bisulfite reagent to create bisulfite-converted epithelial cell-specific DNA.
3 . The method of claim 2 , wherein said epithelial cell-specific DNA comprises ZDHHC1 DNA.
4 . The method of claim 3 , wherein said bisulfite-converted epithelial cell-specific DNA comprises a DNA strand comprising the nucleotide sequence of SEQ ID NO:33.
5 . A method for monitoring a disease state in a subject, the method comprising the steps of:
a) obtaining a first blood product sample from the subject at a first time point;
b) initiating a treatment protocol, where said treatment protocol comprises therapeutic intervention;
c) obtaining a second blood product sample from the subject at a second time point, wherein said second time point is after initiation of said treatment protocol; and
d) assaying said first blood product sample and said second blood product sample for an amount of an epithelial cell-specific DNA, and
e) generating a patient record reporting a difference in the amount of epithelial cell-specific DNA between said first blood product sample and said second blood product,
wherein a difference in the amount of epithelial cell-specific DNA between said first blood product sample and said second blood product sample is indicative of a change in the disease state in said subject.
6 . The method of claim 5 , wherein said treatment protocol comprises one or more of surgery, drug therapy, chemotherapy, immunotherapy, nutritional therapy, radiation therapy, temperature therapy, and physical therapy.
7 . The method of claim 5 , wherein a difference in the amount of epithelial cell-specific DNA between said first blood product sample and said second blood product sample is indicative of recurrence, progression, or regression of the disease state in said subject.
8 . The method of claim 5 , wherein said disease state is cancer.
9 . The method of claim 8 , wherein said cancer is metastatic cancer.
10 . The method of claim 5 , wherein said blood product is plasma.
11 . A composition, comprising:
a complex of a ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of said oligonucleotide is hybridized to said ZDHHC1 nucleic acid.
12 . The composition of claim 54 , wherein said ZDHHC1 nucleic acid is bisulfite-converted ZDHHC1 nucleic acid.
13 . A composition comprising a strand of DNA comprising the nucleotide sequence of SEQ ID NO:33 or comprising the nucleotide sequence of SEQ ID NO:27.
14 . The composition of claim 13 , further comprising a detection probe oligonucleotide, wherein the detection probe oligonucleotide comprises a region that is complementary to a portion of said strand of DNA.
15 . The composition of claim 14 , wherein the detection probe oligonucleotide comprises a region that is complementary to a portion of SEQ ID NO:27.
16 . The composition of claim 15 , wherein said detection probe oligonucleotide comprises a reporter molecule.
17 . The composition of claim 16 , where said reporter molecule comprises a fluorophore.
18 . The composition of claim 14 , wherein said detection probe oligonucleotide comprises a flap sequence.
19 . The composition of claim 14 , further comprising a FRET cassette.
20 . The composition of claim 18 , further comprising a FEN-1 endonuclease.
21 . The composition of claim 14 , further comprising a thermostable DNA polymerase.
22 . A reaction mixture comprising the composition of claim 13 .
23 . The reaction mixture of claim 22 , further comprising one or more of a primer, reporter oligonucleotide, a thermostable DNA polymerase, a FEN-1 endonuclease, and a FRET cassette.
24 . A kit, comprising:
a) at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to bisulfite-converted ZDHHC1 DNA; and
b) bisulfite reagent.
25 . The kit of claim 24 , comprising at least one oligonucleotide that comprises a region that is complementary to a portion of SEQ ID NO:27.
26 . The kit of claim 24 , wherein said oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an invasive oligonucleotide.
27 . A method of producing an amplified product, the method comprising:
a) treating DNA from a blood or blood product sample with a bisulfite reagent to produce bisulfite-converted DNA;
b) amplifying a region of said bisulfite-converted DNA using a pair of primers, wherein said amplifying produces amplified product having a sequence comprising a region of SEQ ID NO:27.
28 . The method of claim 27 , further comprising a step of detecting the amplified product with a detection probe.
29 . The method of claim 28 , wherein said detection probe comprises a reporter molecule.
30 . The method of claim 28 , wherein said detection probe comprises a flap sequence.