COMPOSITIONS AND METHODS FOR CHARACTERIZING ZDHHC1 DNA
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 - 30 . (canceled)
31 . A method of producing an amplified product from a stool sample from a human subject, the method comprising:
a) in a clarified stool supernatant sample, forming a complex comprising:
i) a target sequence-specific capture reagent comprising a particle covalently attached to a target-specific capture probe oligonucleotide complementary to methylated human ZDHHC1 DNA at a site within SEQ ID NO:26 or its complement, and
ii) a strand of methylated ZDHHC1 DNA comprising SEQ ID NO:26 or its complement from a sample from a human subject, wherein cytosine-phosphate-guanine (CpG) dinucleotides in the methylated ZDHHC1 DNA are methylated;
wherein the target-specific capture probe oligonucleotide is specifically hybridized to the strand of methylated ZDHHC1 DNA at a site within SEQ ID NO:26 or its complement;
b) separating the complex from the clarified stool supernatant sample to form a separated sample comprising the strand of methylated ZDHHC1 DNA; and
c) amplifying a region of ZDHHC1 DNA from the separated sample using a pair of primers that hybridize within a region of ZDHHC1 DNA corresponding to SEQ ID NO: 26 or its complement and that are extended by a DNA polymerase to produce an amplified product.
32 . The method of claim 31 , further comprising detecting the amplified product.
33 . The method of claim 32 , wherein detecting the amplified product comprises a quantitative amplification reaction.
34 . The method of claim 33 , wherein the quantitative amplification reaction comprises real-time fluorescence detection.
35 . The method of claim 33 , wherein detecting the amplified product comprises detecting hybridization of amplified product to a detection probe.
36 . The method of claim 35 , wherein the detection probe comprises a flap sequence.
37 . The method of claim 35 , wherein the detection probe comprises a reporter molecule.
38 . The method of claim 37 , where the reporter molecule comprises a fluorophore.
39 . The method of claim 35 , wherein detecting hybridization of the amplified product to the detection probe comprises use of a FEN-1 endonuclease.
40 . The method of claim 35 , wherein detecting hybridization of the amplified product to the detection probe comprises use of a FRET cassette.
41 . The method of claim 33 , wherein detecting the amplified product comprises using one or more of nucleic acid sequencing and mass-based separation.
42 . The method of claim 31 , further comprising prior to step c) a step of treating DNA from separated sample with bisulfite, wherein treatment with bisulfite converts un-methylated dC bases in the DNA from the separated sample to dU bases.
43 . The method of claim 31 , wherein the particle is a magnetic particle.
44 . The method of claim 31 , wherein the target-specific capture probe oligonucleotide has a complete length of between 15 and 100 nucleotides.
45 . The method of claim 44 , wherein the complete length of the target-specific capture probe oligonucleotide is hybridized to the methylated ZDHHC1 DNA within SEQ ID NO:26.
46 . The method of claim 31 , wherein the target-specific capture probe oligonucleotide is covalently attached to the particle by a non-nucleic acid chemical linkage.
47 . The method of claim 46 wherein the non-nucleic acid chemical linkage comprises a multi-carbon chain.
48 . The method of claim 47 , wherein the multi-carbon chain is attached to a 5′ nucleotide of the target-specific capture probe oligonucleotide.
49 . The method of claim 31 , wherein the clarified stool supernatant sample comprises guanidine thiocyanate.
50 . The method of claim 31 , wherein the clarified stool supernatant sample is pretreated to remove assay inhibitors.